CN111089977A - Method for detecting oncolytic virus effectiveness by using organoid - Google Patents
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Abstract
The present disclosure relates to a method for detecting the effectiveness of an oncolytic virus using an organoid, the method comprising: a. mixing and culturing the oncolytic virus and a tumor organoid for 12-96h to obtain a first culture, and detecting the replication level of the oncolytic virus in the first culture; b. mixing and culturing the oncolytic virus and tumor organoid for 12-96h to obtain a second culture, and detecting the killing rate of the oncolytic virus to tumor cells in the second culture; c. culturing oncolytic virus, immune cells and tumor organoid for 2-8h to obtain a third culture, detecting the level of cytokine in the third culture and calculating the expression promoting capacity of the cytokine of the oncolytic virus, wherein the cytokine comprises interleukin-2 and/or gamma-interferon; if the replication level, the killing rate to tumor cells and the cytokine expression promoting ability of the test oncolytic virus are all higher than those of the reference oncolytic virus, indicating that the effectiveness of the test oncolytic virus is higher than that of the reference oncolytic virus. The detection method disclosed by the invention can be used for more truly detecting the effectiveness of the oncolytic virus.
Description
Technical Field
The disclosure relates to the technical field of biomedicine, in particular to a method for detecting the effectiveness of oncolytic virus by using organoids.
Background
Oncolytic viruses are specialized viruses formed by genetic engineering of naturally occurring, less pathogenic viruses. Oncolytic viruses can selectively infect tumor cells using aberrant signaling pathways in the tumor cells (such as inactivation or defects of oncogenes) and multiply in numbers within the tumor cells to destroy the tumor cells, but they cannot infect normal cells. Meanwhile, oncolytic virus can also stimulate immune response, attract more immune cells to continuously kill residual tumor cells. Oncolytic viruses are used as biological agents for tumor therapy and need to be screened for their effectiveness and safety in the treatment of tumor patients.
In the related art, the effectiveness of oncolytic virus is tested by using a monolayer cell line obtained by 2D culture of tumor cells or a PDX model.
However, the oncolytic virus effectiveness data obtained by the detection method is greatly different from the oncolytic virus effectiveness data obtained in the clinical research stage. The drug effectiveness and safety of the oncolytic virus serving as a biological drug are closely related to the tumor microenvironment and the reaction of the patient's own immune system to the drug, so that a model and a detection method which can simulate the tumor in the patient body more truly are urgently needed, the effectiveness of the oncolytic virus is judged, and the oncolytic virus drug with higher clinical transformation value is screened out.
Disclosure of Invention
The invention provides a method for detecting the effectiveness of oncolytic virus by utilizing organoids, wherein the organoids used in the method can more truly simulate the heterogeneity of tumors in patients and the drug reaction condition.
In order to achieve the above objects, the present disclosure provides a method for detecting the effectiveness of an oncolytic virus using an organoid, the method comprising:
a. performing mixed culture on the oncolytic virus and a tumor organoid for 12-96h to obtain a first culture, and detecting the replication level of the oncolytic virus in the first culture;
b. performing mixed culture on the oncolytic virus and a tumor organoid for 12-96h to obtain a second culture, and detecting the killing rate of the oncolytic virus to tumor cells in the second culture;
c. mixedly culturing the oncolytic virus, immune cells and tumor organoids for 2-8h to obtain a third culture, detecting the level of cytokines in the third culture and calculating the cytokine expression promoting capacity of the oncolytic virus, wherein the cytokines comprise interleukin-2 and/or gamma-interferon;
if the replication level, killing rate against tumor cells, and cytokine expression-promoting capacity of the test oncolytic virus are all higher than those of the reference oncolytic virus, indicating that the effectiveness of the test oncolytic virus is higher than that of the reference oncolytic virus.
Optionally, step a further comprises a step of performing a first pre-culture on the tumor organoid, and then performing mixed culture on the tumor organoid after the first pre-culture and the oncolytic virus; the first pre-culture comprises: mixing the tumor organoid with temperature-sensitive hydrogel and tumor organoid culture solution, and culturing for 12-96h, wherein the dosage of the temperature-sensitive hydrogel is 500-3000 mu L and the dosage of the tumor organoid culture solution is 2000-3000 mu L relative to the tumor cells in 10000 tumor organoids of 5000-; when the tumor organoid and the oncolytic virus are cultured in a mixed way, the dosage of the oncolytic virus is 0.01-1 MOI;
step b also comprises the step of carrying out second pre-culture on the tumor organoid, and then carrying out mixed culture on the tumor organoid subjected to second pre-culture and the oncolytic virus; the second pre-culture comprises: mixing the tumor organoid with the temperature-sensitive hydrogel and the tumor organoid culture solution, and culturing for 12-96h, wherein the dosage of the temperature-sensitive hydrogel is 30-80 mu L and the dosage of the tumor organoid culture solution is 100-150 mu L relative to the tumor cells in 1000 tumor organoids with 300-; when the tumor organoid and the oncolytic virus are cultured in a mixed way, the dosage of the oncolytic virus is 0.01-1 MOI;
in step c, the mixed culture of the oncolytic virus, the immune cell and the tumor organoid comprises the following steps: c1, will contain immune cellsInoculating cell suspension of cells and tumor organoid in culture dish, adding tumor organoid culture solution, and culturing for 2-8 hr to obtain mixed culture solution, wherein the concentration of immunocyte and tumor cell in the cell suspension is 0.1-1 × 107individual/mL, immune cell: tumor organoid cells ═ (2-8): 1, the amount of said tumor organoid culture medium is 2-3mL, relative to 0.5-2mL of said cell suspension; c2, mixing the mixed culture solution with the oncolytic virus for culturing for 3-5h, wherein the dosage of the oncolytic virus is 1-100 MOI.
Optionally, in step a, said detecting the level of replication of the oncolytic virus in said first culture comprises:
a1, obtaining the oncolytic virus in the first culture to obtain a virus to be detected;
a2, culturing the host cells of the oncolytic virus and the virus to be detected for 12-96h in a mixed way, and then detecting the virus titer of the virus to be detected in the host cells; wherein the higher the viral titer of said test virus in said host cell, the higher the level of replication of the oncolytic virus in said first culture.
Optionally, in step a1, the obtaining the oncolytic virus in the first culture to obtain a test virus includes:
and (3) performing freeze-thawing treatment on the first culture for 2-8 times, then performing centrifugation and collecting supernatant to obtain virus liquid to be detected, wherein the virus liquid to be detected contains the virus to be detected.
Optionally, in step b, detecting the killing rate of the oncolytic virus on the tumor cells in the second culture by using a CCK8 kit;
in step c, the cytokine level in the third culture is detected using an ELISA kit.
Optionally, the immune cells of step c are isolated from the peripheral blood of the patient.
Optionally, the mixed culture described in steps a, b and c is performed in a tumor organoid culture solution comprising DMEM/F12 medium and growth factors; the growth factor comprises at least one of Glutamax, HEPES, Gastrin, nicotinamide, A83-01, Noggin, n-acetyl cysteine, streptomycin, EGF and BSA.
Optionally, in the growth factor, the concentration of Glutamax is 0.5-2%, the concentration of HEPES is 5-15mM, the concentration of Gastrin is 5-15nM, the concentration of nicotamide is 5-15mM, the concentration of A83-01 is 250-600ng/mL, the concentration of Noggin is 50-150ng/mL, the concentration of n-Acetylcystine is 0.5-2mM, the concentration of streptomycin is 50-150 μ g/mL, the concentration of EGF is 50-150ng/mL, and the concentration of BSA is 0.001-0.005 mg/mL.
Optionally, the method for preparing a tumor organoid comprises:
mixing the tumor cells, the temperature-sensitive hydrogel and the tumor organoid culture solution, culturing, and replacing the culture medium every 2-4 days until the tumor organoid with the diameter not less than 0.5mm is observed under a microscope.
Optionally, the dosage of the temperature-sensitive hydrogel is 1500-2000 μ L and the dosage of the tumor organoid culture solution is 2000-3000 μ L relative to 20000 tumor cells.
Through the technical scheme, the tumor organoid is used as the tumor cell model for detecting the tumor tissue efficacy of the oncolytic virus, and the tumor organoid can better reflect the biological characteristics of the tumor tissue in the patient body, so the detection result of the method can more truly reflect the efficacy of the oncolytic virus in the tumor tissue in the patient body.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The present disclosure provides a method for detecting the effectiveness of an oncolytic virus using an organoid, the method comprising: a. performing mixed culture on the oncolytic virus and a tumor organoid for 12-96h to obtain a first culture, and detecting the replication level of the oncolytic virus in the first culture; b. performing mixed culture on the oncolytic virus and a tumor organoid for 12-96h to obtain a second culture, and detecting the killing rate of the oncolytic virus to tumor cells in the second culture; c. mixedly culturing the oncolytic virus, immune cells and tumor organoids for 2-8h to obtain a third culture, detecting the level of cytokines in the third culture and calculating the cytokine expression promoting capacity of the oncolytic virus, wherein the cytokines comprise interleukin-2 and/or gamma-interferon; if the replication level, killing rate against tumor cells, and cytokine expression-promoting capacity of the test oncolytic virus are all higher than those of the reference oncolytic virus, indicating that the effectiveness of the test oncolytic virus is higher than that of the reference oncolytic virus.
The inventor of the present disclosure finds that the reason why the oncolytic virus validity data obtained by the existing oncolytic virus validity detection method is greatly different from the oncolytic virus validity data obtained in the clinical research stage is that: the tumor cell model adopted in the existing oncolytic virus validity detection method can not truly simulate the biological characteristics of the tumor tissue in the body of a patient, so that the existing oncolytic virus validity detection process can not truly simulate the action process of the oncolytic virus in the body of the patient, for example, a monolayer cell line obtained by 2D culture of tumor cells used in the conventional method for detecting the effectiveness of an oncolytic virus, it does not reflect tumor heterogeneity or mimic the microenvironment in the patient, and with the increasing passage of conventional tumor cell lines, tumor cells often exhibit different biological properties than primary tumor cells, such as mutations in the genotype, faster phenotypic growth, or increased sensitivity to specific drugs, thus, conventional tumor cell lines do not truly mimic the biological properties of tumor tissue in a patient. The inventors of the present disclosure have attempted to use tumor organoids as tumor cell models for the detection of the efficacy of oncolytic viruses, and have unexpectedly found that the detection results thereof enable a more realistic detection of the efficacy of oncolytic viruses on tumor tissue in patients.
In the technical scheme, the replication level of the oncolytic virus in the first culture reflects the capacity of continuously replicating and proliferating after the oncolytic virus infects the tumor cells, the higher the replication level of the oncolytic virus in the first culture is, the higher the capacity of continuously replicating and proliferating after the oncolytic virus infects the tumor cells is, and the oncolytic virus with the strong capacity of continuously replicating and proliferating after infecting the tumor cells can continuously replicate and generate more oncolytic cells in the tumor cells, so that the effect of killing tumor tissues by the oncolytic cells is further improved; the killing rate of the oncolytic virus in the second culture to the tumor cell reflects the capacity of the oncolytic virus to kill the tumor cell after infecting the tumor cell, and the higher the killing rate of the oncolytic virus in the second culture to the tumor cell is, the stronger the capacity of the oncolytic virus to kill the tumor cell after infecting the tumor cell is; the higher the level of interleukin-2 and/or gamma-interferon in the third culture is, the stronger the expression capacity of the cytokine of the oncolytic virus is, and the stronger the capacity of the cytokine of the host immune system to generate the anti-tumor immune effect after the oncolytic virus infects the tumor cells is. The method disclosed by the invention can comprehensively characterize the effectiveness of the oncolytic virus in killing tumor cells through the three detection aspects.
The method adopts the tumor organoid as a tumor cell model for detecting the tumor tissue efficacy of the oncolytic virus, and the tumor organoid can better reflect the biological characteristics of the tumor tissue in the patient body, so the detection result of the method can more truly reflect the efficacy of the oncolytic virus on the tumor tissue oncolytic effect in the patient body.
According to the present disclosure, preferably, the step a further comprises the step of performing a first pre-culture on the tumor organoid, and then performing mixed culture on the tumor organoid after the first pre-culture and the oncolytic virus; the first pre-culture comprises: mixing the tumor organoid with temperature-sensitive hydrogel and tumor organoid culture solution, and culturing for 12-96h, wherein the dosage of the temperature-sensitive hydrogel is 500-3000 mu L and the dosage of the tumor organoid culture solution is 2000-3000 mu L relative to the tumor cells in 10000 tumor organoids of 5000-; will be swollenWhen the tumor organs and the oncolytic virus are subjected to mixed culture, the dosage of the oncolytic virus is 0.01-1 MOI; step b also comprises the step of carrying out second pre-culture on the tumor organoid, and then carrying out mixed culture on the tumor organoid subjected to second pre-culture and the oncolytic virus; the second pre-culture comprises: mixing the tumor organoid with the temperature-sensitive hydrogel and the tumor organoid culture solution, and culturing for 12-96h, wherein the dosage of the temperature-sensitive hydrogel is 30-80 mu L and the dosage of the tumor organoid culture solution is 100-150 mu L relative to the tumor cells in 1000 tumor organoids with 300-; when the tumor organoid and the oncolytic virus are cultured in a mixed way, the dosage of the oncolytic virus is 0.01-1 MOI; in step c, the mixed culture of the oncolytic virus, the immune cell and the tumor organoid comprises the following steps: c1, inoculating cell suspension containing immune cells and tumor organoid in a culture dish, adding tumor organoid culture solution, and culturing for 2-8h to obtain mixed culture solution, wherein the concentration of immune cells and tumor cells in the cell suspension is 0.1-1 × 107individual/mL, immune cell: tumor organoid cells ═ (2-8): 1, the amount of said tumor organoid culture medium is 2-3mL, relative to 0.5-2mL of said cell suspension; c2, mixing the mixed culture solution with the oncolytic virus for culturing for 3-5h, wherein the dosage of the oncolytic virus is 1-100 MOI.
In light of the present disclosure, the method of detecting the level of replication of the oncolytic virus in the first culture described in step a can be conventional in the art, and any method capable of detecting the level of replication of the oncolytic virus can be used in the present disclosure. Preferably, in step a, said detecting the level of replication of the oncolytic virus in said first culture comprises: a1, obtaining the oncolytic virus in the first culture to obtain a virus to be detected; a2, culturing the host cells of the oncolytic virus and the virus to be detected for 12-96h in a mixed way, and then detecting the virus titer of the virus to be detected in the host cells; wherein the higher the viral titer of said test virus in said host cell, the higher the level of replication of the oncolytic virus in said first culture.
Preferably, in step a1, the obtaining the oncolytic virus in the first culture to obtain a test virus comprises: and (3) performing freeze-thawing treatment on the first culture for 2-8 times, then performing centrifugation and collecting supernatant to obtain virus liquid to be detected, wherein the virus liquid to be detected contains the virus to be detected. Wherein the lowest temperature of the freeze-thaw treatment is-20 to-80 ℃, and the highest temperature is 10 to 25 ℃; the conditions of the centrifugation include: the centrifugal speed is 5000 plus 10000rpm/min, and the centrifugal time is 30-60 min.
Preferably, in step b, the killing rate of the tumor cells by the oncolytic virus in the second culture can be detected by using a CCK8 kit; in step c, the cytokine level in the third culture can be detected using an ELISA kit.
Preferably, the immune cells in step c are isolated from the peripheral blood of the patient. The method for separating the immune cells from the peripheral blood of the patient can be a conventional method in the field, and is not described in detail herein.
According to the present disclosure, the mixed culture in steps a, b and c is performed in a tumor organoid culture solution, the composition of which can be selected within a wide range, for example, the tumor organoid culture solution of the present disclosure can include DMEM/F12 medium and growth factors; the growth factor may include at least one of Glutamax, HEPES, Gastrin, nicotinamide, A83-01, Noggin, n-acetyl cysteine, streptomycin, EGF and BSA.
According to the present disclosure, the concentration of each of the growth factors may vary within a wide range, for example, the concentration of Glutamax is 0.5-2%, the concentration of HEPES is 5-15mM, the concentration of Gastrin is 5-15nM, the concentration of nicotinamide is 5-15mM, the concentration of A83-01 is 250-600ng/mL, the concentration of Noggin is 50-150ng/mL, the concentration of n-Acetylcysteine is 0.5-2mM, the concentration of streptomycin is 50-150 μ g/mL, the concentration of EGF is 50-150ng/mL, and the concentration of BSA is 0.001-0.005 mg/mL.
In accordance with the present disclosure, methods of making tumor organoids contemplated by the present disclosure may be conventional in the art, for example, methods of making tumor organoids contemplated by the present disclosure include: mixing the tumor cells, the temperature-sensitive hydrogel and the tumor organoid culture solution, culturing, and replacing the culture medium every 2-4 days until the tumor organoid with the diameter not less than 0.5mm is observed under a microscope.
Preferably, in the above method for preparing a tumor organoid, the amount of the temperature-sensitive hydrogel may be 1500-2000 μ L and the amount of the tumor organoid culture solution may be 2000-3000 μ L relative to 20000 tumor cells.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
The materials, reagents, instruments and equipment involved in the embodiments of the present disclosure may be obtained commercially, unless otherwise specified.
Where specific experimental temperatures are not noted in the examples of the present disclosure, the experimental temperatures are all room temperature (20-25 ℃). The sources of reagents used in the examples of the disclosure are as follows:
DMEM/F12 medium was purchased from HyClone, USA; cosmo temperature-sensitive hydrogels were purchased from Cosmo Bio, Japan; fetal bovine serum albumin (FBS) was purchased from inner mongolia jinyuan kang bioengineering ltd; penicillin and streptomycin are purchased from Shanghai biological engineering Co., Ltd; collagen hydrolase (Collagenase) was purchased from Sigma- -Aldrich, USA.
The tumor cell culture solution used in the embodiments of the present disclosure is configured autonomously, and includes: DMEM/F12 medium and growth factors such as 1% Glutamax (Gibco, 35050061), 10mM HEPES (Gibco, 15630080), 10nM Gastrin (Sigma, G9145), 10mM nicotinamide (Sigma, N06365), 500ng/mL A83-01(Tocris, 2939), 100ng/mL Noggin (Peprotech, 120-10C), 1mM N-Acetycysteine (Sigma, A9165), 100. mu.g/mL streptomycin, 50ng/mL EGF (Peprotech, AF-100-15), and 0.4G/100mL BSA (Sigma, 48 9046-8).
Examples
1. Culturing tumor organoids
The newly taken lung cancer tumor puncture tissue or operation tissue sample is preserved in a tumor organoid culture solution and transported to a laboratory within 48 hours by a 4 ℃ cold chain.
Removing the tumor organoid culture solution, and placing the sample in a 6cm culture dish; preparing a DMEM/F12 culture medium containing 2% FBS and 2mg/ml collagen hydrolase as an enzymolysis solution, filtering and sterilizing the enzymolysis solution, carrying out warm bath at 37 ℃, adding the enzymolysis solution into a culture dish containing a sample, wherein 1g of the sample is correspondingly added with 8ml of the enzymolysis solution; shearing the sample tissue blocks into minced meat by using sterilized scissors, transferring the sheared tissue blocks into a 24-pore plate by using a 10ml pipette, putting the 24-pore plate into a 37 ℃ constant-temperature incubator for enzymolysis for 2 hours, and blowing and beating the enzymolysis tissue by using a pipettor every 30 minutes in the enzymolysis process to promote the enzymolysis effect; and then, putting the mixed solution after enzymolysis into a cell grinding sieve, adding enzymolysis liquid for secondary grinding enzymolysis, collecting filtrate after full grinding, centrifuging the enzymolysis liquid for 3min under the centrifugal condition of 300g, removing supernatant, precipitating, and adding 10ml of tumor organoid culture solution for resuspension to obtain tumor cells.
The tumor cells with the cell number of 20000 are planted in a 96-well plate containing 40 mul Cosmo temperature-sensitive hydrogel in each well, 150 mul tumor organoid culture solution is added in each well, the culture is placed in a constant-temperature incubator at 37 ℃ for culture, and the culture medium is replaced every 3 days in the culture process until the lung cancer tumor organoids with the diameter larger than 0.5mm are observed under a microscope.
2. Detecting the level of replication of oncolytic viruses upon infection of tumor cells in a tumor organoid
Inoculating tumor organoid cell suspension in a 6-well plate to ensure that the number of tumor cells is 6000 per well, wherein each well contains 500 mu l of Cosmo temperature-sensitive hydrogel and 1.5mL of tumor organoid culture solution; putting the inoculated 6-hole plate into a constant-temperature incubator at 37 ℃ for pre-culturing for 96 hours, taking out, and infecting tumor organoids with 0.01-1MOI (Newcastle disease virus NDV oncolytic virus is taken as oncolytic virus to be detected and adenovirus ProstAtak oncolytic virus is taken as reference oncolytic virus) for 24 hours, 48 hours and 72 hours respectively; collecting organoid cells and cell supernatant generated by culture, freezing and thawing for 3 times, and centrifugally collecting supernatant to obtain virus liquid to be detected; inoculating a suspension (100 μ L/well) of host cells (such as DF1 cells as host cells of NDV) of the oncolytic virus into a 96-well plate, and placing the suspension into an incubator for pre-culture for 24 hours;diluting the virus liquid to be tested by 10 times (10)-1-10-11) And adding 8 multiple wells of the virus of each dilution gradient into each row of the 96-well plate respectively, culturing for 5-7 days at 37 ℃, observing pathological changes, calculating virus titer, and accurately calculating the titer by a Reed-Muench two-stage method. The test results are shown in Table 1.
3. Detecting the killing rate of oncolytic virus to tumor cells in tumor organoids
Inoculating a tumor organoid cell suspension in a 96-well plate so that each well contains 400-700 tumor cells, wherein each well contains 40 mu l of Cosmo temperature-sensitive hydrogel and 150 mu l of organoid culture solution; putting the inoculated 96-well plate into an incubator for pre-culture for 96 hours, taking out, and infecting tumor organs respectively for 24 hours, 48 hours and 72 hours by using 0.01-1MOI oncolytic virus (NDV oncolytic virus of Newcastle disease virus is used as oncolytic virus to be detected, and adenovirus ProstAtak oncolytic virus is used as reference oncolytic virus); cell viability was measured with CCK8 Kit (Cell Counting Kit 8), 10. mu.L of CCK8 solution was added to each well at 23, 47 and 71 hours of culture, incubation was continued for 1 hour, absorbance at 450nM was measured with a microplate reader, and tumor Cell killing rate (%) by oncolytic virus was calculated. The test results are shown in Table 2.
4. Testing the ability of oncolytic viruses to induce the host immune system to produce anti-tumor immunity
Centrifuging newly-extracted peripheral blood of a patient for 10min under the centrifugation condition of 800 Xg/min, inactivating upper layer plasma, centrifuging and sucking supernatant, transferring to a centrifuge tube for later use, adding physiological saline into a lower layer sediment of the blood to form a physiological saline blood sample mixed solution, putting an equal volume of lymphocyte separated liquid into a new centrifuge tube, slowly adding the physiological saline blood sample mixed solution to the upper layer of the lymphocyte separated liquid, centrifuging for 20min under the condition of 18-20 ℃ and 800 Xg/min, taking a middle white membrane layer, placing the middle white membrane layer into a new centrifuge tube, immediately adding PBS with three times volume to wash twice to obtain PBMCs, placing the PBMCs into a 1640 culture medium containing 100ng/mL CD3, 1000IU/mL IL-2 and 1ng/mL IL-1 α, and amplifying to the cell number of 0.1-1X 107Per mL, obtaining enough immune cells.
Inoculating 0.1-1X 10 in 6cm dishes7individual/mL of immune cells and tumor organoid cells, wherein the immune cells: tumor organoid cells ═ 3: 1, adding 4ml of tumor organoid culture solution, and putting the tumor organoid culture solution into an incubator for pre-culture for 24 hours; infecting the cultured mixed cells at 37 ℃ for 4 hours with 10MOI oncolytic virus (NDV oncolytic virus of newcastle disease virus, as a test oncolytic virus, and adenovirus ProstAtak oncolytic virus, as a reference oncolytic virus); and (4) sucking the supernatant, detecting the concentration of the cytokines interleukin-2 and the gamma-interferon in the cell supernatant according to the specification of an ELISA kit, and calculating the cytokine expression promoting capacity of the oncolytic virus. The test results are shown in Table 3.
Comparative example 1
The procedure was followed as in example 2 except that the lung cancer tumor organoids in step 2 were replaced with the a549 cell line 2D-cultured for lung cancer, and the normal embryonic lung fibroblast cell line MRC-5 was used as a negative control, and placed in an incubator and cultured for 24 hours, followed by infection with 0.01-1MOI oncolytic virus (newcastle disease virus NDV oncolytic virus as the oncolytic virus to be tested and adenovirus ProstAtak oncolytic virus as the reference oncolytic virus), and the replication level of the oncolytic virus was examined. The results are shown in Table 1.
Comparative example 2
The culture was performed according to the method of example 3, except that the lung cancer tumor organoids in step 2 were replaced with an a549 cell line of 2D-cultured lung cancer, and the normal embryonic lung fibroblast cell line MRC-5 was used as a negative control, and after 24 hours of culture in an incubator, the tumor cells were infected with 0.01 to 1MOI oncolytic virus (newcastle disease virus NDV oncolytic virus was used as the oncolytic virus to be tested, and adenovirus ProstAtak oncolytic virus was used as the reference oncolytic virus), and the killing rate of the oncolytic virus against the tumor cells was examined. The results are shown in Table 2.
Comparative example 3
The culture was performed according to the method of example 3, except that the lung cancer tumor organoids in step 2 were replaced with an a549 cell line of 2D-cultured lung cancer, and a normal embryonic lung fibroblast cell line MRC-5 was used as a negative control, and after being placed in an incubator and cultured for 24 hours, the cells were infected with 0.01 to 1MOI oncolytic virus (newcastle disease virus NDV oncolytic virus was used as an oncolytic virus to be tested, and adenovirus ProstAtak oncolytic virus was used as a reference oncolytic virus), the concentrations of cytokines interleukin-2 and γ -interferon in the supernatant were measured, and the cytokine pro-expression ability of the oncolytic virus was calculated. The results are shown in Table 3.
TABLE 1
As can be seen from table 1, both NDV oncolytic virus and ProstAtak oncolytic virus can replicate and amplify in both a549 cell line and lung cancer organoids, but both NDV oncolytic virus and ProstAtak oncolytic virus replicate at a weaker level than in a549 cell line, indicating that the lung cancer organoids have a tumor microenvironment that can affect the replication of oncolytic virus in tumor cells. In lung cancer organoids, NDV oncolytic virus replication capacity is weaker than that of ProstAtak oncolytic virus; in the a549 cell line, NDV oncolytic virus replication capacity is superior to that of the ProstAtak oncolytic virus.
TABLE 2
It can be seen from table 2 that both NDV oncolytic virus and ProstAtak oncolytic virus have killing effects on tumor cells in a549 cell line and lung cancer organoids, but both NDV oncolytic virus and ProstAtak oncolytic virus have a weaker killing ability on tumor cells in lung cancer organoids than on tumor cells in a549 cell line, indicating that the tumor microenvironment of lung cancer organoids can influence the killing ability of oncolytic virus on lung cancer cells. In lung cancer organoids, NDV oncolytic virus has a weaker oncolytic capacity than ProstAtak oncolytic virus; in the a549 cell line, NDV oncolytic virus has an oncolytic capacity significantly better than that of ProstAtak oncolytic virus.
TABLE 3
As can be seen from table 3, both NDV oncolytic virus and ProstAtak oncolytic virus can induce immune cells to produce cytokines in a549 cell lines and lung cancer organoids, but both NDV oncolytic virus and ProstAtak oncolytic virus have weaker ability to induce immune cells to produce cytokines in lung cancer organoids than their ability to induce immune cells to produce cytokines in a549 cell lines, which indicates that the tumor microenvironment of lung cancer organoids can influence the oncolytic virus to induce immune cells to produce cytokines, so the test result of the ability of oncolytic virus to induce immune cells to produce cytokines in tumor tissues of different patients can be more truly reflected. In lung cancer organoids, NDV oncolytic virus has a weaker ability to induce immune cells to produce cytokines than ProstAtak oncolytic virus; in the A549 cell line, the capacity of NDV (Newcastle disease virus) to induce immune cells to generate cytokines is obviously superior to that of ProstAtak oncolytic virus.
Test example 1
NDV (Newcastle disease virus) is taken as an oncolytic virus to be detected, adenovirus ProstAtak oncolytic virus is taken as a reference oncolytic virus, 10000pfu of NDV oncolytic virus and ProstAtak oncolytic virus are respectively injected into a PDX mouse, the tumor volume reduction ratio of the PDX mouse is observed after 30 days, the average survival period of the PDX mouse is calculated after 6 months, and the result is shown in Table 4.
The method for establishing the PDX mouse model comprises the following steps: a PDX mouse model was obtained by transplanting tumor tissue of a tumor patient into a severe immunodeficiency mouse (NSG) by a subcutaneous transplantation method and growing the tumor tissue in the mouse.
TABLE 4
| Oncolytic virus | Tumor volume reduction ratio% | Mean survival time of mice, day |
| NDV oncolytic virus | 7.36 | 93±2 |
| ProstAtak oncolytic virus | 15.82 | 122±3 |
In lung cancer organoids, the virus replication level, the killing rate to tumor cells and the cytokine expression promoting capacity of NDV (Newcastle disease virus) oncolytic virus are all weaker than those of ProstAtak oncolytic virus, and the verification of a PDX mouse animal model is obtained;
in A549 cell line, the virus replication level, the killing rate to tumor cells and the cytokine expression promoting capacity of NDV (Newcastle disease virus) are all superior to those of ProstAtak oncolytic virus, and the difference of the in vivo verification result of the NDV oncolytic virus and the in vivo verification result of a PDX mouse animal model is larger.
The above experiments illustrate that: by using the organoid as an in vitro cell model for detecting the effectiveness of the oncolytic virus, the detection result can more truly reflect the effectiveness of the oncolytic virus on tumor tissues in a patient.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (10)
1. A method for detecting the effectiveness of an oncolytic virus using an organoid, the method comprising:
a. performing mixed culture on the oncolytic virus and a tumor organoid for 12-96h to obtain a first culture, and detecting the replication level of the oncolytic virus in the first culture;
b. performing mixed culture on the oncolytic virus and a tumor organoid for 12-96h to obtain a second culture, and detecting the killing rate of the oncolytic virus to tumor cells in the second culture;
c. mixedly culturing the oncolytic virus, immune cells and tumor organoids for 2-8h to obtain a third culture, detecting the level of cytokines in the third culture and calculating the cytokine expression promoting capacity of the oncolytic virus, wherein the cytokines comprise interleukin-2 and/or gamma-interferon;
if the replication level, killing rate against tumor cells, and cytokine expression-promoting capacity of the test oncolytic virus are all higher than those of the reference oncolytic virus, indicating that the effectiveness of the test oncolytic virus is higher than that of the reference oncolytic virus.
2. The method of claim 1, wherein step a further comprises the step of subjecting the tumor organoid to a first pre-culture, followed by co-culturing the tumor organoid after the first pre-culture with the oncolytic virus; the first pre-culture comprises: mixing the tumor organoid with temperature-sensitive hydrogel and tumor organoid culture solution, and culturing for 12-96h, wherein the dosage of the temperature-sensitive hydrogel is 500-3000 mu L and the dosage of the tumor organoid culture solution is 2000-3000 mu L relative to the tumor cells in 10000 tumor organoids of 5000-; when the tumor organoid and the oncolytic virus are cultured in a mixed way, the dosage of the oncolytic virus is 0.01-1 MOI;
step b also comprises the step of carrying out second pre-culture on the tumor organoid, and then carrying out mixed culture on the tumor organoid subjected to second pre-culture and the oncolytic virus; the second pre-culture comprises: mixing the tumor organoid with the temperature-sensitive hydrogel and the tumor organoid culture solution, and culturing for 12-96h, wherein the dosage of the temperature-sensitive hydrogel is 30-80 mu L and the dosage of the tumor organoid culture solution is 100-150 mu L relative to the tumor cells in 1000 tumor organoids with 300-; when the tumor organoid and the oncolytic virus are cultured in a mixed way, the dosage of the oncolytic virus is 0.01-1 MOI;
in step c, the mixed culture of the oncolytic virus, the immune cell and the tumor organoid comprises the following steps: c1, inoculating cell suspension containing immune cells and tumor organoid in a culture dish, adding tumor organoid culture solution, and culturing for 2-8h to obtain mixed culture solution, wherein the concentration of immune cells and tumor cells in the cell suspension is 0.1-1 × 107individual/mL, immune cell: tumor organoid cells ═ (2-8): 1, the amount of said tumor organoid culture medium is 2-3mL, relative to 0.5-2mL of said cell suspension; c2, mixing the mixed culture solution with the oncolytic virus for culturing for 3-5h, wherein the dosage of the oncolytic virus is 1-100 MOI.
3. The method of claim 1, wherein in step a, said detecting the level of replication of the oncolytic virus in said first culture comprises:
a1, obtaining the oncolytic virus in the first culture to obtain a virus to be detected;
a2, culturing the host cells of the oncolytic virus and the virus to be detected for 12-96h in a mixed way, and then detecting the virus titer of the virus to be detected in the host cells; wherein the higher the viral titer of said test virus in said host cell, the higher the level of replication of the oncolytic virus in said first culture.
4. The method of claim 3, wherein in step a1, said obtaining the oncolytic virus in the first culture to obtain a test virus comprises:
and (3) performing freeze-thawing treatment on the first culture for 2-8 times, then performing centrifugation and collecting supernatant to obtain virus liquid to be detected, wherein the virus liquid to be detected contains the virus to be detected.
5. The method according to claim 1, wherein in step b, the killing rate of tumor cells by the oncolytic virus in the second culture is detected by using a CCK8 kit;
in step c, the cytokine level in the third culture is detected using an ELISA kit.
6. The method of claim 1, wherein the immune cells in step c are isolated from the peripheral blood of the patient.
7. The method according to any one of claims 1 to 6, wherein said mixed culture of steps a, b and c is carried out in a tumor organoid culture medium comprising DMEM/F12 medium and growth factors; the growth factor comprises at least one of Glutamax, HEPES, Gastrin, nicotinamide, A83-01, Noggin, n-acetyl cysteine, streptomycin, EGF and BSA.
8. The method as claimed in claim 7, wherein the concentration of Glutamax is 0.5-2%, the concentration of HEPES is 5-15mM, the concentration of Gastrin is 5-15nM, the concentration of nicotamide is 5-15mM, the concentration of A83-01 is 250-600ng/mL, the concentration of Noggin is 50-150ng/mL, the concentration of n-Acetycysteine is 0.5-2mM, the concentration of streptomycin is 50-150 μ g/mL, the concentration of EGF is 50-150ng/mL, and the concentration of BSA is 1-5mg/mL in the growth factor.
9. The method of any one of claims 1-6, wherein the method of preparing the tumor organoid comprises:
mixing the tumor cells, the temperature-sensitive hydrogel and the tumor organoid culture solution, culturing, and replacing the culture medium every 2-4 days until the tumor organoid with the diameter not less than 0.5mm is observed under a microscope.
10. The method as claimed in claim 9, wherein the temperature-sensitive hydrogel is used in an amount of 1500-2000 μ L and the tumor organoid culture fluid is used in an amount of 2000-3000 μ L, respectively, with respect to 20000 tumor cells.
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