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Cancers
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Photodynamic Therapy (PDT) in Oncology
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Photodynamic Therapy (PDT) in Oncology

A special issue of Cancers (ISSN 2072-6694).

Deadline for manuscript submissions: closed (31 January 2020) | Viewed by 77744

Special Issue Editors

Prof. Dr. Ángele Juarranz

E-Mail Website
Guest Editor
Department of Biology, Faculty of Sciences, Universidad Autónoma de Madrid, 28049 Madrid, Spain
Interests: photocarcinogenesis; non-melanoma skin cancer; photodynamic therapy; in vitro and in vivo models
Special Issues, Collections and Topics in MDPI journals
Dr. Yolanda Gilaberte

E-Mail Website
Guest Editor
Department of Dermatology, University Hospital Miguel Servet, IIS Aragón, 50009 Zaragoza, Spain
Interests: photoprotection; photodynamic therapy; non-melanoma skin cancer; atopic dermatitis; photodermatology
Special Issues, Collections and Topics in MDPI journals
Dr. Salvador González

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Guest Editor
Department of Medicine and Medical Specialties, Faculty of Medicine, Alcalá de Henares University, 28805 Madrid, Spain
Interests: skin; photoprotection; skin cancer; inflammation; natural products; confocal microscopy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Photodynamic therapy (PDT) is a minimally invasive therapeutic modality approved for treatment of several types of cancer and non-oncological disorders. PDT is able to selectively destroy tumours accessible to light, being used in dermatology for the treatment of non-melanoma skin cancers (basal cell carcinoma and Bowen disease) and precancerous lesions (actinic keratosis) as well as for the treatment of head and neck cancer, endoscopically accessible tumours such as pulmonary, bladder, gastrointestinal and gynaecological neoplasms. Moreover, interstitial PDT has been proposed for solid tumors such as brain and prostate cancer. Additionally, PDT can be used for cancer diagnose being theragnosis a promising technique based for targeted fluorescent imaging and PDT. Although photofrin, aminolevulinic acid and its ester derivatives are the main compounds used in clinical trials, newer photosensitizers and delivery tools are being evaluated. From the first articles published by the group of Dougherty, T.J. that describe the advantages and applications of PDT in the eighties, many investigations the mechanisms of action, new photosensitizers and new cancer applications have been performed.

This special Issue on Cancers is focused on photodynamic therapy and it would include original articles on aspects related with treatment of cancer. In particular, research on photochemical mechanisms, new photosensitizers and delivery tools, cellular and tissue targets, cellular response (cell death and survival), vascular damage and immune response by using different cellular and animal models. Translational work describing the value of PDT alone or in combination with other treatment modalities in cancer treatment will also be included.

Prof. Dr. Ángeles Juarranz
Dr. Yolanda Gilaberte
Dr. Salvador González
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cancers is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • photodynamic therapy
  • photosensitizers
  • action mechanisms
  • in vitro and in vivo models
  • applications in cancer
  • delivery tools
  • resistance mechanisms

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Published Papers (16 papers)

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18 pages, 4499 KiB  
Article
Efficacy of 5-Aminolevulinic Acid in Photodynamic Detection and Photodynamic Therapy in Veterinary Medicine
by Tomohiro Osaki, Inoru Yokoe, Yuji Sunden, Urara Ota, Tomoki Ichikawa, Hideo Imazato, Takuya Ishii, Kiwamu Takahashi, Masahiro Ishizuka, Tohru Tanaka, Liming Li, Masamichi Yamashita, Yusuke Murahata, Takeshi Tsuka, Kazuo Azuma, Norihiko Ito, Tomohiro Imagawa and Yoshiharu Okamoto
Cancers 2019, 11(4), 495; https://doi.org/10.3390/cancers11040495 - 7 Apr 2019
Cited by 31 | Viewed by 5761
Abstract
5-Aminolevulinic acid (5-ALA), a commonly used photosensitizer in photodynamic detection (PDD) and therapy (PDT), is converted in situ to the established photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. To extend 5-ALA-PDT application, we evaluated the PpIX fluorescence induced by exogenous 5-ALA [...] Read more.
5-Aminolevulinic acid (5-ALA), a commonly used photosensitizer in photodynamic detection (PDD) and therapy (PDT), is converted in situ to the established photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. To extend 5-ALA-PDT application, we evaluated the PpIX fluorescence induced by exogenous 5-ALA in various veterinary tumors and treated canine and feline tumors. 5-ALA-PDD sensitivity and specificity in the whole sample group for dogs and cats combined were 89.5 and 50%, respectively. Notably, some small tumors disappeared upon 5-ALA-PDT. Although single PDT application was not curative, repeated PDT+/−chemotherapy achieved long-term tumor control. We analyzed the relationship between intracellular PpIX concentration and 5-ALA-PDT in vitro cytotoxicity using various primary tumor cells and determined the correlation between intracellular PpIX concentration and 5-ALA transporter and metabolic enzyme mRNA expression levels. 5-ALA-PDT cytotoxicity in vitro correlated with intracellular PpIX concentration in carcinomas. Ferrochelatase mRNA expression levels strongly negatively correlated with PpIX accumulation, representing the first report of a correlation between mRNA expression related to PpIX accumulation and PpIX concentration in canine tumor cells. Our findings suggested that the results of 5-ALA-PDD might be predictive for 5-ALA-PDT therapeutic effects for carcinomas, with 5-ALA-PDT plus chemotherapy potentially increasing the probability of tumor control in veterinary medicine. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
►▼ Show Figures

Figure 1

Figure 1
<p>Representative images of photodynamic detection-positive cases. (<b>a</b>–<b>c</b>) Canine thyroid carcinoma. The red fluorescence at 635 nm is evident in the cancerous lesions but not in the necrotic tumor lesions. Scale bar: 2 cm. (<b>d</b>–<b>f</b>) Inguinal lymph node metastasis in a mammary gland tumor. The red fluorescence at 635 nm is evident in the metastatic lesions but not in the non-metastatic regions. Scale bar: 3 mm. (<b>a</b>,<b>d</b>) White light images; (<b>b</b>,<b>e</b>) fluorescence light images; (<b>c</b>,<b>f</b>) hematoxylin and eosin-stained images.</p> Full article ">Figure 2
<p>Representative images of photodynamic detection cases. The upper images comprise the white light images and the lower images are the fluorescence images. (<b>a</b>) Positive case of osteosarcoma of the distal femur. The red fluorescence at 635 nm is evident in the cancerous lesions but not in the necrotic tumor lesions. (<b>b</b>) Negative case of a subcutaneous granulomatous inflammation. Red fluorescence was not observed. (<b>c</b>) False-negative case of splenic hemangiosarcoma with a large quantity of blood. Although tumor cells were observed by histological examination, there was no peak for photosensitizer protoporphyrin IX (PpIX) in the fluorescence spectrum. (<b>d</b>) False-positive case of lymphatic follicles. The red fluorescence and follicles occur at the same location, which might be related to the infiltration of inflammatory cells.</p> Full article ">Figure 3
<p>Interstitial photodynamic therapy and intra-arterial chemotherapy injection for case No. 10. A 12 year old, mixed-breed dog presented with swelling of the left frontal sinus. The dog was orally administered 40 mg/kg of 5-ALA. After 4 h, the tumor was interstitially irradiated with 630 nm laser light emitted (150 mW, 270 J) by a diode laser under general anesthesia. The dog also received weekly doses of 100 mg/m<sup>2</sup> carboplatin and 10 mg/m<sup>2</sup> doxorubicin. An over-the-needle intravenous catheter was inserted through the skin directly into the tumor under computed tomography imaging guidance. The cylindrical fiber (white arrow) could then be passed through an external cylinder of the catheter (white arrowhead). The fiber was placed inside a 14 gauge needle and inserted into the tissue. Doxorubicin hydrochloride was administered with a Huber needle through the skin into the port (yellow arrow).</p> Full article ">Figure 4
<p>Angiographic computed tomography images of case No. 10. (<b>a</b>) The dog was diagnosed with adenocarcinoma of the left paranasal sinus on Day 1. Opacities consistent with soft tissue could be observed in the images of the left nasal cavity and frontal sinus, and the forehead bone was significantly absorbed (red circle). (<b>b</b>) The tumor slightly decreased in size on Day 14 (red circle). (<b>c</b>) Complete remission was achieved on Day 56. (<b>d</b>) Imaging and histological evaluation showed no progression of disease during treatment on Day 304. The dog survived for 718 days after initial presentation.</p> Full article ">Figure 5
<p>Photodynamic therapy and intra-arterial chemotherapy injection for case No. 11. An 11 year old, mixed-breed dog presented with hematuria and polyuria. The dog was diagnosed with transitional cell carcinoma of the lower urinary tract and treated with photodynamic therapy as in case No. 10 along with intra-arterial chemotherapy. An optical fiber placed in an 8Fr versatile catheter was inserted into the urethra (white arrow). The fiber tip was placed at the tumor site under ultrasound and the tumor was irradiated (150 mW, 270 J) under sedation.</p> Full article ">Figure 6
<p>Double contrast images of case No. 11. (<b>a</b>) The tumor size of approximately 2 cm in diameter at the trigone of the bladder could be observed on Day 1 (red circle). The tumor slightly decreased in size on Day 56 (red circle) (<b>b</b>) and was markedly reduced on day 196 (<b>c</b>). The dog survived for 393 days after initial presentation.</p> Full article ">Figure 7
<p>Cytotoxicity of 5-ALA-photodynamic therapy (PDT). A total of 15 primary tumor cell lines (Carcinomas: (<b>a</b>) IUT, (<b>b</b>) Jack, (<b>c</b>) Gal, (<b>d</b>) YDP, (<b>e</b>) HDC, (<b>f</b>) LuBi, (<b>g</b>) JDM, (<b>h</b>) FBC, and (<b>i</b>) SNP; Sarcomas: (<b>j</b>) SGR, (<b>k</b>) KLC, (<b>l</b>) DML, (<b>m</b>) YCC, (<b>n</b>) ITP, and (<b>o</b>) HTR) were incubated with different concentrations of ALA (0, 0.03, 0.1, 0.3, 1 mM) and irradiated for 0 or 500 s. Cell viability was examined using Cell Counting Kit 8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Data are expressed as the means ± S.D. (<span class="html-italic">n</span> = 6).</p> Full article ">Figure 8
<p>Relationship between the effects of photodynamic therapy (PDT) and the intracellular PpIX concentration in carcinomas (<b>a</b>) and sarcomas (<b>b</b>). Carcinomas: IUT (blue closed circle), Jack (blue open circle), Gal (blue closed triangle), YDP (blue open triangle), HDC (blue closed diamond), LuBi (blue open diamond), JDM (blue closed square), FBC (blue open square), and SNP (blue cross); Sarcomas: SGR (orange closed circle), KLC (orange open circle), DML (orange closed triangle), YCC (orange open triangle), ITP (orange closed square), and HTR (orange open square). Correlation of 5-ALA-PDT cytotoxicity at 1 mM with intracellular PpIX concentration was determined using Pearson correlation analysis.</p> Full article ">Figure 9
<p>Correlation of intracellular PpIX concentration and relative mRNA levels in carcinomas. (<b>a</b>) Peptide transporter 1 (PEPT1), (<b>b</b>) peptide transporter 2 (PEPT2), (<b>c</b>) GABA transporter 2 (GAT2), (<b>d</b>) taurine transporter (TAUT), (<b>e</b>) H+/amino acid transporter 1 (PAT1), (<b>f</b>) ATP-binding cassette transporter G2 (ABCG2), (<b>g</b>) ATP-binding cassette transporter B6 (ABCB6, (<b>h</b>) hydroxymethylbilane synthase (HMBS), (<b>i</b>) uroporphyrinogen III synthase (UROS), (<b>j</b>) uroporphyrinogen III decarboxylase (UROD), (<b>k</b>) coproporphyrinogen oxidase (CPOX), (<b>l</b>) ferrochelatase (FECH), (<b>m</b>) heme oxigenase 1 (HO-1). Carcinomas: IUT (blue closed circle), Jack (blue open circle), Gal (blue closed triangle), YDP (blue open triangle), HDC (blue closed diamond), LuBi (blue open diamond), JDM (blue closed square), FBC (blue open square), and SNP (blue cross); Sarcomas: SGR (orange closed circle), KLC (orange open circle), DML (orange closed triangle), YCC (orange open triangle), ITP (orange closed square), and HTR (orange open square). Pearson’s correlation analysis (<span class="html-italic">n</span> = 9).</p> Full article ">Figure 10
<p>Correlation of intracellular PpIX concentration and relative mRNA levels in sarcomas. (<b>a</b>) Peptide transporter 1 (PEPT1), (<b>b</b>) peptide transporter 2 (PEPT2), (<b>c</b>) GABA transporter 2 (GAT2), (<b>d</b>) taurine transporter (TAUT), (<b>e</b>) H+/amino acid transporter 1 (PAT1), (<b>f</b>) ATP-binding cassette transporter G2 (ABCG2), (<b>g</b>) ATP-binding cassette transporter B6 (ABCB6), (<b>h</b>) hydroxymethylbilane synthase (HMBS), (<b>i</b>) uroporphyrinogen III synthase (UROS), (<b>j</b>) uroporphyrinogen III decarboxylase (UROD), (<b>k</b>) coproporphyrinogen oxidase (CPOX), (<b>l</b>) ferrochelatase (FECH), (<b>m</b>) heme oxigenase 1 (HO-1). Carcinomas: IUT (blue closed circle), Jack (blue open circle), Gal (blue closed triangle), YDP (blue open triangle), HDC (blue closed diamond), LuBi (blue open diamond), JDM (blue closed square), FBC (blue open square), and SNP (blue cross); Sarcomas: SGR (orange closed circle), KLC (orange open circle), DML (orange closed triangle), YCC (orange open triangle), ITP (orange closed square), and HTR (orange open square). Pearson’s correlation analysis (<span class="html-italic">n</span> = 6).</p> Full article ">
19 pages, 8840 KiB  
Article
A Basic Study of Photodynamic Therapy with Glucose-Conjugated Chlorin e6 Using Mammary Carcinoma Xenografts
by Tomohiro Osaki, Shota Hibino, Inoru Yokoe, Hiroaki Yamaguchi, Akihiro Nomoto, Shigenobu Yano, Yuji Mikata, Mamoru Tanaka, Hiromi Kataoka and Yoshiharu Okamoto
Cancers 2019, 11(5), 636; https://doi.org/10.3390/cancers11050636 - 8 May 2019
Cited by 21 | Viewed by 5744
Abstract
By using the Warburg effect—a phenomenon where tumors consume higher glucose levels than normal cells—on cancer cells to enhance the effect of photodynamic therapy (PDT), we developed a new photosensitizer, glucose-conjugated chlorin e6 (G-Ce6). We analyzed the efficacy of PDT with G-Ce6 against [...] Read more.
By using the Warburg effect—a phenomenon where tumors consume higher glucose levels than normal cells—on cancer cells to enhance the effect of photodynamic therapy (PDT), we developed a new photosensitizer, glucose-conjugated chlorin e6 (G-Ce6). We analyzed the efficacy of PDT with G-Ce6 against canine mammary carcinoma (CMC) in vitro and in vivo. The pharmacokinetics of G-Ce6 at 2, 5, and 20 mg/kg was examined in normal dogs, whereas its intracellular localization, concentration, and photodynamic effects were investigated in vitro using CMC cells (SNP cells). G-Ce6 (10 mg/kg) was administered in vivo at 5 min or 3 h before laser irradiation to SNP tumor-bearing murine models. The in vitro study revealed that G-Ce6 was mainly localized to the lysosomes. Cell viability decreased in a G-Ce6 concentration- and light intensity-dependent manner in the PDT group. Cell death induced by PDT with G-Ce6 was not inhibited by an apoptosis inhibitor. In the in vivo study, 5-min-interval PDT exhibited greater effects than 3-h-interval PDT. The mean maximum blood concentration and half-life of G-Ce6 (2 mg/kg) were 15.19 ± 4.44 μg/mL and 3.02 ± 0.58 h, respectively. Thus, 5-min-interval PDT with G-Ce6 was considered effective against CMC. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
►▼ Show Figures

Figure 1

Figure 1
<p>Glucose-conjugated chlorin e6 (G-Ce6). (<b>a</b>) Chemical structure of G-Ce6. Methyl (7S,8S)-18-ethyl-5-(2-methoxy-2-oxoethyl)-7-(3-methoxy-3-oxopropyl)-2,8,12,17-tetramethyl-13-(1-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) thio) propoxy) ethyl)-7H,8H-porphyrin-3-carboxylate (G-Ce6). (<b>b</b>) White light image of 2 mg/mL G-Ce6 solution (20% Tween 80 in phosphate buffered saline). (<b>c</b>) Fluorescence image of 2 mg/mL G-Ce6 (excitation, 405 nm). (<b>d</b>) UV-vis spectrum for G-Ce6 in DMSO (3 × 10<sup>−6</sup> M). (<b>e</b>) HPLC spectrum for G-Ce6. Column: Kinetex XB-C18 (2.6 μm (particle size), 4.6 mm (internal diameter), 75 mm (length), Phenomenex), 10 mmol/L ammonium acetate/acetonitrile (35:65, <span class="html-italic">v</span>/<span class="html-italic">v</span>) as solvent, and UV detector at 254 nm were used.</p> Full article ">Figure 2
<p>Subcellular localization of glucose-conjugated chlorin e6 in the mitochondria after 6 h of incubation. The subcellular localization of glucose-conjugated chlorin e6 (G-Ce6) was characterized using fluorescence microscopy. (<b>a</b>) Green fluorescence of MitoTracker Green FM stained mitochondria, (<b>b</b>) red fluorescence of G-Ce6 in the same view as (<b>a</b>), (<b>c</b>) combination of (<b>a</b>) and (<b>b</b>) images, and (<b>d</b>) transmission images. Scale bar, 50 μm.</p> Full article ">Figure 3
<p>Subcellular localization of glucose-conjugated chlorin e6 in the lysosome after 6 h of incubation. The subcellular localization of glucose-conjugated chlorin e6 (G-Ce6) was characterized using fluorescence microscopy. (<b>a</b>) Green fluorescence of LysoTracker Yellow HCK-123 stained lysosome, (<b>b</b>) red fluorescence of G-Ce6 in the same view as (<b>a</b>), (<b>c</b>) combination of (<b>a</b>) and (<b>b</b>) images, and (<b>d</b>) transmission images. The images showed that G-Ce6 mainly localized in the lysosomes. Scale bar, 50 μm.</p> Full article ">Figure 4
<p>Photodynamic cytotoxicity in SNP cells. The cells were incubated with various concentrations of (<b>a</b>) mono-L-aspartyl chlorin e6 (NPe6) and (<b>b</b>) glucose-conjugated chlorin e6 (G-Ce6) for 24 h at 37 °C. After washing with fresh media, the cells were irradiated with 650-nm laser light (10 mW/cm<sup>2</sup>; 0, 1, 5, and 15 J/cm<sup>2</sup>). The viability of SNP cells at 24 h after photodynamic therapy, along with IC<sub>50</sub> values, were determined. (<b>a</b>) The IC<sub>50</sub> values of NPe6 at light doses of 1, 5, and 15 J/cm<sup>2</sup> were 75.2, 30.4, and 30.6 μg/mL, respectively. (<b>b</b>) The IC<sub>50</sub> values of G-Ce6 at light doses of 1, 5, and 15 J/cm<sup>2</sup> were 33.4, 10.4, and 1.7 μg/mL, respectively. IC<sub>50</sub>, half-maximal inhibitory concentration. Results are presented as the mean ± standard deviation.</p> Full article ">Figure 5
<p>Morphological changes in SNP cells 4 h after photodynamic therapy (PDT) with glucose-conjugated chlorin e6 (G-Ce6). The cells were incubated with 10 μg/mL G-Ce6 for 24 h. After washing with fresh media, the cells were irradiated with 650-nm laser light (10 mW/cm<sup>2</sup>; 0, 1, 5, and 15 J/cm<sup>2</sup>). (<b>a,f</b>) Control, (<b>b,g</b>) 0 J/cm<sup>2</sup>, (<b>c,h</b>) 1 J/cm<sup>2</sup>, (<b>d,i</b>) 5 J/cm<sup>2</sup>, (<b>e,j</b>) 15 J/cm<sup>2</sup>. Upper panel: transmitted light images (a–e). Lower panel: fluorescence images (f–j). The cells were stained with Hoechst 33342 dye 4 h after laser irradiation. No signs of apoptosis were observed in the control and 0 J/cm<sup>2</sup> PDT groups (<b>a</b>,<b>b</b>). In the 1 J/cm<sup>2</sup> PDT group, the cells showed membrane blebbing (<b>c</b>). In the 5 J/cm<sup>2</sup> PDT group, the cells showed shrinkage (<b>d</b>). Cells in the 5 and 15 J/cm<sup>2</sup> PDT groups displayed nuclear condensation (<b>i</b>,<b>j</b>).</p> Full article ">Figure 6
<p>Representative images of SNP cells stained with Annexin V-fluorescein isothiocyanate (green) and ethidium homodimer III (red) following photodynamic therapy (PDT). Cells were incubated with 8 μg/mL glucose-conjugated chlorin e6 (G-Ce6) for 24 h. Following washing with fresh medium, the cells were irradiated with 650-nm laser light (10 mW/cm<sup>2</sup>; 0, 1, 5, or 15 J/cm<sup>2</sup>). Following 4 h of PDT, the cells were stained using the Promokine Apoptotic/Necrotic Cells Detection kit. The images depict (<b>a</b>) 8 μg/mL G-Ce6 and 0 J/cm<sup>2</sup> laser energy, (<b>b</b>) 8 μg/mL G-Ce6 and 1 J/cm<sup>2</sup> laser energy, (<b>c</b>) 8 μg/mL G-Ce6 and 5 J/cm<sup>2</sup> laser energy, and (<b>d</b>) 8 μg/mL G-Ce6 and 15 J/cm<sup>2</sup> laser energy. Scale bar, 100 μm.</p> Full article ">Figure 7
<p>Assessment of apoptosis in the kinetics experiment. Apoptosis induced by glucose-conjugated chlorin e6 (G-Ce6)-mediated photodynamic therapy was determined by an Annexin V-fluorescein isothiocyanate fluorescence study using a live-cell analysis system. The cells were incubated with various concentrations of G-Ce6 (0, 0.064, 0.32, 1.6, or 8.0 μg/mL) for 24 h at 37 °C. After washing with fresh media, the cells were irradiated with 650-nm laser light (10 mW/cm<sup>2</sup>; 5 J/cm<sup>2</sup>). The x-axis represents time (h), and the y-axis represents the total green object integrated intensity. Apoptosis was dependent on the concentration of G-Ce6. GCU, green calibrated unit.</p> Full article ">Figure 8
<p>Analysis of apoptosis. Apoptosis was assessed 4 h after laser irradiation using the Muse<sup>®</sup> Annexin V and Dead Cell Assay kit. The cells were incubated with 8.0 μg/mL glucose-conjugated chlorin e6 for 24 h at 37 °C. After washing with fresh media, the cells were irradiated with 650-nm laser light (10 mW/cm<sup>2</sup>; 0, 1, 5, 15 J/cm<sup>2</sup>). Data were analyzed using Dunn’s multiple comparison test (* <span class="html-italic">p</span> &lt; 0.05; control vs. 15 J/cm<sup>2</sup>). The results are presented as the mean ± standard deviation.</p> Full article ">Figure 9
<p>Reactive oxygen species assay. Reactive oxygen species (ROS) generation was assessed 4 h after laser irradiation using the Muse<sup>®</sup> Oxidative Stress kit. The cells were then incubated with 8.0 μg/mL glucose-conjugated chlorin e6 for 24 h at 37 °C. After washing with fresh media, the cells were irradiated with 650-nm laser light (10 mW/cm<sup>2</sup>; 0, 1, 5, 15 J/cm<sup>2</sup>). Data were analyzed using Dunn’s multiple comparison test. (** <span class="html-italic">p</span> &lt; 0.01; 5 J/cm<sup>2</sup> vs. 15 J/cm<sup>2</sup>). The results are presented as the mean ± standard deviation.</p> Full article ">Figure 10
<p>Cytotoxicity in the presence or absence of a pan-caspase inhibitor. SNP cells were incubated with 8 μg/mL glucose-conjugated chlorin e6 for 24 h in the presence (+) or absence (−) of 10 μM Z-VAD-FMK. After rinsing with fresh medium, the cells were irradiated with 650-nm laser light (10 mW/cm<sup>2</sup>; 5 J/cm<sup>2</sup>). (<b>a</b>) The percentage of apoptotic cells; and (<b>b</b>) cell viability (%). The results are presented as the mean ± standard deviation.</p> Full article ">Figure 11
<p>Intratumoral localization of glucose-conjugated chlorin e6 in SNP tumors 5 min and 3 h after administration. (<b>a</b>) Glucose-conjugated chlorin e6 (G-Ce6) fluorescence was observed within the tumor blood vessel 5 min after administration. (<b>b</b>) G-Ce6 fluorescence was apparent in both tumor interstitial tissue and tumor cells 3 h following administration. Scale bar, 50 μm.</p> Full article ">Figure 12
<p>Changes in tumor volume in response to photodynamic therapy (PDT). (<b>a</b>) The 5-min-interval PDT (<span class="html-italic">n</span> = 4). (<b>b</b>) The 3-h-interval PDT (<span class="html-italic">n</span> = 3). Data were analyzed using Bonferroni’s multiple comparison test. The results are presented as the mean ± standard deviation. Data were analyzed using Bonferroni’s multiple comparison test (** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 13
<p>The mean tumor weight 25 days after photodynamic therapy (PDT). (<b>a</b>) The 5-min-interval PDT. (<b>b</b>) The 3-h-interval PDT. The results are presented as the mean ± standard deviation.</p> Full article ">Figure 14
<p>Representative hematoxylin &amp; eosin stained images of the SNP tumors after photodynamic therapy (PDT). (<b>a</b>,<b>d</b>) Untreated tumors. Tumor cells infiltrated the dermis and necrotic tumor cells occupied the center region of the tumors (white arrows). (<b>b</b>,<b>e</b>) At 24 h after the 5-min-interval PDT. Widespread tumor cells appeared necrotic (black arrows). (<b>c</b>,<b>f</b>) At 24 h following the 3-h-interval PDT. Tumor cells at the superficial tumor tissue appeared necrotic (black arrows), whereas deep-seated tumor cells appeared intact. Widespread tumor cells appeared necrotic. (<b>a</b>–<b>c</b>) Scale bar = 200 μm. (<b>d</b>–<b>f</b>) Scale bar = 50 μm.</p> Full article ">Figure 15
<p>The mean concentrations of glucose-conjugated chlorin e6 (G-Ce6) in the plasma after intravenous administration of three doses of G-Ce6. The volume of G-Ce6 required to achieve the desired doses was diluted with 0.5% saline to obtain a total injection volume of 10 mL. The full injection volume was administered intravenously over 10 min at a rate of 60 mL/h; 2 mg/kg (<span class="html-italic">n</span> = 6), 5 mg/kg (<span class="html-italic">n</span> = 3), and 20 mg/kg (<span class="html-italic">n</span> = 1).</p> Full article ">
32 pages, 8011 KiB  
Article
Comparison of Cellular Death Pathways after mTHPC-mediated Photodynamic Therapy (PDT) in Five Human Cancer Cell Lines
by Carsten Lange, Christiane Lehmann, Martin Mahler and Patrick J. Bednarski
Cancers 2019, 11(5), 702; https://doi.org/10.3390/cancers11050702 - 21 May 2019
Cited by 39 | Viewed by 6174
Abstract
One of the most promising photosensitizers (PS) used in photodynamic therapy (PDT) is the porphyrin derivative 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC, temoporfin), marketed in Europe under the trade name Foscan®. A set of five human cancer cell lines from head and neck [...] Read more.
One of the most promising photosensitizers (PS) used in photodynamic therapy (PDT) is the porphyrin derivative 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC, temoporfin), marketed in Europe under the trade name Foscan®. A set of five human cancer cell lines from head and neck and other PDT-relevant tissues was used to investigate oxidative stress and underlying cell death mechanisms of mTHPC-mediated PDT in vitro. Cells were treated with mTHPC in equitoxic concentrations and illuminated with light doses of 1.8–7.0 J/cm2 and harvested immediately, 6, 24, or 48 h post illumination for analyses. Our results confirm the induction of oxidative stress after mTHPC-based PDT by detecting a total loss of mitochondrial membrane potential (Δψm) and increased formation of ROS. However, lipid peroxidation (LPO) and loss of cell membrane integrity play only a minor role in cell death in most cell lines. Based on our results, apoptosis is the predominant death mechanism following mTHPC-mediated PDT. Autophagy can occur in parallel to apoptosis or the former can be dominant first, yet ultimately leading to autophagy-associated apoptosis. The death of the cells is in some cases accompanied by DNA fragmentation and a G2/M phase arrest. In general, the overall phototoxic effects and the concentrations as well as the time to establish these effects varies between cell lines, suggesting that the cancer cells are not all dying by one defined mechanism, but rather succumb to an individual interplay of different cell death mechanisms. Besides the evaluation of the underlying cell death mechanisms, we focused on the comparison of results in a set of five identically treated cell lines in this study. Although cells were treated under equitoxic conditions and PDT acts via a rather unspecific ROS formation, very heterogeneous results were obtained with different cell lines. This study shows that general conclusions after PDT in vitro require testing on several cell lines to be reliable, which has too often been ignored in the past. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Graphical abstract

Graphical abstract
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<p>Dark (■) and light-induced (□) loss of cellular viability after mTHPC treatment as assessed by MTT viability assay. The five cell lines A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) were treated with mTHPC for 24 h in concentrations ranging from 0.001–5.0 µM and kept in the dark (dark toxicity) or illuminated with a light dose of 1.8 J/cm<sup>2</sup> (light-induced toxicity). MTT assay was carried out 24 h post illumination and the absorbance of the reduced formazan was measured at λ = 570 nm. The percentage of cell viability was calculated by dividing the absorbance for the treated group by the absorbance in the solvent dark control. IC<sub>50</sub> and IC<sub>90</sub> values were calculated by using Prism 6. Results are presented as means ± SD from at least three independent experiments.</p> Full article ">Figure 2
<p>Dark (●, 6 h; ■, 24 h) and light-induced (○, 6 h; □, 24 h) cytotoxicity after mTHPC-PDT as assessed by LDH release assay. The five cell lines A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) were treated with mTHPC for 24 h in concentrations ranging from 0.01–10.0 µM and kept in the dark (dark toxicity) or illuminated with a light dose of 1.8 J/cm<sup>2</sup> or 3.5 J/cm<sup>2</sup> (light-induced toxicity). LDH release assay was carried out 24 or 6 h post illumination, respectively, and the absorbance of the reduced INT was measured at λ = 490 nm. The percentage of cytotoxicity was calculated by dividing the absorbance for the treated group by the absorbance at maximum LDH release. Results are presented as means ± SD from at least three independent experiments.</p> Full article ">Figure 3
<p>Reactive oxygen species (ROS) formation after mTHPC-PDT in A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (D), and SISO (<b>E</b>) cells. Cells were treated with solvent (SC) or equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>) of mTHPC between 0.02–0.3 µM, illuminated with 1.8 J/cm<sup>2</sup>, 3.5 J/cm<sup>2</sup>, and 7.0 J/cm<sup>2</sup> or left in the dark, stained with H<sub>2</sub>DCF-DA and DCF fluorescence intensity was measured directly after illumination. Flow cytometric analysis of the single cell population was carried out using the FITC channel (λ<sub>Ex/Em</sub> = 488/525–550 nm). Fluorescence intensity was plotted with reference to non-illuminated, solvent-treated cells (fluorescence intensity was set to 1.0). Cells treated with 1.0–2.0 mM H<sub>2</sub>O<sub>2</sub> for 10 min were used as positive control. Data presented as means ± SD from at least three independent experiments. (** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 4
<p>Analysis of LPO after mTHPC-PDT in A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) cells. Cells were treated with solvent (SC) or equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>) of mTHPC between 0.02–0.3 µM, illuminated with 1.8 J/cm<sup>2</sup> or left in the dark, stained with BODIPY<sup>665/676</sup> and fluorescence intensity was measured 6 (left), 24 (middle), or 48 h (right) after illumination. Flow cytometric analysis of the single cell population was carried out using the APC channel (λ<sub>Ex/Em</sub> = 635/655–730 nm). Fluorescence intensity was plotted with reference to non-illuminated, solvent-treated cells (fluorescence intensity was set to 1.0). Cells treated with 0.4–3.0 mM <span class="html-italic">t</span>-BHP for 24 h (A-427: 0.2–0.4 mM <span class="html-italic">t</span>-BHP for 4 h) were used as positive control. Data presented as means ± SD from at least three independent experiments. (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 5
<p>Evaluation of mitochondrial membrane potential (Δ<span class="html-italic">ψ</span><sub>m</sub>) after mTHPC-PDT in A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) cells. Cells were treated with solvent (SC) or equitoxic concentrations of mTHPC between 0.07–0.3 µM (equal to the respective IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>), illuminated with 1.8 J/cm<sup>2</sup> or left in the dark, and stained with the cationic dye JC-1 after an incubation period of 6 h post illumination or incubation in the dark. JC-1 monomers and aggregates were visualized with a fluorescence microscope equipped with a 63× oil/1.4 NA objective. JC-1 aggregates within active mitochondria are shown in red, whereas cytosolic JC-1 monomers display a green fluorescence. A decrease in red fluorescence indicates a decline in Δ<span class="html-italic">ψ</span><sub>m</sub>, which is a sign of early apoptosis. Fluorescence images were captured with the FITC filter cube (green; λ<sub>Ex/Em</sub> = 460–500/512–542 nm) and the RHOD filter cube (red; λ<sub>Ex/Em</sub> = 541–551/565–605 nm). Solvent-treated and non-illuminated cells served as the negative control with active mitochondria. As a positive control, cells were treated with 50 µM CCCP, a mitochondrial oxidative phosphorylation uncoupling agent.</p> Full article ">Figure 6
<p>Induction of apoptosis, measured by phosphatidylserine externalization after mTHPC-PDT in A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) cells. Cells were treated with either solvent (SC) or equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>) of mTHPC between 0.02–0.3 µM, illuminated with 1.8 J/cm<sup>2</sup> or left in the dark, stained with Annexin V-FITC and propidium iodide (PI) and analyzed 6 (left), 24 (middle), or 48 h (right) after photodynamic treatment. Flow cytometric analysis of the single cell population was carried out using the FITC channel (λ<sub>Ex/Em</sub> = 488/525–550 nm) for the detection of Annexin V-positive cells and the PI channel (λ<sub>Ex/Em</sub> = 488/655–730 nm) for PI-positive cells. The percentage of apoptotic cells is plotted on the left axis, while late-apoptotic cells can be seen on the right axis in opposite direction. Non-illuminated, solvent-treated cells served as the reference sample; cells treated with 0.5–5.0 µM DOXO were used as positive control. Data presented as means ± SD from at least three independent experiments. (<sup>*</sup> <span class="html-italic">p</span> &lt; 0.05; <sup>**</sup> <span class="html-italic">p</span> &lt; 0.01; <sup>***</sup> <span class="html-italic">p</span> &lt; 0.001; <sup>****</sup> <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 7
<p>Poly(ADP-ribose) polymerase (PARP) cleavage in A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) cells after mTHPC-PDT. Cells were treated with either solvent (SC) or equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>) of mTHPC between 0.02–0.3 µM, illuminated with 1.8 J/cm<sup>2</sup> or left in the dark, and total protein extracts were harvested 24 h after illumination and western blotting performed. Representative blots are shown and the % cleaved PARP relative to PARP was evaluated by densitometric analysis. GAPDH was used as a loading control. Data presented as dot plots from at least three independent experiments. Solvent-treated, non-illuminated cells served as the reference group. (** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 8
<p>Caspase 3-activation in A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) cells after photodynamic treatment. Cells were treated with either solvent (SC) or equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>) of mTHPC between 0.02–0.3 µM, illuminated with 1.8 J/cm<sup>2</sup> or left in the dark, and total protein extracts were harvested 24 h after illumination and western blotting performed. Representative blots are shown and % cas 3 relative to pro-cas 3 was evaluated by densitometric analysis. GAPDH was used as a loading control. Data presented as dot plots from at least three independent experiments. Solvent-treated, non-illuminated cells served as the reference group. (<sup>**</sup> <span class="html-italic">p</span> &lt; 0.01; <sup>***</sup> <span class="html-italic">p</span> &lt; 0.001; <sup>****</sup> <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 9
<p>Cell cycle analyses of A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) cells after mTHPC-PDT. Cells were treated with either solvent (SC) or equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a> of mTHPC between 0.02–0.3 µM, illuminated with 1.8 J/cm<sup>2</sup> or left in the dark, stained with PI and analyzed 6 (left), 24 (middle), or 48 h (right) after photodynamic treatment. Flow cytometric analysis of the single cell population was carried out using the PI channel (λ<sub>Ex/Em</sub> = 488/655–730 nm). Cells were assigned to either sub G<sub>1</sub> (black, fragmented DNA, apoptotic), G<sub>0</sub>/G<sub>1</sub> (green), S (red), or G<sub>2</sub>/M (blue) phase. Data presented as means ± SD from at least three independent experiments. Solvent-treated, non-illuminated cells served as the reference group. (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 10
<p>Formation of LC3-II in A-427 (A), BHY (B), KYSE-70 (C), RT-4 (D) and SISO (E) cells after photodynamic treatment. Cells were treated with either solvent (SC) or equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>) of mTHPC between 0.02–0.3 µM, illuminated with 1.8 J/cm<sup>2</sup> or left in the dark, and total protein extracts were harvested 6 (●) or 24 h (○) after illumination and western blotting performed. Representative blots are shown and the amount of LC3-II relative to a solvent-treated, non-illuminated reference control (LC3-II level for this sample was set to 1.0) was evaluated by densitometric analysis. GAPDH was used for normalization. Data presented as dot plots from at least three independent experiments. (** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 11
<p>Formation of LC3-II in A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) cells after photodynamic treatment in absence or presence of lysosomal protease inhibitors pepstatin A (PSA) and E-64d for autophagic flux detection or PI3K inhibitor wortmannin (Wort) for blocking of autophagic sequestration, respectively. Experiments were carried out with selected mTHPC concentrations that led to an increase of LC3-II levels (<a href="#cancers-11-00702-f010" class="html-fig">Figure 10</a>). Cells were treated with equitoxic concentrations (IC<sub>50</sub> or IC<sub>90</sub> in <a href="#cancers-11-00702-t001" class="html-table">Table 1</a>) of mTHPC that led to elevated LC3-II levels in the absence of the particular inhibitor. Only selected concentrations were tested for inhibitor pretreatment was carried out with 100 µM PSA and 10 µg/mL E-64d for 4 h or with 2 µM wortmannin for 1 h before PDT and cells were then illuminated with 1.8 J/cm<sup>2</sup> or left in the dark. Total protein extracts were harvested 24 h after illumination and western blotting performed. Representative blots are shown and the amount of LC3-II was evaluated by densitometric analysis. GAPDH was used for normalization. Data presented as dot plots from at least three independent experiments.</p> Full article ">Figure A1
<p>Representative flow cytometric analysis of ROS formation in SISO cells. Cells were treated with solvent (red and yellow), H<sub>2</sub>O<sub>2</sub> (black), and the IC<sub>50</sub> (green) or IC<sub>90</sub> (blue) of mTHPC. Afterwards, cells were kept in the dark (left) or were illuminated with 1.8 J/cm<sup>2</sup> (right) and stained with H<sub>2</sub>DCF-DA directly after illumination. For each sample, 10,000 events were counted and gated for the single cell population (not shown). DCF fluorescence was detected with the FITC channel (λ<sub>Ex/Em</sub> = 488/525–550 nm) of a MACS Quant flow cytometer. Data were analyzed with the MACS Quantify Software.</p> Full article ">Figure A2
<p>Representative flow cytometric analysis of LPO in SISO cells. Cells were treated with solvent (red and yellow), <span class="html-italic">t</span>-BHP (black), and the IC<sub>50</sub> (green) or IC<sub>90</sub> (blue) of mTHPC. Afterwards, cells were kept in the dark (left) or illuminated with 1.8 J/cm<sup>2</sup> (right) and stained with BODIPY<sup>665/676</sup> 24 h after illumination. For each sample, 10,000 events were counted and gated for the single cell population (not shown). BODIPY<sup>665/676</sup> fluorescence was detected with the APC channel (λ<sub>Ex/Em</sub> = 635/655–730 nm) of a MACS Quant flow cytometer. Data were analyzed with the MACS Quantify Software.</p> Full article ">Figure A3
<p>Representative dot plots after flow cytometric analysis of apoptosis induction in SISO cells. Cells were treated with solvent, DOXO, or the IC<sub>90</sub> of mTHPC. Afterwards, cells were kept in the dark or illuminated with 1.8 J/cm<sup>2</sup> and stained with Annexin V-FITC/PI 24 h after illumination. For each sample, 10,000 events were counted and gated for the single cell population (not shown). Unstained, vital cells appear in the lower left quadrant, Annexin V-FITC (apoptotic) cells in the upper left quadrant and Annexin V-FITC- and PI-positive (late-apoptotic) cells appear in the upper right part. The FITC channel (λ<sub>Ex/Em</sub> = 488/525–550 nm) of a MACS Quant flow cytometer was used for the detection of Annexin V-positive cells and the PI channel (λ<sub>Ex/Em</sub> = 488/655–730 nm) was used for the PI signal. Data were analyzed with the MACS Quantify Software.</p> Full article ">Figure A4
<p>Representative histograms after flow cytometric analysis of cell cycle distribution in SISO cells. Cells were treated with solvent, and the IC<sub>50</sub> or IC<sub>90</sub> of mTHPC. Afterwards, cells were kept in the dark or illuminated with 1.8 J/cm<sup>2</sup>. Fixation and PI staining were carried out 48 h after illumination. For each sample, 10,000 events were counted and gated for the single cell population (not shown) and cells were assigned either sub G<sub>1</sub> (fragmented DNA, apoptotic), G<sub>0</sub>/G<sub>1</sub>, S, or G<sub>2</sub>/M phase. The PI channel (λ<sub>Ex/Em</sub> = 488/655–730 nm) of a MACS Quant flow cytometer was used for the detection of PI fluorescence. Data were analyzed with the MACS Quantify Software.</p> Full article ">Figure A5
<p>Dark (●, 6 h; ■, 24 h) and light-induced (○, 6 h; □, 24 h) loss of cellular viability after mTHPC treatment as assessed by the MTT viability assay (with remaining cells from LDH release assay). The five cell lines A-427 (<b>A</b>), BHY (<b>B</b>), KYSE-70 (<b>C</b>), RT-4 (<b>D</b>), and SISO (<b>E</b>) were treated with mTHPC for 24 h in concentrations ranging from 0.01–10.0 µM and kept in the dark (dark toxicity) or illuminated with a light dose of 1.8 J/cm<sup>2</sup> or 3.5 J/cm<sup>2</sup> (light-induced toxicity). MTT assay was carried out 6 h or 24 h post illumination and the absorbance of the reduced formazan was measured at λ = 570 nm. The percentage of cell viability was calculated by dividing the absorbance for the treated group by the absorbance in the solvent dark control. Results presented as means ± SD from at least three independent experiments.</p> Full article ">
22 pages, 953 KiB  
Review
Photochemical Internalization: Light Paves Way for New Cancer Chemotherapies and Vaccines
by Lara Šošić, Pål Kristian Selbo, Zuzanna K. Kotkowska, Thomas M. Kündig, Anders Høgset and Pål Johansen
Cancers 2020, 12(1), 165; https://doi.org/10.3390/cancers12010165 - 9 Jan 2020
Cited by 33 | Viewed by 6522
Abstract
Photochemical internalization (PCI) is a further development of photodynamic therapy (PDT). In this report, we describe PCI as a potential tool for cellular internalization of chemotherapeutic agents or antigens and systematically review the ongoing research. Eighteen published papers described the pre-clinical and clinical [...] Read more.
Photochemical internalization (PCI) is a further development of photodynamic therapy (PDT). In this report, we describe PCI as a potential tool for cellular internalization of chemotherapeutic agents or antigens and systematically review the ongoing research. Eighteen published papers described the pre-clinical and clinical developments of PCI-mediated delivery of chemotherapeutic agents or antigens. The studies were screened against pre-defined eligibility criteria. Pre-clinical studies suggest that PCI can be effectively used to deliver chemotherapeutic agents to the cytosol of tumor cells and, thereby, improve treatment efficacy. One Phase-I clinical trial has been conducted, and it demonstrated that PCI-mediated bleomycin treatment was safe and identified tolerable doses of the photosensitizer disulfonated tetraphenyl chlorin (TPCS2a). Likewise, PCI was pre-clinically shown to mediate major histocompatibility complex (MHC) class I antigen presentation and generation of tumor-specific cytotoxic CD8+ T-lymphocytes (CTL) and cancer remission. A first clinical Phase I trial with the photosensitizer TPCS2a combined with human papilloma virus antigen (HPV) was recently completed and results are expected in 2020. Hence, photosensitizers and light can be used to mediate cytosolic delivery of endocytosed chemotherapeutics or antigens. While the therapeutic potential in cancer has been clearly demonstrated pre-clinically, further clinical trials are needed to reveal the true translational potential of PCI in humans. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Figure 1

Figure 1
<p>Photochemical internalization. The drug is co-administered with the photosensitizer. The photosensitizer accumulates in cell membranes and the drug is taken up through endocytosis. ROS are generated during illumination, which leads to disruption of the endocytic membrane and release of the drug into the cytosol (modified with courtesy from PCI Biotech: <a href="http://pcibiotech.no/what-is-pci/" target="_blank">http://pcibiotech.no/what-is-pci/</a>).</p> Full article ">Figure 2
<p>Antigen uptake, processing, and T-cell presentation in PCI-based vaccination. Photosensitizer and antigen are endocytosed into an antigen-presenting cell (APC). The photosensitizer is attached to the endosomal membrane and the antigen is contained in the endosomal lumen. After a wash-out period, where excess photosensitizer dissociates from the outer plasma membrane, light exposure causes endosomal eruption and cytosolic release of antigen for proteasomal degradation and MHC class-I presentation to CD8 T cells. In the absence of the photosensitizer and light, endosomes mature and fuse with lysosomes for MHC class-II presentation of digested antigens to CD4 T cells.</p> Full article ">Figure 3
<p>PRISMA flow diagram for systematic selection and review of studies.</p> Full article ">
18 pages, 2947 KiB  
Article
In-Vivo Optical Monitoring of the Efficacy of Epidermal Growth Factor Receptor Targeted Photodynamic Therapy: The Effect of Fluence Rate
by Wei Peng, Henriette S. de Bruijn, Timo L. M. ten Hagen, Kristian Berg, Jan L. N. Roodenburg, Go M. van Dam, Max J. H. Witjes and Dominic J. Robinson
Cancers 2020, 12(1), 190; https://doi.org/10.3390/cancers12010190 - 13 Jan 2020
Cited by 12 | Viewed by 4147
Abstract
Targeted photodynamic therapy (PDT) has the potential to improve the therapeutic effect of PDT due to significantly better tumor responses and less normal tissue damage. Here we investigated if the efficacy of epidermal growth factor receptor (EGFR) targeted PDT using cetuximab-IRDye700DX is fluence [...] Read more.
Targeted photodynamic therapy (PDT) has the potential to improve the therapeutic effect of PDT due to significantly better tumor responses and less normal tissue damage. Here we investigated if the efficacy of epidermal growth factor receptor (EGFR) targeted PDT using cetuximab-IRDye700DX is fluence rate dependent. Cell survival after treatment with different fluence rates was investigated in three cell lines. Singlet oxygen formation was investigated using the singlet oxygen quencher sodium azide and singlet oxygen sensor green (SOSG). The long-term response (to 90 days) of solid OSC-19-luc2-cGFP tumors in mice was determined after illumination with 20, 50, or 150 mW·cm−2. Reflectance and fluorescence spectroscopy were used to monitor therapy. Singlet oxygen was formed during illumination as shown by the increase in SOSG fluorescence and the decreased response in the presence of sodium azide. Significantly more cell death and more cures were observed after reducing the fluence rate from 150 mW·cm−2 to 20 mW·cm−2 both in-vitro and in-vivo. Photobleaching of IRDye700DX increased with lower fluence rates and correlated with efficacy. The response in EGFR targeted PDT is strongly dependent on fluence rate used. The effectiveness of targeted PDT is, like PDT, dependent on the generation of singlet oxygen and thus the availability of intracellular oxygen. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Figure 1

Figure 1
<p>Cell survival of OSC-19-luc2-cGFP (black bar), scc-U2 (striped bar), and scc-U8 (white bar) treated with cetuximab-IRDye700DX using a 24 h DLI and illuminated with different fluence rates to a fluence of 15 J·cm<sup>−2</sup> for OSC-19-luc2-cGFP and scc-U2 and 7 J·cm<sup>−2</sup> for scc-U8. † statistically significant from all other fluence rates investigated with the same cell line with <span class="html-italic">p</span> &lt; 0.05, ‡ statistically significant from all other fluence rates investigated with the same cell line with <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 2
<p>Cell survival of OSC-19-luc2-cGFP (black bar), scc-U2 (dashed bar), and scc-U8 (white bar) cells treated with either NaN<sub>3</sub> only, cetuximab-IRDye700DX mediated photodynamic therapy (PDT) or the combination using a 24 h DLI and illuminated with 20 mW·cm<sup>−2</sup> to a fluence of 15 J·cm<sup>−2</sup> for OSC-19-luc2-cGFP and scc-U2 and 7 J·cm<sup>−2</sup> for scc-U8. ‡ statistically significant from PDT only with <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>Examples of 2D images and 3D surface plots from images collected pre and post PDT of scc-U2 and scc-U8 cells incubated with cetuximab-IRDye700DX for 24 h and singlet oxygen sensor green (SOSG) for 2 h. White arrows point to some of the endo/lysosomes showing cetuximab-IRDye700DX fluorescence. Blue arrow point to blebs formed in response to the PDT treatment. Bar is 20 µm. Height of the peaks correspond to the fluorescence intensities of SOSG (green).</p> Full article ">Figure 4
<p>Example of a set of fluorescence images recorded of scc-U8 cells incubated with cetuximab-IRDye700DX for 24 h and SOSG for 2 h, illuminated with 20 mW·cm<sup>−2</sup> to a fluence of 7 J·cm<sup>−2</sup>. (<b>a</b>) transmission image of cells pre illumination. (<b>b</b>) Cetuximab-IRDye700DX fluorescence (red) and SOSG fluorescence (green) at the start of illumination. (<b>c</b>) Cetuximab-IRDye700DX fluorescence (red) and SOSG fluorescence (green) at the end of illumination. (<b>d</b>) Zoomed in image of region shown in image c. White bar is 200 µm and black bar is 50 µm. Blue arrows point to examples of blebs formed in response to the PDT treatment. (<b>e</b>) The rate of SOSG-EP fluorescence increase in counts per J·cm<sup>−2</sup> during illumination with either 20 or 150 mW·cm<sup>−2</sup> in scc-U2 (orange) and scc-U8 (violet) cells. Open circles express the weighted mean for a set of images as shown in a-c and the solid bar expresses the weighted mean over 3–5 sets of images. (<b>f</b>) The rate of cetuximab-IRDye700DX photobleaching in counts per J·cm<sup>−2</sup> during illumination with either 20 or 150 mW·cm<sup>−2</sup> in scc-U2 (orange) and scc-U8 (violet) cells. Open circles express the weighted mean for a set of images as shown in a-c and the solid bar expresses the weighted mean over 3–5 sets of images.</p> Full article ">Figure 5
<p>Reflectance measurements during PDT using different fluence rates. (<b>a</b>) Example of a collected reflectance spectrum recorded pre illumination (cyan), fitted spectrum (black solid line), and scattering background (dashed line) to determine StO<sub>2</sub>, BVF, and VD. (<b>b</b>) Weighted mean StO<sub>2</sub> determined during PDT at 20 mW·cm<sup>−2</sup> (blue diamonds), 50 mW·cm<sup>−2</sup> (red squares), and 150 mW·cm<sup>−2</sup> (green circles). (<b>c</b>) Weighted mean blood volume fraction (BVF) determined during PDT at 20 mW·cm<sup>−2</sup> (blue diamonds), 50 mW·cm<sup>−2</sup> (red squares), and 150 mW·cm<sup>−2</sup> (green circles). (<b>d</b>) Weighted mean vessel diameter (VD) determined during PDT at 20 mW·cm<sup>−2</sup> (blue diamonds), 50 mW·cm<sup>−2</sup> (red squares), and 150 mW·cm<sup>−2</sup> (green circles). No significant differences in vessel density and blood volume fraction were observed between tumors in each group.</p> Full article ">Figure 6
<p>Fluorescence measurements during PDT using different fluence rates. (<b>a</b>) Example of a collected fluorescence spectrum recorded during illumination (azure blue) and the IRDye700DX basis spectrum (black). (<b>b</b>) Weighted mean intrinsic fluorescence intensity determined during PDT at 20 mW·cm<sup>−2</sup> (blue diamonds), 50 mW·cm<sup>−2</sup> (red squares), and 150 mW·cm<sup>−2</sup> (green circles). (<b>c</b>) Weighted mean of reciprocal of normalized fluorescence during the first 5 J·cm<sup>−2</sup> delivered at 20 mW·cm<sup>−2</sup> (blue diamonds), 50 mW·cm<sup>−2</sup> (red squares), and 150 mW·cm<sup>−2</sup> (green circles) and their corresponding linear regression line.</p> Full article ">Figure 7
<p>Effect of PDT using different fluence rates on the growth of OSC-19-luc2-cGFP tumor. (<b>a</b>) Relative tumor volume in time after treatment with control (black), 20 mW·cm<sup>−2</sup> (blue diamonds), 50 mW·cm<sup>−2</sup> (red squares), and 150 mW·cm<sup>−2</sup> (green circles). (<b>b</b>) Kaplan–Meier plot of the percentage of tumors that didn’t grow to more than 200% of the treatment volume after treatment with control (black), 20 mW·cm<sup>−2</sup> (blue), 50 mW·cm<sup>−2</sup> (red), and 150 mW·cm<sup>−2</sup> (green).</p> Full article ">Figure 8
<p>Schematic drawing of the illumination and single fiber spectroscopy set-up.</p> Full article ">
12 pages, 5680 KiB  
Article
Selective Targeting of Cancer Stem Cells (CSCs) Based on Photodynamic Therapy (PDT) Penetration Depth Inhibits Colon Polyp Formation in Mice
by Jun Ki Kim, Mi Ran Byun, Chi Hoon Maeng, Yi Rang Kim and Jin Woo Choi
Cancers 2020, 12(1), 203; https://doi.org/10.3390/cancers12010203 - 14 Jan 2020
Cited by 10 | Viewed by 3707
Abstract
Targeting cancer stem cells (CSCs) without damaging normal stem cells could contribute to the development of novel radical cancer therapies. Cells expressing leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) constitute a cancer-causing population in the colon; therefore, targeting of Lgr5+ cells is expected [...] Read more.
Targeting cancer stem cells (CSCs) without damaging normal stem cells could contribute to the development of novel radical cancer therapies. Cells expressing leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) constitute a cancer-causing population in the colon; therefore, targeting of Lgr5+ cells is expected to provide an opportunity to mitigate colon cancer. However, the expression of Lgr5 in normal stem cells makes it difficult to prove the efficacy of therapies targeted exclusively at Lgr5+ cancer cells. We used a modified photodynamic therapy technique involving cellular radiative transfer between green fluorescent protein (GFP)-expressing cells and a rose bengal photosensitizer. After treatment, tumors containing GFP-Lgr5+ cells were observed to be significantly suppressed or retarded with little effect on GFP-Lgr5+ stem cells at the crypt bottom. Lgr5+ CSCs were specifically eradicated in situ, when localized based on the depth from the colon lumen, revealing the potential preventive efficacy of Lgr5-targeted therapy on tumor growth. This study supports the idea that Lgr5+ cells localized near the colon luminal surface are central to colorectal cancer. With further development, the targeting of localized Lgr5+ cancer stem cells, which this study demonstrates in concept, may be feasible for prevention of colon cancer in high-risk populations. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Figure 1

Figure 1
<p>Locations of noncancerous and cancerous leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5+) cells in the colon. (<b>A</b>) Schematic depicting the length of the colon crypt and locations of colon stem cells. (<b>B</b>) Confocal image and H&amp;E (Hematoxylin and Eosin) staining of the Lgr5-EGFP-IRES-creERT2 knock-in mouse model. Scale bar: 50 µm (<b>C</b>) Schematic of the novel photodynamic therapy (PDT) method applied to selectively eliminate cancer stem cells while minimizing the effects on normal stem cells at the crypt bottom. This is possible because of the limited penetration depth of the 473-nm laser and the propensity of cancer stem cells and normal stem cells to localize in different parts of the colon. Following the novel PDT method, cancer stem cells were selectively destroyed and tumor formation was decreased.</p> Full article ">Figure 2
<p>Selection of an appropriate laser power to avoid damage of normal stem cells. (<b>A</b>) Photodynamic therapy (PDT) incident light power attenuation was measured with a power meter for transmission through 200 μm of colon tissue. (<b>B</b>) Cells survived at 2 mW/cm<sup>2</sup>; however, there was significant cell death at 7 mW/cm<sup>2</sup> for three types of green fluorescent protein (GFP) cells in vitro. (<b>C</b>) Formation and growth of colon organoids were compared after irradiation treatment at 0, 2, and 7 mW/cm<sup>2</sup>. The organoids were counted (upper left panel). The number of organoids did not vary at 2 mW/cm<sup>2</sup> as compared to the non-treated organoids, but the number significantly decreased following treatment at 7 mW/cm<sup>2</sup>. (40x magnification). ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>Therapeutic effect of Photodynamic therapy (PDT) on Lgr5+ cells. (<b>A</b>) Cylindrical diffuse fibers with a diameter of 600 µm for uniform delivery of light into the mouse colonic epithelium (<b>B</b>) Mouse colon irradiation via the anus using cylindrical diffuse fibers. (<b>C</b>) Representative in vivo bright-field images of the colon of wild-type (upper column) and green fluorescent protein (GFP)-Lgr5 (lower column) mouse models obtained using the Coloview system. (<b>D</b>) Representative images of isolated colons after the experiment. Right panel shows the immunohistochemical analysis of Ki-67 in control (untreated) and laser-irradiated (473-nm laser) colon of GFP-Lgr5 mouse. Scale bar: 200 µm (<b>E</b>) Measurement of polyp number under various conditions of PDT. ns, not significant; **, <span class="html-italic">p</span> &lt; 0.01. The data presented in the graph indicate a significant reduction in colon polyps when photosensitized cancer stem cells were subjected to PDT treatment.</p> Full article ">Figure 4
<p>Wavelength-based deep-tissue damage. (<b>A</b>) Differences between tumor necrosis factor (TNF)-α and Ki-67 before and after illumination. Arrows indicate TNFα-positive cells. The right panel indicates the q uantitative analysis of Ki-67- and TNFα-positive cells. (<b>B</b>) Specific death signal in the circumferential eGFP+ area after rose Bengal (RB) administration and laser irradiation. Scale bar: 100 µm.</p> Full article ">
16 pages, 2507 KiB  
Communication
CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro
by Elisa Gaio, Claudia Conte, Diletta Esposito, Elena Reddi, Fabiana Quaglia and Francesca Moret
Cancers 2020, 12(2), 278; https://doi.org/10.3390/cancers12020278 - 23 Jan 2020
Cited by 49 | Viewed by 4372
Abstract
The presence of rare but highly tumorigenic cancer stem cells (CSCs) within the tumors is recognized as one of the major reasons of failure of conventional chemotherapies, mainly attributed to the development of drug resistance and increasing metastatic potential. Here, we propose a [...] Read more.
The presence of rare but highly tumorigenic cancer stem cells (CSCs) within the tumors is recognized as one of the major reasons of failure of conventional chemotherapies, mainly attributed to the development of drug resistance and increasing metastatic potential. Here, we propose a therapeutic strategy based on the simultaneous delivery of docetaxel (DTX) and the photosensitizer meso-tetraphenyl chlorine disulfonate (TPCS2a) using hyaluronic acid (HA) coated polymeric nanoparticles (HA-NPs) for the targeting and killing of CD44 over-expressing breast cancer (BC) cells, both differentiated and CSCs (CD44high/CD24low population), thus combining chemotherapy and photodynamic therapy (PDT). Using the CD44high MDA-MB-231 and the CD44low MCF-7 cells, we demonstrated the occurrence of CD44-mediated uptake of HA-NPs both in monolayers and mammosphere cultures enriched in CSCs. Cell treatments showed that combination therapy using co-loaded NPs (HA@DTX/TPCS2a-NPs) had superior efficacy over monotherapies (HA@DTX-NPs or HA@TPCS2a-NPs) in reducing the self-renewal capacity, measured as mammosphere formation efficiency, and in eradicating the CSC population evaluated with aldehyde dehydrogenase activity assay and CD44/CD24 immunostaining. In summary, these in vitro studies demonstrated for the first time the potential of the combination of DTX-chemotherapy and TPCS2a-PDT for killing CSCs using properly designed NPs. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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<p>CD44-mediated endocytosis of HA@TPCS<sub>2a</sub>-NPs. (<b>a</b>) Uptake of HA@TPCS<sub>2a</sub>-NPs (50 μg/mL NPs) in MDA-MB-231 and MCF-7 cells after 2 h of incubation at 37 °C in medium with or without 10 mg/mL of free HA. (<b>b</b>) Concentration-dependent uptake of HA@TPCS<sub>2a</sub>-NPs in MDA-MB-231 and MCF-7 cells after 24 h of incubation at 37 °C. Data are expressed as means ± SD of at least three independent experiments, carried out in triplicate; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.001 (Student’s t test).</p> Full article ">Figure 2
<p>Cytotoxicity of single and combined treatments in differentiated MCF-7 cells cultured as monolayers. (<b>a</b>) Dose-response curves of cells incubated for 24 h with single drugs or their combination loaded in HA-NPs and irradiated with 1 J/cm<sup>2</sup> of red light (600–800 nm) when PDT was part of the treatment. After additional 24 h in drug-free medium, cell viability was measured with the MTS assay. Total drug concentration is referred to DTX + TPCS<sub>2a</sub> concentration. Data are expressed as mean percentage ± SD of at least three independent experiments, carried out in triplicate. (<b>b</b>) Plots of combination index (CI) vs. fraction affected (Fa) relative to cells treated with HA@DTX/TPCS<sub>2a</sub>-NPs loaded with DTX and TPCS<sub>2a</sub> in the 1:35 (blue) or 1:5 (red) molar ratio.</p> Full article ">Figure 3
<p>Mammosphere formation efficiency (MFE) after HA-NPs treatments. Percentage MFE measured in first generation mammospheres generated from MDA-MB-231 (<b>a</b>) and MCF-7 (<b>c</b>) monolayered cells incubated for 24 h with HA@DTX-NPs, HA@TPCS<sub>2a</sub>-NPs and HA@DTX/TPCS<sub>2a</sub>-NPs, irradiated with 1 J/cm<sup>2</sup>, and re-seeded in non-adherent conditions to allow formation of spheres (protocol 1). Percentage MFE measured in second generation mammospheres generated from first generation mammospheres of MDA-MB-231 (<b>b</b>) and MCF-7 (<b>d</b>) exposed to drug-loaded NPs for 24 h, irradiated with 1 J/cm<sup>2</sup>, and re-seeded in non-adherent conditions (protocol 2). MFE was evaluated after 7 and 4 days from re-seeding for MCF-7 and MDA-MB-231, respectively. Data are expressed as mean ± S.D. of at least two independent experiments, carried out in triplicate; * <span class="html-italic">p</span> &lt; 0.05 (one-way ANOVA, Bonferroni’s correction).</p> Full article ">Figure 4
<p>ALDEFLUOR assay in MCF-7 (<b>a</b>) and MDA-MB-231 (<b>b</b>) cells. Percentages of ALDH positive cells (<b>a</b>,<b>b</b>) and exemplificative flow cytometry plots (<b>c</b>) measured in first and second-generation mammospheres treated with drugs in HA-NPs. First generation mammospheres were incubated for 24 h with HA-NPs, irradiated with 1 J/cm<sup>2</sup> of red light and re-seeded in non-adherent conditions to allow formation of II-generation spheres. Seven (MCF-7) or 4 (MDA-MB-231) days after the reseed, the population of ALDH<sup>high</sup> cells was evaluated by gating the ALDH<sup>high</sup> cells, (green population and percentages) whose fluorescence in flow cytometry plots (DEAB−) exceeded that of the negative controls (DEAB+) stained with the ALDH inhibitor DEAB to control background fluorescence. Flow cytometry plots are referred to mammospheres treated with drug doses of 0.02 μg/mL DTX, 0.1 μg/mL TPCS<sub>2a</sub>, 0.12 μg/mL DTX + TPCS<sub>2a</sub>. Data are expressed as mean ± S.D. of at least two independent experiments; * <span class="html-italic">p</span> &lt; 0.05 (one-way ANOVA, Bonferroni’s correction).</p> Full article ">Figure 5
<p>Uptake of HA@TPCS<sub>2a</sub>-NPs in MDA-MB-231 and MCF-7 first generation mammospheres incubated for 2 or 24 h with 50 μg/mL HA@TPCS<sub>2a</sub>-NPs (TPCS<sub>2a</sub> dose 1.25 μg/mL). Column 1: Bright field images; Column 2: TPCS<sub>2a</sub> fluorescence at the equatorial plane of mammospheres; Column 3: Three-dimensional reconstruction of TPCS<sub>2a</sub> fluorescence distribution and intensity in the equatorial plane of the mammospheres; Column 4: Maximum projection obtained from the superimposition of 20 different acquired focal planes. Scale bar unit in the images is µm.</p> Full article ">
17 pages, 1660 KiB  
Review
Sensitive Photodynamic Detection of Adult T-cell Leukemia/Lymphoma and Specific Leukemic Cell Death Induced by Photodynamic Therapy: Current Status in Hematopoietic Malignancies
by Takashi Oka, Ken-ichi Matsuoka and Atae Utsunomiya
Cancers 2020, 12(2), 335; https://doi.org/10.3390/cancers12020335 - 2 Feb 2020
Cited by 9 | Viewed by 4560
Abstract
Adult T-cell leukemia/lymphoma (ATL), an aggressive type of T-cell malignancy, is caused by the human T-cell leukemia virus type I (HTLV-1) infections. The outcomes, following therapeutic interventions for ATL, have not been satisfactory. Photodynamic therapy (PDT) exerts selective cytotoxic activity against malignant cells, [...] Read more.
Adult T-cell leukemia/lymphoma (ATL), an aggressive type of T-cell malignancy, is caused by the human T-cell leukemia virus type I (HTLV-1) infections. The outcomes, following therapeutic interventions for ATL, have not been satisfactory. Photodynamic therapy (PDT) exerts selective cytotoxic activity against malignant cells, as it is considered a minimally invasive therapeutic procedure. In PDT, photosensitizing agent administration is followed by irradiation at an absorbance wavelength of the sensitizer in the presence of oxygen, with ultimate direct tumor cell death, microvasculature injury, and induced local inflammatory reaction. This review provides an overview of the present status and state-of-the-art ATL treatments. It also focuses on the photodynamic detection (PDD) of hematopoietic malignancies and the recent progress of 5-Aminolevulinic acid (ALA)-PDT/PDD, which can efficiently induce ATL leukemic cell-specific death with minor influence on normal lymphocytes. Further consideration of the ALA-PDT/PDD system along with the circulatory system regarding the clinical application in ATL and others will be discussed. ALA-PDT/PDD can be promising as a novel treatment modality that overcomes unmet medical needs with the optimization of PDT parameters to increase the effectiveness of the tumor-killing activity and enhance the innate and adaptive anti-tumor immune responses by the optimized immunogenic cell death. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Figure 1
<p>Diagnosis of adult T-cell leukemia/lymphoma (ATL) and the therapeutic strategy. ATL is divided into two types in order to decide treatment strategy; one is an aggressive type and the other is an indolent type. The aggressive types are acute, lymphoma, and unfavorable chronic, while the indolent types include the favorable chronic and smoldering types. Therapeutic strategies are decided based on these classifications. Allo-HCT, allogeneic hematopoietic cell transplantation; ATL-G-CSF, combination chemotherapy consisting of vincristine, vindesine, doxorubicin, mitoxantrone, cyclophosphamide, etoposide, ranimustine, and prednisone with granulocyte-colony stimulating factor support; AZT⁄IFN-α, zidovudine and interferon-α; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP14 is performed every 2 weeks, and CHOP21 is performed every 3 weeks); CR, complete remission; hyper-CVAD, cyclophosphamide, vincristine, doxorubicin, and dexamethasone; MAC, myeloablative conditioning; mEPOCH, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) with modifications; PD, progressive disease; PR, partial remission; PS, performance status; RIC, reduced-intensity conditioning; SD, stable disease; VCAP−AMP−VECP, vincristine, cyclophosphamide, doxorubicin and prednisone (VCAP)-doxorubicin, ranimustine and prednisone (AMP)−vindesine, etoposide, carboplatin, and prednisone (VECP). <a href="#cancers-12-00335-f001" class="html-fig">Figure 1</a> was reproduced and modified from <a href="#cancers-12-00335-f001" class="html-fig">Figure 1</a> in Utsunomiya et al. (<span class="html-italic">Cancer Science</span>, 2015) [<a href="#B19-cancers-12-00335" class="html-bibr">19</a>]. Reprint is permitted by <span class="html-italic">Cancer Science</span>.</p> Full article ">Figure 2
<p>Dynamic changes of Flow cytometry (FCM) profiles during onset and progression of ATL. (<b>A</b>) FCM profiles with the Protoporphyrin IX (PpIX)/TSLC1 parameters indicating the dynamic changes during the onset and progression of ATL. (b–d) asymptomatic carrier (AC) peripheral blood mononuclear cells (PBMCs) profiles showed three patterns: Low-risk ACs (similar to healthy profile), medium-risk ACs (intermediate profile), and high-risk ACs (similar to smoldering ATL profile). (<b>B</b>) Leukemia Risk Index (LRI) and Inflammatory Reaction Index (IRI) changes in healthy donors, ACs and three types of ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). (a) ATL showed onset and progression-dependent increase in LRI. AC PBMCs were classified into three categories (low-, medium-, and high-risk) according to the LRI values, corresponding to the typical FCM profiles in A). (b) HAM/TSP showed high IRI values. This figure was reproduced and modified from <a href="#cancers-12-00335-f003" class="html-fig">Figure 3</a> in Oka et al. (<span class="html-italic">Scientific Reports</span> 2018) [<a href="#B59-cancers-12-00335" class="html-bibr">59</a>]. Reprint is permitted by <span class="html-italic">Scientific Reports</span>.</p> Full article ">Figure 3
<p>FCM analyses of chronic ATL patient PBMCs before and after photodynamic therapy (PDT). After PDT treatment, cells were labeled with PI, tumor suppressor in lung cancer 1 (TSLC1)-Alexa647, and Annexin V-FITC and analyzed. (<b>A</b>) (d) FCM analyses showed that 98.7% of TSLC1(+) ATL leukemic cells were TSLC1(+)/Annexin V(+) dead cells (red), whereas 77.5% of TSLC1(-) normal cells were TSLC1(-)/Annexin V(-) live cells (blue) after aminolevulinic acid (ALA)-PDT treatment, indicating that ALA-PDT induced highly-specific ATL leukemia cell death with minimal damage to normal PBMCs. FCM analyses of chronic ATL patient specimens before and after ALA-PDT treatment. (<b>B</b>) (a,b) Chronic ATL PBMCs incubated in 1 mM 5ALA for 48 h showing 2 peaks corresponding to the normal and ATL leukemic cells in TSLC1-FITC and PpIX profiles. (c,d) After 10 min of light exposure-treatment, the ATL leukemic cell peak completely disappeared and only the normal cell peak remained. This figure is reproduced, modified from Figures 5 and 6 in Oka et al. (<span class="html-italic">Scientific Reports</span> 2018) [<a href="#B59-cancers-12-00335" class="html-bibr">59</a>] Reprint is permitted by <span class="html-italic">Scientific Reports</span>.</p> Full article ">Figure 4
<p>Model of ALA-PDT for hematopoietic malignancies using extracorporeal circulation system.</p> Full article ">
22 pages, 6159 KiB  
Article
Selective Killing of Activated T Cells by 5-Aminolevulinic Acid Mediated Photodynamic Effect: Potential Improvement of Extracorporeal Photopheresis
by Sagar Darvekar, Petras Juzenas, Morten Oksvold, Andrius Kleinauskas, Toril Holien, Eidi Christensen, Trond Stokke, Mouldy Sioud and Qian Peng
Cancers 2020, 12(2), 377; https://doi.org/10.3390/cancers12020377 - 6 Feb 2020
Cited by 13 | Viewed by 3914
Abstract
Extracorporeal photopheresis (ECP), a modality that exposes isolated leukocytes to the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light, is used to treat conditions such as cutaneous T-cell lymphoma and graft-versus-host disease. However, the current procedure of ECP has limited selectivity and efficiency; and [...] Read more.
Extracorporeal photopheresis (ECP), a modality that exposes isolated leukocytes to the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light, is used to treat conditions such as cutaneous T-cell lymphoma and graft-versus-host disease. However, the current procedure of ECP has limited selectivity and efficiency; and produces only partial response in the majority of treated patients. Additionally, the treatment is expensive and time-consuming, so the improvement for this modality is needed. In this study, we used the concept of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor of an endogenously synthesized photosensitizer protoporphyrin IX (PpIX) in combination with blue light to explore the possibility of targeting activated human blood T cells ex vivo. With various T-cell activation protocols, a high ALA-induced PpIX production took place in activated CD3+, CD4+CD25+, and CD8+ T cell populations with their subsequent killing after blue light exposure. By contrast, resting T cells were much less damaged by the treatment. The selective and effective killing effect on the activated cells was also seen after co-cultivating activated and resting T cells. Under our clinically relevant experimental conditions, ALA-PDT killed activated T cells more selectively and efficiently than 8-MOP/UV-A. Monocyte-derived dendritic cells (DCs) were not affected by the treatment. Incubation of ALA-PDT damaged T cells with autologous DCs induced a downregulation of the co-stimulatory molecules CD80/CD86 and also upregulation of interleukin 10 (IL-10) and indoleamine 2,3-dioxygenase expression, two immunosuppressive factors that may account for the generation of tolerogenic DCs. Overall, the data support the potential use of ALA-PDT strategy for improving ECP by selective and effective killing of activated T cells and induction of immune tolerance. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>A</b> and <b>B</b>) Cell cycle and mitosis measurements. PBMCs from healthy donor were activated in vitro with anti-CD3/CD28 antibodies for three days. Resting and activated cells were fixed in methanol and stored at −20 °C until use. Cells were stained with rabbit anti-phospho-Histone H3 followed by Alexa fluor 647 donkey anti-rabbit IgG and Hoechst 33258 dye before the measurements of cell cycle and mitosis by flow cytometry. (<b>C</b>) Resting and activated cells were divided into two groups and stained for CD4/CD25/Ki-67 or CD3/CD8/Ki-67 + fixable viability dye. (<b>D</b>) ALA-induced PpIX in resting and activated CD3<sup>+</sup> T cells. Histograms of PpIX fluorescence in resting cells and anti-CD3/CD28 activated cells with (24 h of ALA incubation) and without ALA were compared; (<b>E</b>) effects of T cell activation protocols on ALA-induced PpIX production. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 for three days or overnight with phytohemagglutinin (PHA) or cell stimulation cocktail (CSC), and then incubated with ALA for 24 h before the measurement of the PpIX content in CD3<sup>+</sup> T cells by flow cytometry. The geometric mean fluorescence (Geo. MFI) intensity of PpIX was used to compare various groups. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 2
<p>Effects of the parameters affecting ALA-induced PpIX production. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 antibodies for 3 days. (<b>A</b>) effects of different ALA incubation time intervals on PpIX production in resting and anti-CD3/CD28 activated CD3<sup>+</sup> T cells; (<b>B</b>) the effect of cell density on ALA-induced PpIX production; (<b>C</b>) the effect of temperature (RT and 37 °C) on ALA-PpIX production in resting, anti-CD3/IL-2 activated and anti-CD3/CD28 activated PBMCs.</p> Full article ">Figure 3
<p>ALA-PDT mediated apoptosis and necrosis of CD3<sup>+</sup>CD25<sup>+</sup> T cells in PBMCs. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 antibodies for three days, followed by ALA incubation (3 mM) for 1 h in the dark. The cells were then irradiated with LED blue light (30 mW/cm<sup>2</sup>, 9 J/cm<sup>2</sup>). Apoptosis and necrosis at 0, 1, 8, and 24 h after irradiation were measured by flow cytometry with annexin V/fixable viability dye staining. The cells with both negative annexin V and fixable viability dye staining were considered as viable cells. (<b>A</b>) Representative data from resting PBMCs; (<b>B</b>) Representative data from activated PBMCs.</p> Full article ">Figure 3 Cont.
<p>ALA-PDT mediated apoptosis and necrosis of CD3<sup>+</sup>CD25<sup>+</sup> T cells in PBMCs. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 antibodies for three days, followed by ALA incubation (3 mM) for 1 h in the dark. The cells were then irradiated with LED blue light (30 mW/cm<sup>2</sup>, 9 J/cm<sup>2</sup>). Apoptosis and necrosis at 0, 1, 8, and 24 h after irradiation were measured by flow cytometry with annexin V/fixable viability dye staining. The cells with both negative annexin V and fixable viability dye staining were considered as viable cells. (<b>A</b>) Representative data from resting PBMCs; (<b>B</b>) Representative data from activated PBMCs.</p> Full article ">Figure 4
<p>Comparison between ALA/Blue light and 8-MOP/UV-A. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 antibodies for three days. The resting and activated cells were incubated with ALA (3 mM) or 8-MOP (1 µM) for 1 h in the dark at 37 °C, and then exposed to the in-house built LED blue light or UV-A light. The CD3<sup>+</sup> cell survivals were measured at 20 h after irradiation with flow cytometry using annexin-V and fixable viability dye. (<b>A</b>) Survivals of CD3<sup>+</sup> T cells after 8-MOP/UV-A; (<b>B</b>) Survivals of CD3<sup>+</sup> T cells after ALA-PDT.</p> Full article ">Figure 5
<p>ALA-induced PpIX production and PDT of CD4<sup>+</sup>CD25<sup>+</sup> T cells. Healthy donor PBMCs were either activated in vitro with anti-CD3/IL-2 or anti-CD3/CD28 or kept non-activated (resting) for three days. (<b>A</b>) geometric mean fluorescence intensity (Geo. MFI) of PpIX in CD4<sup>+</sup>CD25<sup>+</sup> cells incubated with 3 mM ALA for 1 h at 37 °C after anti-CD3/IL-2 or anti-CD3/CD28 activation; (<b>B</b>, <b>C</b> and <b>D</b>) resting and activated cells were incubated with 3 mM ALA for 1 h and then exposed to the in-house built LED blue light as indicated. The survivals of CD4<sup>+</sup>CD25<sup>+</sup> T cells were measured before light or 20 h after light exposure with flow cytometry as described in <a href="#cancers-12-00377-f003" class="html-fig">Figure 3</a>; (<b>B</b>) for resting cells; while (<b>C</b> and <b>D</b>) for activated cells with anti-CD3/IL-2 and anti-CD3/CD28, respectively. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>ALA-induced PpIX production and PDT of CD8<sup>+</sup> T cells. Healthy donor PBMCs were either activated in vitro with anti-CD3/IL-2 or anti-CD3/CD28 or kept non-activated (resting) for 3 days. (<b>A</b>) geometric mean fluorescence intensity (Geo. MFI) of PpIX in CD8<sup>+</sup> cells incubated with 3 mM ALA for 1 h at 37 °C after anti-CD3/IL-2 or anti-CD3/CD28 activation; (<b>B</b>, <b>C</b> and <b>D</b>) resting and activated cells were incubated with 3 mM ALA for 1 h and then exposed to the in-house built LED blue light as indicated. The survivals of CD8<sup>+</sup> T cells were measured before light or 20 h after light exposure with flow cytometry as described in <a href="#cancers-12-00377-f003" class="html-fig">Figure 3</a>. (<b>B</b>) for resting cells, while (<b>C</b> &amp; <b>D</b>) for activated cells with anti-CD3/IL-2 and anti-CD3/CD28, respectively. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 7
<p>ALA-PDT of mixed populations of resting and activated cells. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 antibodies for three days. The activated T cells were then labeled with anti-human CD25-FITC antibody. The resting and CD25-FITC labeled activated T cells were mixed in certain ratios as indicated. The mixed cells were incubated with 3 mM ALA for 1 h at 37 °C and then irradiated with the LED blue light at 0.9 J/cm<sup>2</sup> or 1.8 J/cm<sup>2</sup>. The cell survivals were measured 2 h after light irradiation with flow cytometry as described in <a href="#cancers-12-00377-f003" class="html-fig">Figure 3</a>. The control samples without light are also included. (<b>A</b>) Mixture of 1% CD25-FITC labelled activated T cells with 99% resting PBMCs. (<b>B</b>) Mixture of 5% CD25-FITC labelled activated T cells with 95% resting PBMCs. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 8
<p>Monocyte-derived dendritic cells (DCs) resistant towards ALA-PDT. CellTiter Glo<sup>®</sup> (CTG) luminescent cell viability assay was performed on DCs and Jurkat cells. Up to 3 × 10<sup>5</sup> DCs or Jurkat cells/well were seeded in the Corning<sup>®</sup> 96-well white polystyrene microplates and incubated at 37 °C for 24 h. The cells were then incubated with ALA at various concentrations for 1 h and irradiated with the LED blue light at 1.8 J/cm<sup>2</sup>. The cell survivals were measured at 24 h after light exposure. (<b>A</b>) for DCs, (<b>B</b>) for Jurkat cells, (<b>C</b>) for pure CD11c<sup>+</sup> DCs, and (<b>D</b>) for CD11c<sup>+</sup> DCs isolated from anti-CD3/CD28 activated PBMCs.</p> Full article ">Figure 9
<p>ALA-PDT treated T cells induce tolerogenic DCs. (<b>A</b>) monocyte-derived immature DCs (iDC) were co-cultured with autologous ALA-PDT damaged CD4<sup>+</sup> T cells and then stimulated with LPS for 48 h as illustrated; (<b>B</b>) a representative example of expression of the co-stimulatory molecules CD80 and CD86 analyzed by flow cytometry; (<b>C</b>) IL-10 content in the culture supernatants measured by ELISA. The data is a representative of three independent experiments.</p> Full article ">Figure 10
<p>ALA-PDT treated T cells induce expression of IDO. (<b>A</b>) monocyte-derived immature DCs were incubated with control or PDT-treated autologous CD4<sup>+</sup> T cells at a ratio of 1/2 (4 &amp; 6) or 1/4 (5 &amp; 7) (as indicated) overnight and then stimulated with LPS (50 ng/mL) for further 48 h. The IDO expression was analyzed by Western immunoblotting. Data is a representative of three independent experiments; (<b>B</b>) normalized OD values of IDO expression with β-actin; (<b>C</b>) the Kynurenine contents in the supernatants of activated cells treated with ALA alone or plus light were measured with ELISA; (<b>D</b>) a simple schematic illustration of the suggested mechanism on how ALA-PDT mediated over-expressed IDO in iDCs induces an immune-suppressive effect with the downregulation of CD80 and CD86 and increased expression of IL-10 and Kynurenine.</p> Full article ">Figure A1
<p>LED lamp with its emission spectrum and surface light distribution. For details refer to point 4.8 in materials and methods section.</p> Full article ">Figure A2
<p>Sizes of cells and mitochondria in resting (<b>A</b>) and activated PBMCs (<b>B</b>). Resting and activated PBMCs without ALA treatment were seeded on MatTek glass bottom Petri dishes. Mitochondria were stained with 1 μM Rhodamine 123 (Rh123). For imaging, a Zeiss Axiovert 40CFL microscope with a 100× objective (1.25 NA) was used. For Rh123 detection, the filter combination was composed of a 450–490 nm (peak at 475–485 nm) excitation BP filter and a &gt;515 nm emission LP filter.</p> Full article ">Figure A3
<p>Dark toxicity of ALA on resting and activated CD3<sup>+</sup> T cells. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 antibodies for three days. The resting and activated PBMCs were incubated with 0, 3, and 10 mM ALA for 1 h in the dark. The cells labeled with anti-CD3, fixable viability dye, and annexin V were analyzed by flow cytometry as described in <a href="#cancers-12-00377-f003" class="html-fig">Figure 3</a>.</p> Full article ">
22 pages, 2638 KiB  
Article
Role of Polymer Micelles in the Delivery of Photodynamic Therapy Agent to Liposomes and Cells
by Laure Gibot, Maxime Demazeau, Véronique Pimienta, Anne-Françoise Mingotaud, Patricia Vicendo, Fabrice Collin, Nathalie Martins-Froment, Stéphane Dejean, Benjamin Nottelet, Clément Roux and Barbara Lonetti
Cancers 2020, 12(2), 384; https://doi.org/10.3390/cancers12020384 - 7 Feb 2020
Cited by 17 | Viewed by 3480
Abstract
The use of nanocarriers for hydrophobic photosensitizers, in the context of photodynamic therapy (PDT) to improve pharmacokinetics and bio-distribution, is well-established. However, the mechanisms at play in the internalization of nanocarriers are not well-elucidated, despite its importance in nanocarrier design. In this study, [...] Read more.
The use of nanocarriers for hydrophobic photosensitizers, in the context of photodynamic therapy (PDT) to improve pharmacokinetics and bio-distribution, is well-established. However, the mechanisms at play in the internalization of nanocarriers are not well-elucidated, despite its importance in nanocarrier design. In this study, we focus on the mechanisms involved in copolymer poly(ethylene oxide)-block-poly(ε-caprolactone) PEO-PCL and poly(ethylene oxide)-block-poly styrene PEO-PS micelles - membrane interactions through complementary physico-chemical studies on biomimetic membranes, and biological experiments on two-dimensional (2D) and three-dimensional (3D) cell cultures. Förster Resonance Energy Transfer measurements on fluorescently-labelled lipid vesicles, and flow cytometry on two cancerous cell lines enabled the evaluation in the uptake of a photosensitizer, Pheophorbide a (Pheo), and copolymer chains towards model membranes, and cells, respectively. The effects of calibrated light illumination for PDT treatment on lipid vesicle membranes, i.e., leakage and formation of oxidized lipids, and cell viability, were assessed. No significant differences were observed between the ability of PEO-PCL and PEO-PS micelles in delivering Pheo to model membranes, but Pheo was found in higher concentrations in cells in the case of PEO-PCL. These higher Pheo concentrations did not correspond to better performances in PDT treatment. We demonstrated that there are subtle differences in PEO-PCL and PEO-PS micelles for the delivery of Pheo. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Full article ">Figure 1
<p>The transfer of Pheophorbide from PEO-PCL and PEO-PS micelles to Large Unilamellar Vesicles LUVs. (<b>a</b>) Determination of Pheo association constant, <math display="inline"><semantics> <mrow> <msub> <mi>K</mi> <mrow> <mi>a</mi> <mi>s</mi> </mrow> </msub> </mrow> </semantics></math>, for the polymer micelles assessed by fluorescence experiments. Statistical analysis by t-test, *** = <span class="html-italic">p</span> &lt; 0.001 (<b>b</b>). The transfer of Pheo from PBS solution (free Pheo) and Pheo loaded copolymer micelles to liposomes assessed by Förster Resonance Energy Transfer. <math display="inline"><semantics> <mrow> <msub> <mi>K</mi> <mrow> <mi>t</mi> <mi>r</mi> </mrow> </msub> </mrow> </semantics></math> is the constant of Pheo transfer in the different conditions. Statistical analysis by One-way Anova followed by Tukey’s multiple comparisons test. ns = non-significant; Data are represented as the mean value ± SEM.</p> Full article ">Figure 2
<p>Pheophorbide fluorescence in human tumor cells exposed to Pheo loaded PEO-PCL and PEO-PS micelles. (<b>a</b>) Quantification by flow cytometry of Pheo fluorescence in HCT-116 when incubated over 24 h with Pheo in PBS (free Pheo) or Pheo loaded PEO-PCL or PEO-PS micelles. (<b>b</b>) Quantification by flow cytometry of Pheo fluorescence in A375 cells when incubated over 24 h with Pheo in PBS (free Pheo) or Pheo loaded PEO-PCL or PEO-PS micelles. <span class="html-italic">n</span> = 4. Data are represented as the mean value ± SEM.</p> Full article ">Figure 3
<p>Transfer of polymers from micelles to LUVs. (<b>a</b>) Analysis of Rhodamine labelled PEO-PCL transfer from PEO-PCL micelles to NBD-LUVs, assessed by FRET. (<b>b</b>) Carboxyfluoresceine leakage from DOPC LUVs alone and DOPC LUVs challenged with free Pheo and with the PEO-PCL and PEO-PS micelles.</p> Full article ">Figure 4
<p>Internalization of PEO-PCL by human tumor cells. (<b>a</b>) Comparison of cell penetration kinetics of Pheo loaded PEO-PCL micelles and rhodamine labelled PEO-PCL micelles, quantified by flow cytometry in HCT-116. (<b>b</b>) Comparison of cell penetration kinetics of Pheo loaded PEO-PCL micelles and rhodamine labelled PEO-PCL micelles, quantified by flow cytometry in HCT-116. <span class="html-italic">n</span> = 4. Data are represented as the mean value ± SEM.</p> Full article ">Figure 5
<p>Analysis of Pheo loaded micelles interactions with liposomes under light irradiation. (<b>a</b>) The permeability of liposomes was quantified by fluorescence through carboxyfluoresceine (CBF) leakage. Dashed bar indicates light irradiation. LUV = Large Unilamellar Vesicle. (<b>b</b>) P = permeability constant. (<b>c</b>) Singlet oxygen quantum yield of free Pheo of Pheo loaded micelles quantified by spectrophotometric analysis. (<b>d</b>) Determination of oxidation rate constant from fitted UPLC-MS data. *** = <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>Assessment of tumor cell viability after PDT treatment with Pheo loaded PEO-PCL and PEO-PS micelles in 2D monolayers and flow cytometry analysis of Pheo levels in cells. (<b>a</b>) Cell viability was quantified 24 h after PDT treatment on monolayers using prestoblue assay. <span class="html-italic">n</span> = 3, <span class="html-italic">n</span> &gt; 15. (<b>b</b> and <b>c</b>) After 30 min of incubation with Pheo loaded micelles or free Pheo, cells were analyzed by flow cytometry for positively labelled cells percentage (<b>b</b>) and the fluorescence intensity of Pheo in positively labelled cells (<b>c</b>). Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test. ns = non-significant; **** = <span class="html-italic">p</span> &lt; 0.0001. Data are represented as the mean value ± SEM.</p> Full article ">Figure 7
<p>Assessment of tumor cell viability after PDT treatment with Pheo loaded PEO-PCL and PEO-PS micelles on 3D spheroids. (<b>a</b>) Growth curve of tumor spheroids after PDT treatment. <span class="html-italic">n</span> = 6. (<b>b)</b> Cell viability assessed 6 days after PDT treatment by intracellular ATP quantification on spheroids. <span class="html-italic">n</span> = 6. Statistical analysis by one (<b>b</b>) or two (<b>a</b>) –way ANOVA followed by Tukey’s multiple comparisons test. ns = non-significant; * = <span class="html-italic">p</span> &lt; 0.1, ** = <span class="html-italic">p</span> &lt; 0.05, **** = <span class="html-italic">p</span> &lt; 0.0001. Data are represented as the mean value ± SEM.</p> Full article ">Figure A1
<p>Morphological aspect of 2D monolayers and 3D tumor spheroids produced with human colorectal HCT-116 and human melanoma A375 cell lines.</p> Full article ">
24 pages, 6543 KiB  
Article
Photochemically-Induced Release of Lysosomal Sequestered Sunitinib: Obstacles for Therapeutic Efficacy
by Judith Jing Wen Wong, Maria Brandal Berstad, Ane Sofie Viset Fremstedal, Kristian Berg, Sebastian Patzke, Vigdis Sørensen, Qian Peng, Pål Kristian Selbo and Anette Weyergang
Cancers 2020, 12(2), 417; https://doi.org/10.3390/cancers12020417 - 11 Feb 2020
Cited by 15 | Viewed by 4732
Abstract
Lysosomal accumulation of sunitinib has been suggested as an underlying mechanism of resistance. Here, we investigated if photochemical internalization (PCI), a technology for cytosolic release of drugs entrapped in endosomes and lysosomes, would activate lysosomal sequestered sunitinib. By super-resolution fluorescence microscopy, sunitinib was [...] Read more.
Lysosomal accumulation of sunitinib has been suggested as an underlying mechanism of resistance. Here, we investigated if photochemical internalization (PCI), a technology for cytosolic release of drugs entrapped in endosomes and lysosomes, would activate lysosomal sequestered sunitinib. By super-resolution fluorescence microscopy, sunitinib was found to accumulate in the membrane of endo/lysosomal compartments together with the photosensitizer disulfonated tetraphenylchlorin (TPCS2a). Furthermore, the treatment effect was potentiated by PCI in the human HT-29 and the mouse CT26.WT colon cancer cell lines. The cytotoxic outcome of sunitinib-PCI was, however, highly dependent on the treatment protocol. Thus, neoadjuvant PCI inhibited lysosomal accumulation of sunitinib. PCI also inhibited lysosomal sequestering of sunitinib in HT29/SR cells with acquired sunitinib resistance, but did not reverse the resistance. The mechanism of acquired sunitinib resistance in HT29/SR cells was therefore not related to lysosomal sequestering. Sunitinib-PCI was further evaluated on HT-29 xenografts in athymic mice, but was found to induce only a minor effect on tumor growth delay. In immunocompetent mice sunitinib-PCI enhanced areas of treatment-induced necrosis compared to the monotherapy groups. However, the tumor growth was not delayed, and decreased infiltration of CD3-positive T cells was indicated as a possible mechanism behind the failed overall response. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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<p>Close proximity of photosensitizer and sunitinib in endo/lysosomal membranes results in photochemical damage of sunitinib and lack of enhanced cytotoxicity with “light after” sunitinib-photochemical internalization (PCI). Representative fluorescence microscopy images of (<b>a</b>) intracellular co-localization (yellow) of sunitinib (green) and LysoTracker Red (red) after 24 h 2 µM sunitinib incubation in live HT-29 cells and (<b>b</b>) LysoTracker Green (green) and TPCS<sub>2a</sub> (red) co-localization (yellow) after 18 h 0.4 µg/mL TPCS<sub>2a</sub> incubation and 4 h wash (left) followed by 60 s blue light exposure (right). Blue: Hoechst 33342 stained nucleus. Scale bars: 20 µm. Cellular viability (MTT) of HT-29 cells (<b>c</b>) post-PCI “light after” procedure of 1 µM sunitinib (48 + 18 h incubation) with blue light at LD<sub>50</sub> (~60 s) or (<b>d</b>) post-PCI “light after” procedure of 8 µM sunitinib (48 + 18 h incubation) with 90 s red light (mean of three experiments ± S.E.) or (<b>e</b>) 0.5 µM rGel using 60 s blue light (representative experiment of three, mean of triplicates ± S.D.). 60 s blue light ≈ 0.58 J/cm<sup>2</sup>, 90 s red light ≈ 0.54 J/cm<sup>2</sup>. (<b>f</b>) Superresolution (structured illumination microscopy, SIM) images of 2 µM sunitinib (green) and 0.4 µg/mL TPCS<sub>2a</sub> (red) in live HT-29/SR cells after 18 h TPCS<sub>2a</sub> incubation and 4 h chase. Co-localization indicated in yellow. Images are presented with maximum intensity projection of seven z-sections. One single z-section is presented for the enlarged images. Scale bars: 2 µm and 200 nm (enlarged) (<b>g</b>) Representative fluorescence emission spectra of 0.15 µg/mL TPCS<sub>2a</sub>, 1.5 µM sunitinib, and the combination in phosphate-buffered saline (PBS) with 1% fetal bovine serum (FBS) before and after blue light exposure (≈18.9 J/cm<sup>2</sup>) at pH 7. Data in the table are presented as decrease in peak intensity (%) after light exposure (mean of three experiments ± S.E.). n.s.: not significant. Statistical significance calculated with Student’s test (two-tailed <span class="html-italic">p</span> value).</p> Full article ">Figure 2
<p>“Light first” sunitinib-PCI induces a synergistic cytotoxic treatment response. Cellular viability (MTT) of HT-29 cells post-PCI “light first” of (<b>a</b>) sunitinib at increasing concentrations (representative of three experiments, mean of triplicates ± S.D.) or (<b>b</b>) 8 µM sunitinib (data are mean of three independent experiments ± S.E.) exposed to 90 s red light. (<b>c</b>) PCI “light first” of sunitinib at increasing concentrations evaluated with clonogenic assay (60 s blue light, representative experiment of three, mean of triplicates ± S.D.). (<b>d</b>) Cellular viability (MTT) of CT26.WT cells post-PCI “light first” of sunitinib exposed to 40 s blue light. Sun: sunitinib. Statistical significance calculated with Student’s test (two-tailed <span class="html-italic">p</span> value) where *** indicates <span class="html-italic">p</span> ≤ 0.001 and ** <span class="html-italic">p</span> ≤ 0.01. Cells were incubated with sunitinib for 72 h after light-exposure (<b>e</b>) Representative live cell fluorescence microscopy images of “light after” PCI of 2 µM sunitinib and (<b>f</b>) “light first” PCI of 6 µM sunitinib before and 1 h after blue light exposure (60 s). Sunitinib (green), TPCS<sub>2a</sub> (red), Hoechst-stained nucleus (blue). Co-localization indicated in yellow. Scale bar: 20 µm. 60 s blue light ≈ 0.58 J/cm<sup>2</sup>, 90 s red light ≈ 0.54 J/cm<sup>2.</sup></p> Full article ">Figure 3
<p>“Light first” sunitinib-PCI enhance cytotoxicity in HT-29/SR cells but cannot overcome sunitinib resistance. (<b>a</b>) Relative viability 72 h after sunitinib incubation measured by MTT. The HT-29/SR cells had been exposed to sunitinib for 3 months and were seeded in sunitinib-free medium. The graph is a representative experiment of five, mean of triplicates ± S.D. Sunitinib resistance in HT-29/SR cells was verified with (<b>b</b>) clonogenic assay and (<b>c</b>) proliferative capacity. The data points show % confluence at different sunitinib concentrations using IncuCyte live-cell analysis system. HT-29/SR cells were seeded out without sunitinib present. (<b>d</b>) Live cell fluorescence image of HT-29/SR showing co-localization (yellow) of sunitinib (green) and LysoTracker Red (red). Nucleus stained with Hoechst 33342 (blue). HT-29/SR cells were continuously incubated with sunitinib. Scale bar = 20 µm. (<b>e</b>) Evaluation of sunitinib accumulation in HT-29 (72 h incubation) and HT-29/SR (long-term sunitinib exposure) cells with flow cytometry. HT-29/SR cells were continuously incubated with sunitinib. Median sunitinib fluorescence intensities in live and single cells (mean of three experiments ± S.E.). (<b>f</b>) Photochemical treatment (photosensitizer and light) response of HT-29 and HT-29/SR evaluated with MTT assay post-90 s red light exposure (representative experiment of three, mean of triplicates ± S.D.). Cellular viability of sunitinib in HT-29/SR cells using (<b>g</b>) “light after” with 8 µM sunitinib or (<b>h</b>) “light first” protocol assessed by MTT post-90 seconds red light exposure, respectively (representative data based on three independent experiments, mean of triplicates ± S.D.). HT-29/SR cells were seeded in sunitinib-free medium. (<b>i</b>) Cell viability after PCI “light after” of 0.5 µM rGel as assessed by MTT post-60 seconds blue light exposure (representative experiment of three, mean of triplicates ± S.D.). 60 s blue light ≈ 0.58 J/cm<sup>2</sup>, 90 s red light ≈ 0.54 J/cm<sup>2</sup> NT: no treatment, Sun: sunitinib. Statistical significance calculated with Student’s test (two-tailed <span class="html-italic">p</span> value) where *** indicates <span class="html-italic">p</span> ≤ 0.001, ** <span class="html-italic">p</span> ≤ 0.01 and * <span class="html-italic">p</span> ≤ 0.05, n.s.: not significant.</p> Full article ">Figure 4
<p>Sunitinib-PCI treatment response of HT-29 xenografts in athymic Nude-Foxn1<sup>nu</sup> mice Intra-tumoral distribution of sunitinib (green) and TPCS<sub>2a</sub> (red) (<b>a</b>) before and (<b>b</b>) 30 min post-light exposure of HT-29 tumors treated with the Sun1- PCI protocol. Error bars: 20 µm Co-localization (yellow) indicated with white arrows. Kaplan-Meier plots illustrating overall treatment response following (<b>c</b>) Sun1- and (<b>d</b>) Sun2-PCI, * indicates significance compared to no treatment. Statistical significance established by pairwise long-rank analysis. (<b>e</b>) Mean estimated time to reach endpoint in each treatment group. SE: standard error. (<b>f</b>) Average tumor size in the indicated treatment groups at day 6 (left) and day 10 (right) post-light exposure. Statistical significance with asterisk where *** indicates <span class="html-italic">p</span> ≤ 0.001 and * <span class="html-italic">p</span> ≤ 0.05. (<b>g</b>) Table of <span class="html-italic">p</span> values is shown in cases where the difference in tumor size between the treatment groups is significant (<span class="html-italic">p</span> ≤ 0.05) at day 6 and day 10. Significant difference established by one-way ANOVA test followed by pair wise multiple comparison procedure (Holm-Sidak). NT: No treatment, n.s.: not significant.</p> Full article ">Figure 5
<p>Sunitinib-PCI treatment response of CT26.WT allografts in BALB/c mice. Kaplan-Meier plots illustrating treatment responses following (<b>a</b>) Sun1- and (<b>b</b>) Sun2-PCI, where asterisk indicates significance compared to no treatment. (<b>c</b>) Mean estimated time to reach endpoint in each treatment group. (<b>d</b>) Average tumor size in the indicated treatment groups at day 4 (upper panel) and day 7 (lower panel) post-light exposure. (<b>e</b>) Table of <span class="html-italic">p</span> values is shown in cases where the difference in tumor size between the treatment groups is significant (<span class="html-italic">p</span> ≤ 0.05) at day 4 and day 7. Significant difference established by one-way ANOVA test followed by pairwise multiple comparison procedure (Holm-Sidak). NT: No treatment, n.s.: not significant.</p> Full article ">Figure 6
<p>IHC of CT26. WT tumor tissue sections from BALB/c mice treated with the Sun-2-PCI procedure. Representative images of CT26.WT tumor sections, following PS + light, Sun2 or Sun2-PCI treatment. (<b>a</b>) H&amp;E stain (N: necrotic V: viable) and (<b>b</b>) CD3 stain. (<b>c</b>) Quantification of CD3 staining based on three ROIs in each tumor (two tumors in each group). Mean ± S.E. Significant difference established by one-way ANOVA test followed by pair wise multiple comparison procedure. (<b>d</b>) Ki-67 stain and (<b>e</b>) CD31 stain. Arrows indicate intact (white) and collapsed (yellow) vessels. Magnification in overview 20×, scale bar: 500 µm. ROI: region of interest.</p> Full article ">
23 pages, 7218 KiB  
Article
TLD1433 Photosensitizer Inhibits Conjunctival Melanoma Cells in Zebrafish Ectopic and Orthotopic Tumour Models
by Quanchi Chen, Vadde Ramu, Yasmin Aydar, Arwin Groenewoud, Xue-Quan Zhou, Martine J. Jager, Houston Cole, Colin G. Cameron, Sherri A. McFarland, Sylvestre Bonnet and B. Ewa Snaar-Jagalska
Cancers 2020, 12(3), 587; https://doi.org/10.3390/cancers12030587 - 4 Mar 2020
Cited by 35 | Viewed by 8316
Abstract
The ruthenium-based photosensitizer (PS) TLD1433 has completed a phase I clinical trial for photodynamic therapy (PDT) treatment of bladder cancer. Here, we investigated a possible repurposing of this drug for treatment of conjunctival melanoma (CM). CM is a rare but often deadly ocular [...] Read more.
The ruthenium-based photosensitizer (PS) TLD1433 has completed a phase I clinical trial for photodynamic therapy (PDT) treatment of bladder cancer. Here, we investigated a possible repurposing of this drug for treatment of conjunctival melanoma (CM). CM is a rare but often deadly ocular cancer. The efficacy of TLD1433 was tested on several cell lines from CM (CRMM1, CRMM2 and CM2005), uveal melanoma (OMM1, OMM2.5, MEL270), epidermoid carcinoma (A431) and cutaneous melanoma (A375). Using 15 min green light irradiation (21 mW/cm2, 19 J.cm−2, 520 nm), the highest phototherapeutic index (PI) was reached in CM cells, with cell death occurring via apoptosis and necrosis. The therapeutic potential of TLD1433 was hence further validated in zebrafish ectopic and newly-developed orthotopic CM models. Fluorescent CRMM1 and CRMM2 cells were injected into the circulation of zebrafish (ectopic model) or behind the eye (orthotopic model) and 24 h later, the engrafted embryos were treated with the maximally-tolerated dose of TLD1433. The drug was administrated in three ways, either by (i) incubating the fish in drug-containing water (WA), or (ii) injecting the drug intravenously into the fish (IV), or (iii) injecting the drug retro-orbitally (RO) into the fish. Optimally, four consecutive PDT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW/cm2, 114 J.cm−2, 520 nm). This PDT protocol was not toxic to the fish. In the ectopic tumour model, both systemic administration by IV injection and RO injection of TLD1433 significantly inhibited growth of engrafted CRMM1 and CRMM2 cells. However, in the orthotopic model, tumour growth was only attenuated by localized RO injection of TLD1433. These data unequivocally prove that the zebrafish provides a fast vertebrate cancer model that can be used to test the administration regimen, host toxicity and anti-cancer efficacy of PDT drugs against CM. Based on our results, we suggest repurposing of TLD1433 for treatment of incurable CM and further testing in alternative pre-clinical models. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Figure 1

Figure 1
<p>(<b>a</b>) Chemical structure of the PDT photosynthesizer TLD1433. (<b>b</b>) Jablonski diagram showing the formation of singlet oxygen (<sup>1</sup>O<sub>2</sub>) by irradiation of TLD1433 via initial population of metal-to-ligand charge transfer (MLCT) states and intersystem crossing to intra-ligand (IL, ILCT) states.</p> Full article ">Figure 2
<p>Cell viability after TLD1433 treatment of eight tumour cell lines (CRMM1, CRMM2, CM2005.1, OMM1, OMM2.5, MEL270, A431, A375). The green line shows TLD1433 activated by 520 nm light, 21 mW/cm<sup>2</sup>, 19 J.cm<sup>−2</sup> (light-induced toxicity). The dark line shows TLD1433 treatment without light irradiation (dark toxicity). The tumour cells were treated with TLD1433 for 24 h with concentrations ranging from 0.001 µM to 5 µM and kept in the dark, or ranging from 0.0001 µM to 0.025 µM and illuminated with a light dose of 21 mW/cm<sup>2</sup>, 19 J/cm<sup>2</sup>. SRB assay was carried out at 48 h after light irradiation. The absorbance of Sulforhodamine B in solution was measured at 520 nm. Results are presented as means ± SD from three independent experiments with 95% confidence intervals.</p> Full article ">Figure 3
<p>Green light irradiation of TLD1433 induces apoptosis and necrosis in CRMM1 and CRMM2 cells. (<b>A</b>) CRMM1 and (<b>C</b>) CRMM2 were stained with Annexin-V-FITC and Propidium Iodide. The percentages of live, early apoptotic, later apoptotic and necrotic cells in CRMM1 (<b>B</b>) and CRMM2 (<b>D</b>) were counted by FACS. Results are presented as means ± SD from three independent experiments.</p> Full article ">Figure 4
<p>Light toxicity in zebrafish embryos. 2 dpf embryos (<span class="html-italic">n</span> = 30) were exposed to green light (21 mW/cm<sup>2</sup>, 520 nm) for 0, 3, 6, or 12 h. (<b>A</b>) Transmitted light images of the embryos after light irradiation. (<b>B</b>–<b>D</b>) The percentage of mortality, malformation and fish length after various time of light exposure. Results represents the means ± SD from three independent experiments.</p> Full article ">Figure 5
<p>The maximum tolerated dose of TLD1433 in wild type zebrafish embryos administered through three different routes. (<b>A</b>) Schedule of TLD1433 treatment in wild type zebrafish. WA: TLD1433 (2.3 nM, 4.6 nM, 9.2 nM, 11.5 nM, 23 nM) were added to the water containing 10 embryos per well at 2.5, 3.5, 4.5, 5.5 dpf, for 12 h (yellow box). After these treatments, the drug was removed and replaced by egg water followed by 90 min green light irradiation (21 mW/cm<sup>2</sup>, 114J.cm<sup>−2</sup>, 520 nm), depicted as a green lightning bolt. IV or RO: 1 nL of TLD1433 (1.15 mM, 2.3 mM, 4.6 mM, 9.2 mM, 11.5 mM) were injected into the embryos at 3 dpf to 6 dpf every morning, followed by 60 min drug-to-light interval (yellow box) and 90 min green light irradiation (21 mW/cm<sup>2</sup>, 114 J.cm<sup>−2</sup>, 520 nm), depicted as a green lightning bolt. (<b>B</b>) WA, (<b>C</b>) IV, (<b>D</b>) RO. (<b>B</b>–<b>D</b>) Images were made of irradiated (light) and non-irradiated (dark) embryos (<span class="html-italic">n</span> = 30) at 6dpf and the percentages of mortality, malformation and fish length were calculated (shown as means ± SD from three independent experiments). Representative images of embryos under dark and light conditions are shown.</p> Full article ">Figure 5 Cont.
<p>The maximum tolerated dose of TLD1433 in wild type zebrafish embryos administered through three different routes. (<b>A</b>) Schedule of TLD1433 treatment in wild type zebrafish. WA: TLD1433 (2.3 nM, 4.6 nM, 9.2 nM, 11.5 nM, 23 nM) were added to the water containing 10 embryos per well at 2.5, 3.5, 4.5, 5.5 dpf, for 12 h (yellow box). After these treatments, the drug was removed and replaced by egg water followed by 90 min green light irradiation (21 mW/cm<sup>2</sup>, 114J.cm<sup>−2</sup>, 520 nm), depicted as a green lightning bolt. IV or RO: 1 nL of TLD1433 (1.15 mM, 2.3 mM, 4.6 mM, 9.2 mM, 11.5 mM) were injected into the embryos at 3 dpf to 6 dpf every morning, followed by 60 min drug-to-light interval (yellow box) and 90 min green light irradiation (21 mW/cm<sup>2</sup>, 114 J.cm<sup>−2</sup>, 520 nm), depicted as a green lightning bolt. (<b>B</b>) WA, (<b>C</b>) IV, (<b>D</b>) RO. (<b>B</b>–<b>D</b>) Images were made of irradiated (light) and non-irradiated (dark) embryos (<span class="html-italic">n</span> = 30) at 6dpf and the percentages of mortality, malformation and fish length were calculated (shown as means ± SD from three independent experiments). Representative images of embryos under dark and light conditions are shown.</p> Full article ">Figure 6
<p>Development of a new conjunctival melanoma orthotopic tumour model in ZF. (<b>A</b>) Location of CM cell injection, (<b>B</b>) red fluorescent CRMM1 and (C) CRMM2 cells were injected RO into 2 dpf tg(Fli:GFP/Casper) (<span class="html-italic">n</span> = 10) and imaged by fluorescence microscopy at 1 and 4 days post injection (dpi). Relative tumour burden was calculated as fluorescent intensity and area of tumour cells by Image J. Results are means ± SD of three independent experiments.</p> Full article ">Figure 6 Cont.
<p>Development of a new conjunctival melanoma orthotopic tumour model in ZF. (<b>A</b>) Location of CM cell injection, (<b>B</b>) red fluorescent CRMM1 and (C) CRMM2 cells were injected RO into 2 dpf tg(Fli:GFP/Casper) (<span class="html-italic">n</span> = 10) and imaged by fluorescence microscopy at 1 and 4 days post injection (dpi). Relative tumour burden was calculated as fluorescent intensity and area of tumour cells by Image J. Results are means ± SD of three independent experiments.</p> Full article ">Figure 7
<p>Treatment of zebrafish ectopic and orthotopic CM models with TLD1433 through WA. (<b>A</b>) Schedule of tumour injection and TLD1433 administration in zebrafish embryos. Fluorescent CRMM1 and CRMM2 cells were injected at 2 dpf into the Duct of Cuvier (ectopic model) and behind the eye (orthotopic model) and TLD1433 was administered with or without a light treatment following the schedule presented in A. Relative tumour burden estimated as fluorescence intensity and tumour area was calculated by Image J. (<b>B</b>) CRMM1 tumour burden in ectopic model (<span class="html-italic">n</span> ≈ 30). (<b>C</b>) CRMM1 tumour burden in orthotopic model (<span class="html-italic">n</span> ≈ 15). (D) CRMM2 tumour burden in ectopic model (<span class="html-italic">n</span> ≈ 30). (E) CRMM2 tumour burden in orthotopic model (<span class="html-italic">n</span> ≈ 15). Results are presented as means ± SD from three independent experiments. Representative images show CM tumour burden in the head and tail regions in the ectopic model and a localised tumour in the orthotopic model.</p> Full article ">Figure 8
<p>TLD1433 treatment by IV administration in the zebrafish ectopic and orthotopic CM model. (<b>A</b>) Schedule of tumour injection and TLD1433 administration in zebrafish embryos. Relative tumour burden was calculated as described in <a href="#cancers-12-00587-f007" class="html-fig">Figure 7</a>. (<b>B</b>) CRMM1 tumour burden in the ectopic model (<span class="html-italic">n</span> ≈ 30). (<b>C</b>) CRMM1 tumour burden in the orthotopic model (<span class="html-italic">n</span> ≈ 15). (<b>D</b>) CRMM2 tumour burden in the ectopic model (<span class="html-italic">n</span> ≈ 30). (<b>E</b>) CRMM2 tumour burden in the orthotopic model (<span class="html-italic">n</span> ≈ 15). Results are presented as means ± SD from three independent experiments. Representative images show CM tumour burden in the head and tail regions in ectopic model and localised tumours in the orthotopic model.</p> Full article ">Figure 9
<p>TLD1433 treatment by RO administration in the zebrafish ectopic and orthotopic CM model. (<b>A</b>) Schedule of tumour injection and TLD1433 administration in zebrafish embryos. Relative tumour burdens were calculated as described in <a href="#cancers-12-00587-f007" class="html-fig">Figure 7</a>. (<b>B</b>) CRMM1 tumour burden in the ectopic model (<span class="html-italic">n</span> ≈ 30). (<b>C</b>) CRMM1 tumour burden in the orthotopic model (<span class="html-italic">n</span> ≈ 15). (<b>D</b>) CRMM2 tumour burden in the ectopic model (n ≈ 30). (<b>E</b>) CRMM2 tumour burden in the orthotopic model (<span class="html-italic">n</span> ≈ 15). Results are presented as means ± SD from three independent experiments. Representative images show CM tumour burden in the head and tail regions in ectopic model and localised tumour in orthotopic model.</p> Full article ">Figure 10
<p>TUNEL assay of in CRMM1 and CRMM2 orthotopic model after RO of TLD1433. Red fluorescent CRMM1 (<b>A</b>) and CRMM2 (<b>B</b>) cells were injected at 2dpf behind the ZF eye and divided into four groups for drug treatment. RO administration of vehicle control and TLD1433 was performed as described in <a href="#cancers-12-00587-f009" class="html-fig">Figure 9</a>C,E. After dark or light exposure (21 mW/cm<sup>2</sup>, 114 J.cm<sup>−2</sup>, 520 nm) embryos were fixed and TUNEL staining was performed. Representative images of embryos are shown in this figure. (<b>A</b>,<b>B</b>) In TLD1433 light groups nuclear DNA fragmentation by nucleases is detected by co-localization of green (DNA fragments) and red signal of engrafted CM cells, depicted as yellow signal and marked by white arrows. In control dark, control light, TLD1433 dark, there are no positive green apoptotic tumour cells observed. Background green signal in TLD1433 light groups, does not co-localized with cytosolic red signal, which is diminished in degraded cells and TUNEL stains only the DNA breaks in these CM apoptotic cells. Experiment was performed 3 times with a group size of 10 embryos.</p> Full article ">Figure 11
<p>Time line for the SRB assay.</p> Full article ">
19 pages, 4140 KiB  
Article
Metformin as an Adjuvant to Photodynamic Therapy in Resistant Basal Cell Carcinoma Cells
by Marta Mascaraque, Pablo Delgado-Wicke, Cristina Nuevo-Tapioles, Tamara Gracia-Cazaña, Edgar Abarca-Lachen, Salvador González, José M. Cuezva, Yolanda Gilaberte and Ángeles Juarranz
Cancers 2020, 12(3), 668; https://doi.org/10.3390/cancers12030668 - 13 Mar 2020
Cited by 14 | Viewed by 4263
Abstract
Photodynamic Therapy (PDT) with methyl-aminolevulinate (MAL-PDT) is being used for the treatment of Basal Cell Carcinoma (BCC), although resistant cells may appear. Normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, but cancer cells switch this metabolism to aerobic [...] Read more.
Photodynamic Therapy (PDT) with methyl-aminolevulinate (MAL-PDT) is being used for the treatment of Basal Cell Carcinoma (BCC), although resistant cells may appear. Normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, but cancer cells switch this metabolism to aerobic glycolysis (Warburg effect), influencing the response to therapies. We have analyzed the expression of metabolic markers (β-F1-ATPase/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ratio, pyruvate kinase M2 (PKM2), oxygen consume ratio, and lactate extracellular production) in the resistance to PDT of mouse BCC cell lines (named ASZ and CSZ, heterozygous for ptch1). We have also evaluated the ability of metformin (Metf), an antidiabetic type II compound that acts through inhibition of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway to sensitize resistant cells to PDT. The results obtained indicated that resistant cells showed an aerobic glycolysis metabolism. The treatment with Metf induced arrest in the G0/G1 phase and a reduction in the lactate extracellular production in all cell lines. The addition of Metf to MAL-PDT improved the cytotoxic effect on parental and resistant cells, which was not dependent on the PS protoporphyrin IX (PpIX) production. After Metf + MAL-PDT treatment, activation of pAMPK was detected, suppressing the mTOR pathway in most of the cells. Enhanced PDT-response with Metf was also observed in ASZ tumors. In conclusion, Metf increased the response to MAL-PDT in murine BCC cells resistant to PDT with aerobic glycolysis. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Cell survival after Photodynamic Therapy (PDT): Survival of P, 10 G, and 10 GT populations of (<b>a</b>) ASZ and (<b>b</b>) CSZ cell lines subjected to methyl-aminolevulinate (MAL)-PDT and evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazoliumbromide (MTT assay). MTT test was performed 24 h after PDT treatment (0.2 mM MAL for 5 h and subsequently exposed to variable doses of red light). The 10 G population showed the highest resistance to treatment in ASZ cell lines, whereas in CSZ, it was the 10 GT population. Values were represented as mean ± SD (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001) (<span class="html-italic">n</span> = 5).</p> Full article ">Figure 2
<p>Proliferation capacity and metabolic characterization of Basal Cell Carcinoma (BCC) cells: (<b>a</b>) For the clonogenic assay, 50 cells/mL were seeded in each plate of 6 wells, and 7 days later, the colonies formed were stained with 0.2% crystal violet. Colonies were classified in relation to their diameter: small (&lt;1 mm), medium (1–2 mm), and large (&gt;2 mm) (<span class="html-italic">n</span> = 3). (<b>b</b>) Expression of the metabolic markers β-F1-ATPase and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) analyzed by western blot (WB); alphatubulin was used as loading control; and the ratio of β-F1-ATPase/GAPDH indicates the use of glucose by the cells, which was significantly lower in the resistant comparing to that of P cells (<span class="html-italic">n</span> = 5). (<b>c</b>) Pyruvate kinase M2 (PKM2) levels were higher in 10 G of ASZ compared to the P cells (<span class="html-italic">n</span> = 3). (<b>d</b>) Oxygen consumption rate (OCR) measurements over time (min) were determined by using an extracellular flux analyzer after the sequential addition of oligomycin (A), 2,4-Dinitrophenol (DNP) (B), and rotenone + antimycin (C) (<span class="html-italic">n</span> = 4). (<b>e</b>) Oligomycin-sensitive respiration, which represents the activity of oxidative phosphorylation (OXPHOS), was calculated as basal respiration – oligomycin respiration (<span class="html-italic">n</span> = 4). (<b>f</b>) Rates of lactate production determined spectrophotometrically (<span class="html-italic">n</span> = 6). Values were represented as mean ± SD (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 3
<p>Metformin treatment: (<b>a</b>) Cell survival 24 h after Metf treatment in P and resistant populations of BCC cells evaluated by the MTT test. The P cells were dose dependent on Metf, and the resistant were less sensible than P cells. (<b>b</b>) Effect of 24 h treatment of Metf on cell cycle progression in P and resistant populations of ASZ and CSZ cells: Cell cycle distribution was analyzed by flow cytometry. Metf treatment induced a significant increase in the G1-G0 and a decrease in the S phases of all cell populations. (<b>c</b>) Mitochondrial membrane potential determined by JC-1 ratio (J-aggregate fluorescence/J-monomer fluorescence): The green and red fluorescence indicate J-monomers (low mitochondrial membrane potential) and J-aggregate (high mitochondrial membrane potential), respectively (<span class="html-italic">n</span> = 3). (<b>d</b>–<b>e</b>) Expression of the glycolytic markers (β-F1-ATPase/GAPDH ratio and PKM2) analyzed by WB in ASZ (<a href="#cancers-12-00668-f003" class="html-fig">Figure 3</a>d) and CSZ (<a href="#cancers-12-00668-f003" class="html-fig">Figure 3</a>e) cells (<span class="html-italic">n</span> = 5); alfa tubulin was used as loading control. (<b>f</b>) Real-time analysis of OCR in BCC cells after 24 h with 75 µM Metf and the sequential addition of oligomycin (A), 2,4-dinitrophenol (DNP) (B) and rotenone with antimycin (C) to the cells (<span class="html-italic">n</span> = 4). (<b>g</b>) Oligomycin sensitive respiration (OSR) after 24 h with 75 µM Metf, which represents the activity of OXPHOS, was calculated as basal respiration – oligomycin respiration (<span class="html-italic">n</span> = 4). (<b>h</b>) Rates of lactate production determined spectrophotometrically after 24 h with 75 µM Metf (<span class="html-italic">n</span> = 6). Values were represented as mean ± SD (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 4
<p>Combined treatment of Metf and MAL-PDT on cell viability: Cells were treated for 24 h with Metf (25–150 µM) and then subjected to MAL-PDT (0.2 mM MAL and 7.6 J/cm<sup>2</sup> in ASZ cells and 3.8 J/cm<sup>2</sup> in CSZ cells). Cell survival was evaluated by the MTT test. (<b>a</b>) The results obtained showed a decrease in the cell survival after the combined treatment compared to that obtained after Metf or PDT alone in (<b>a</b>) ASZ and (<b>a’</b>) CSZ cell lines. (<b>b</b>) Combined treatment provided a synergistic effect on cell viability in (<b>b</b>) ASZ and (<b>b’</b>) CSZ cell lines. The synergy/antagonism parameter DL (difference in logarithm) was calculated as follows: DL = (log cell survival percentage Metf + log cell survival percentage PDT) – log cell survival percentage combination. Values were represented as mean ± SD (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001) (<span class="html-italic">n</span> = 5).</p> Full article ">Figure 5
<p>Effect of the combined treatment of Metf and MAL-PDT on BCC cell lines: (<b>a</b>) PpIX production was evaluated by flow cytometry after incubation with Metf (24 h, 75 µM), MAL (5 h, 0.2 mM), and Metf and MAL (24 h followed by 5 h, respectively). (<b>b</b>) Expression by western blot of AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway components: pAMPK, AMPK, pAKT, AKT, p-p70S6K and p70S6K after treatments (Metf, MAL-PDT, and Metf plus MAL-PDT). A representative experiment of each marker is shown, and pAMPK/AMPK, pAKT/AKT, and p-p70S6K/p70S6K densitometry graphics of both P and resistant populations of ASZ and CSZ cells are shown. Alfa tubulin was used as loading control. For each cell population, 4 conditions were evaluated: control; 24 h after 75µM Metf; 24 h after PDT treatment (5 h incubation with MAL and 7.6 J/cm<sup>2</sup> in ASZ and 3.8 J/cm<sup>2</sup> in CSZ cells); and combination of Metf and MAL-PDT. Values were represented as mean ± SD (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 6
<p>Effect of the treatments with Metf and/or PDT of ASZ tumors induced in mice: Photographs of the tumors at the time of sacrifice and the evolution of tumor volumes over time after the treatments in (<b>a</b>) P and (<b>b</b>) 10 G ASZ tumors. At day 9, when the tumors reached a volume of 50 mm<sup>3</sup>, they were treated with Metf (200 µg/mL diluted in drinking water along the rest of the experiment). At day 17, when the untreated group reached a volume of 100–200 mm<sup>3</sup>, the tumors were subjected to PDT or to Metf-PDT (2 mM MAL injected in 50 µL PBS, 4 h of incubation, and 25 J/cm<sup>2</sup> of red light). Tumor volume was measured every two days with a caliper. Values were represented as mean ± SD (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001) (<span class="html-italic">n</span> = 3). Scale bar = 10 mm. (<b>c</b>) Representative photographs of tumor sections at the end of the experiments stained with hematoxylin and eosin (H&amp;E) (low and high magnification) and stained with the TUNEL (terminal deoxynucleotide transferase mediated X-dUTP nick end labeling) assay. The H&amp;E showed that the tumors were formed by atypical keratinocytes infiltrating skeletal muscle fibers (asterisk). The treatment with Metf, PDT, and especially Metf + PDT provoked an increment of red blood cell extravasation in the dermis (black arrows) and extensive areas of cell death. The cell death areas were better observed after the TUNEL staining; dead cells appeared fluorescing in green particularly after the combined treatments applied in P tumors. Nuclei were counterstained with Höechst fluorochrome and fluoresced in blue.</p> Full article ">
16 pages, 2752 KiB  
Article
The Potential of Nanobody-Targeted Photodynamic Therapy to Trigger Immune Responses
by Irati Beltrán Hernández, Mathieu L. Angelier, Tommaso Del Buono D’Ondes, Alessia Di Maggio, Yingxin Yu and Sabrina Oliveira
Cancers 2020, 12(4), 978; https://doi.org/10.3390/cancers12040978 - 15 Apr 2020
Cited by 24 | Viewed by 4515
Abstract
Nanobody-targeted photodynamic therapy (NB-PDT) has been recently developed as a more tumor-selective approach rather than conventional photodynamic therapy (PDT). NB-PDT uses nanobodies that bind to tumor cells with high affinity, to selectively deliver a photosensitizer, i.e., a chemical which becomes cytotoxic when excited [...] Read more.
Nanobody-targeted photodynamic therapy (NB-PDT) has been recently developed as a more tumor-selective approach rather than conventional photodynamic therapy (PDT). NB-PDT uses nanobodies that bind to tumor cells with high affinity, to selectively deliver a photosensitizer, i.e., a chemical which becomes cytotoxic when excited with light of a particular wavelength. Conventional PDT has been reported to be able to induce immunogenic cell death, characterized by the exposure/release of damage-associated molecular patterns (DAMPs) from dying cells, which can lead to antitumor immunity. We explored this aspect in the context of NB-PDT, targeting the epidermal growth factor receptor (EGFR), using high and moderate EGFR-expressing cells. Here we report that, after NB-PDT, the cytoplasmic DAMP HSP70 was detected on the cell membrane of tumor cells and the nuclear DAMP HMGB1 was found in the cell cytoplasm. Furthermore, it was shown that NB-PDT induced the release of the DAMPs HSP70 and ATP, as well as the pro- inflammatory cytokines IL- 1β and IL-6. Conditioned medium from high EGFR-expressing tumor cells treated with NB-PDT led to the maturation of human dendritic cells, as indicated by the upregulation of CD86 and MHC II on their cell surface, and the increased release of IL-12p40 and IL-1β. Subsequently, these dendritic cells induced CD4+ T cell proliferation, accompanied by IFNγ release. Altogether, the initial steps reported here point towards the potential of NB-PDT to stimulate the immune system, thus giving this selective-local therapy a systemic reach. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Figure 1

Figure 1
<p>Cell death mechanism induced by nanobody-targeted photodynamic therapy (NB-PDT). Tumor cells were left untreated (NT) or treated with NB-PDT using 7D12-PS (LD50 or LD100) and stained with propidium iodide (PI) for necrotic cells (red) and caspase 3/7 for apoptotic cells (green), controls for necrosis and apoptosis were included. Microscopy images of (<b>a</b>), A431 cells and (<b>b</b>), scc-U8 cells were taken 2 and 18 h after NB-PDT. Top panels depict the transmitted light image and bottom panels the merged images of necrotic and apoptotic cells. Scale bar, 20 µm.</p> Full article ">Figure 2
<p>Cellular localization of heat shock protein 70 (HSP70) and high mobility group box 1 protein (HMGB1) on tumor cells treated with NB-PDT. Tumor cells were left untreated (NT) or treated with NB-PDT using 7D12-PS (LD50 or LD100), and 4 h later stained for HSP70 or HMGB1. Staining of HSP70 (red) was performed on non-permeabilized (<b>a</b>), A431 cells and (<b>b</b>), scc-U8 cells. Intracellular staining of HMGB1 (green) was performed on (<b>c</b>), A431 cells and (<b>d</b>), scc-U8 cells. Cell nuclei were additionally stained with DAPI (blue). Top panels depict only the damage-associated molecular pattern (DAMP) signal, while merged images are shown on the bottom panels. Scale bar, 20 µm.</p> Full article ">Figure 3
<p>Quantification of ATP and HSP70 release from tumor cells after NB-PDT. ATP in the supernatant was detected 4 h after NB-PDT via a luminescence assay and graphs show luminescence values relative to untreated cells for (<b>a</b>), A431 cells and (<b>b</b>), scc-U8 cells. Additionally, released HSP70 was detected 24 h after treatment using ELISA on (<b>c</b>), A431 cells and (<b>d</b>), scc-U8 cells. NT, untreated; LD50, mild cytotoxic NB-PDT; LD100, highly cytotoxic NB-PDT; Light, only light control; NB-PS, only nanobody-photosensitizer (NB-PS) conjugate control. Significance is displayed as * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01.</p> Full article ">Figure 4
<p>Quantification of IL-1β, IL-6 and IL-8 release by tumor cells treated with NB-PDT. A431 or scc-U8 cells were treated with NB-PDT and the concentration of several cytokines in the supernatant was quantified 24 h later. Graphs display the quantification of IL-1β, IL-6 and IL-8 on A431 cells (<b>a</b>, <b>b</b>, and <b>c</b>, respectively) and on scc-U8 cells (<b>d</b>, <b>e</b>, and <b>f</b>, respectively). NT, untreated; LD50, mild cytotoxic NB-PDT; LD100, highly cytotoxic NB-PDT; Light, only light control; NB-PS, only NB-PS conjugate control. Significance is displayed as * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01 and *** <span class="html-italic">p</span> ≤ 0.001.</p> Full article ">Figure 5
<p>Phenotypic maturation and cytokine release of monocyte-derived dendritic cells (moDCs) incubated with supernatant of NB-PDT treated tumor cells. A431 cells were treated with NB-PDT, the supernatant was collected 24 h later and incubated with immature moDCs for another 24 h. Surface marker expression on moDCs was measured with flow cytometry, and cytokine release was assessed by Luminex. (<b>a</b>), Percentage of CD86 positive moDCs. (<b>b</b>), Median fluorescence intensity (MFI) corresponding to MHCII surface expression on moDCs. Each moDC donor (n = 5) is represented by a different symbol and color. ctr, unstimulated DCs; lipopolysaccharide (LPS), LPS-stimulated DCs; NT, untreated tumor cells; LD50, mild cytotoxic NB-PDT; LD100, highly cytotoxic NB-PDT; Light, only light control; NB-PS, only NB-PS conjugate control. Significance is displayed as * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, and *** <span class="html-italic">p</span> ≤ 0.001. C-E, MFI corresponding to the release by moDCs of (<b>c</b>), IL-12p40; (<b>d</b>), IL-1β; and (<b>e</b>), IL-10 (n = 4). No statistical significance was found between groups due to the intrinsic differences between donors.</p> Full article ">Figure 6
<p>Enhanced proliferation and IFNγ release of CD4+ T cells induced by moDCs stimulated with supernatant of NB-PDT treated tumor cells. A431 cells were treated with NB-PDT, the supernatant was collected 24 h later and incubated with immature moDCs for another 24 h. moDCs were then co- incubated with allogeneic CFSE-labeled CD4+ T cells in a 1:10 ratio. After 6 days, CD4+ T cell proliferation was measured with flow cytometry and IFNγ release was assessed by ELISA. (<b>a</b>), Percentage of CD4+ T cells with weak CFSE signal, thus proliferating cells (n = 4). (<b>b</b>), Quantification of released IFNγ by CD4+ T cells (n = 3). Each combination of allogeneic donors is represented by a different symbol and color. ctr, unstimulated DCs; LPS, LPS-stimulated DCs; NT, untreated tumor cells; LD50, mild cytotoxic NB-PDT; LD100, highly cytotoxic NB-PDT; Light, only light control; NB-PS, only NB-PS conjugate control. Significance is displayed as * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01 and *** <span class="html-italic">p</span> ≤ 0.001.</p> Full article ">
10 pages, 1375 KiB  
Brief Report
Cytoplasmic Increase in Hsp70 Protein: A Potential New Biomarker of Early Infiltration of Cutaneous Squamous Cell Carcinoma Arising from Actinic Keratosis
by Montserrat Fernández-Guarino, José Javier Zamorano León, Antonio José López Farré, Maria Luisa González Morales, Ana Isabel Sánchez Adrada, José Barrio Garde, Jose Antonio Arias Navalon and Pedro Jaén Olasolo
Cancers 2020, 12(5), 1151; https://doi.org/10.3390/cancers12051151 - 3 May 2020
Cited by 7 | Viewed by 2709
Abstract
Background: Cutaneous squamous skin cell carcinoma (SCC) is the second most frequent type of non-melanoma skin cancer and is the second leading cause of death by skin cancer in Caucasian populations. However, at present it is difficult to predict patients with poor SCC [...] Read more.
Background: Cutaneous squamous skin cell carcinoma (SCC) is the second most frequent type of non-melanoma skin cancer and is the second leading cause of death by skin cancer in Caucasian populations. However, at present it is difficult to predict patients with poor SCC prognosis. Objective: To identify proteins with expression levels that could predict SCC infiltration in SCC arising from actinic keratosis (SCC-AK). Methods: A total of 20 biopsies from 20 different patients were studied; 10 were SCC-AK samples and 10 were taken from normal skin. Early infiltrated SCC-AK samples were selected based on histological examination, and to determine the expression of proteins, fresh skin samples were processed by two-dimensional electrophoresis. Results: The expression levels of three proteins, namely alpha hemoglobin and heat shock proteins 27 and 70 (Hsp27 and Hsp70, respectively) were significantly increased in SCC-AK samples with respect to normal control skin. However, only the expression level of Hsp70 protein positively correlated with the level of SCC-AK dermis infiltration. Immunohistological examination suggested that increased expression of Hsp70 proteins seemed to mainly occur in the cytoplasm of keratinocytes. The increased cytoplasmic Hsp70 expression in SCC-AK was confirmed by Western blot experiments. Conclusion: Cytoplasmic expression of Hsp70 could be a potential biomarker of early infiltration of SCC arising from AK. Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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Figure 1
<p>Immunohistochemistry analysis showing heat shock protein 70 (Hsp70) expression in control and squamous skin cell carcinoma arising from actinic keratosis (SCC-AK) samples with infiltration levels II and III. Abbreviations: nucleus (N) and cytoplasm (C).</p> Full article ">Figure 2
<p>Representative Western blot experiments showing the expression of Hsp70 protein in cytoplasm from control and SCC-AK samples. β-actin was used as loading control. Bar graphs show the cytoplasmic expression of Hsp70 levels of all the Western blots. Results are represented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05 with respect to control.</p> Full article ">
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Editorial
Photodynamic Therapy (PDT) in Oncology
by Ángeles Juarranz, Yolanda Gilaberte and Salvador González
Cancers 2020, 12(11), 3341; https://doi.org/10.3390/cancers12113341 - 12 Nov 2020
Cited by 26 | Viewed by 3299
Abstract
The issue is focused on Photodynamic Therapy (PDT), which is a minimally invasive therapeutic modality approved for treatment of several types of cancer and non-oncological disorders [...] Full article
(This article belongs to the Special Issue Photodynamic Therapy (PDT) in Oncology)
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