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WO2016146616A1 - Lames de contrôle pour immunohistochimie générées à partir de cultures de lignées de cellules tridimensionnelles - Google Patents

Lames de contrôle pour immunohistochimie générées à partir de cultures de lignées de cellules tridimensionnelles Download PDF

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Publication number
WO2016146616A1
WO2016146616A1 PCT/EP2016/055514 EP2016055514W WO2016146616A1 WO 2016146616 A1 WO2016146616 A1 WO 2016146616A1 EP 2016055514 W EP2016055514 W EP 2016055514W WO 2016146616 A1 WO2016146616 A1 WO 2016146616A1
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WIPO (PCT)
Prior art keywords
cell line
spheroids
cell
tissue
disease
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PCT/EP2016/055514
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English (en)
Inventor
Jean Boyer
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Ventana Medical Systems, Inc.
F. Hoffmann-La Roche Ag
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Application filed by Ventana Medical Systems, Inc., F. Hoffmann-La Roche Ag filed Critical Ventana Medical Systems, Inc.
Publication of WO2016146616A1 publication Critical patent/WO2016146616A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples

Definitions

  • This disclosure relates to control slides for immunohistochemistry, namely, slides generated using three-dimensional cultures of cell lines.
  • tissue expected to display typical staining patterns for the analyte of interest are used as controls.
  • expression patterns can vary between control slides from primary tissues due to, e.g., differences in tissue micro- environments and cellular compositions and genetic variability between subjects.
  • primary tissue is often in short supply and can be difficult to obtain, making it an uneconomical choice for control slides.
  • Xenografts have been used to generate control slides as well.
  • xenografts can be expensive to generate, as they require live animals.
  • the ability to reliably generate xenografts varies from cell line to cell line, with many cell lines being very difficult to use for generation of xenografts.
  • Cell lines have also been used to make control slides. See Xiao et al. However, arrangements of cells resemble of tissue samples cannot be created using traditional two-dimensional cell cultures. Moreover, cells grown in two- dimensional culture do not have the same types of cell-cell and cell-matrix interactions that are experienced by cells in vivo, which can affect intracellular signaling and expression patterns. Additionally, standard two-dimensional cell culture is susceptible to variations resulting from differences in handling between different laboratories, such as differences in culture passage numbers, cell densities at passage, lot to lot characteristics, and other manipulations involved in processing and maintenance of the cell lines.
  • Pinto et al. analyzed three-dimensional cultures of mixed cell colonies microscopically.
  • Pinto's method relies on a MATRIGELTM matrix, which is a matrix based on a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.
  • EHS Engelbreth-Holm-Swarm
  • Cell blocks cultured in MATRIGEL were sandwiched between layers of Histogel and, after hardening, the "sandwiches" were transferred to cassettes for fixation and paraffin embedding. The paraffin-embedded blocks could then be sectioned and stained. However, this process leaves the MATRIGEL matrix intact, which could interfere with accurate staining and reading.
  • the matrix is of mammalian origin, there is a potential for cross-reaction between the matrix and the antibodies.
  • Pinto only analyzed antibody staining using immunofluorescent microscopy, and thus did not demonstrate that their procedure is compatible with the more common brightfield microscopy techniques. Additionally, the procedures used for generating the slides do not result in a sufficient spheroid density for manufacture of consistent and repeatable quality control slides.
  • Kunz-Schughart et al. used an agarose-coated 96 well plate to generate tumor and fibroblast spheroids, which were then combined and co-cultured to study cell-cell interactions between the tumor and fibroblast cells.
  • Kunz-Schughart used only frozen sections for IHC staining, which is incompatible with manufactured slides. Additionally, the procedures used for generating the slides do not result in a sufficient spheroid density for manufacture of consistent and repeatable quality control slides.
  • Graham et al. generated slides from MATRIGEL cell blocks by, inter alia, contacting the cell blocks with human plasma, followed by thrombin to encapsulate the cell block in a thrombin clot.
  • the thrombin clot is then fixed and embedded in paraffin for sectioning.
  • the inclusion of human components to the slide introduces a potential source of non-specific binding.
  • the handling required for using human products, as well as the complexity of the technique itself, makes it very difficult to do on a large scale.
  • the present disclosure includes materials and methods for forming cell blocks useful in generating microscope slides for use in histochemical analysis, such as immunohistochemical and in situ hybridization assay.
  • the resulting cell blocks are particularly useful for generating control slides for diagnostic assays.
  • a method of manufacturing a histological matrix block embedded with a control cell line comprising: (a) culturing a control cell line indicative of a disease or tissue state in an animal origin-free bioscaffold to generate a plurality of spheroids; (b) removing the spheroids from the animal origin-free bioscaffold and concentrating the spheroids; (c) fixing said concentrated spheroids; (d) suspending the fixed spheroids in a porous embedding material compatible with tissue processing for histochemistry; and (e) subjecting the porous embedding material to tissue processing and then embedding the porous embedding material in a histological matrix.
  • the animal origin-free bioscaffold is an alginate matrix having a pore size of from about 50 ⁇ to about 200 ⁇ .
  • the spheroids are removed from the bioscaffold when they have a diameter approximately equal to the pore size of the bioscaffold.
  • the cell line is seeded in the bioscaffold at a density such that the spheroids reach a diameter approximately equal to the pore size within 12 to 14 days of seeding. In other embodiments, the cell line is within the exponential growth phase when the spheroids are removed from the bioscaffold.
  • the spheroids contain at least 90% viable cells as measured by incorporation of calcein-AM dye and ethidium homodimer-1 dye when removed from the bioscaffold.
  • the porous embedding material in which the spheroids are deposited is an agarose-based matrix.
  • the porous embedding material is embedded in a paraffin block.
  • a cell block obtained by the methods disclosed herein is provided.
  • the cell block is a formalin-fixed paraffin- embedded (FFPE) set of concentrated spheroids derived from a control cell line, the concentrated spheroids suspended in an agarose-based porous embedding material prior to embedding.
  • FFPE formalin-fixed paraffin- embedded
  • a method of manufacturing a control slide for a histochemical assay comprising manufacturing the histological matrix embedded with the control cell line as disclosed herein, sectioning the histological matrix block to a size compatible with histochemistry, and affixing said section to a microscope slide.
  • at least 500 slides are manufactured from a single block.
  • control slide is provided, the control slide obtained by the methods as disclosed herein.
  • kits for performing a histochemical assay comprising a cell block or a control slide obtained by a method as disclosed herein and a specific binding entity capable of binding to a biomarker of interest.
  • the kit further comprises at least one additional control slide or cell block as disclosed herein, wherein each control slide or cell block is indicative of a different disease or tissue state for the same tissue.
  • the specific binding entity is a protein-binding entity and the biomarker of interest is a protein characteristic of a disease or tissue state of which the cell line is representative.
  • the specific binding entity is an antibody or antigen-binding fragment thereof, and the biomarker of interest is an antigen characteristic of a disease or tissue state of which the cell line is representative.
  • the specific binding entity is a nucleic acid probe and the biomarker is a nucleic acid characteristic of a disease or tissue state of which the cell line is representative.
  • the kit is useful for calibrating an automated IHC/ISH slide stainer.
  • an immunohistochemical method of assaying a primary tissue sample for an antigen indicative of a disease or tissue state comprising: (a) staining a control slide obtained as disclosed herein and a test slide having a section of the primary tissue sample affixed thereto with an antibody capable of binding to the antigen; and (b) comparing a staining pattern of the control slide to a staining pattern of the test slide.
  • an in situ hybridization method of assaying a primary tissue sample for a disease or tissue state comprising: (a) staining a control slide obtained according to the methods as disclosed herein and a test slide having a section of the primary tissue sample affixed thereto with a nucleic acid probe capable of hybridizing to a biomarker characteristic of a disease or tissue state of which the cell line is representative; and (b) comparing a staining pattern of the control slide to a staining pattern of the test slide.
  • the staining is performed on an automated platform.
  • Fig. 1 is a classification chart of exemplary scaffold-based three-dimensional cell culture methodologies
  • Fig. 2 is a pair of line graphs that demonstrate the growth curves obtained using low and high density seeding in a 3D culture method. Viability represented in the graph is determined using Trypan Blue staining.
  • Fig. 3 is an IHC image of spheroids from a low density-seeded three dimensional culture labeled with calcein AM and EthD-1. Green cells are viable. Red cells are dead. The dead cells are indicated by hatched arrows.
  • Fig. 4 is an IHC image of spheroids from a high density-seeded three dimensional culture labeled with calcein AM and EthD-1. Green cells are viable. Red cells are dead. The dead cells are indicated by hatched arrows.
  • Fig. 5 is image of an IHC assay comparing HT29 2D cultures, HT-29 3D cultures, HT-29 xenografts, and colon adenocarcinoma tissue immunohistochemically labeled for c-Myc, beta-catenin, BRAF V600E, and PTEN and stained with DAB.
  • Fig. 6 illustrates exemplary IHC assays for phospho-AKT using various fixation conditions.
  • A 2-dimensional culture of HT-29 cells fixed using at 4 °C for 2 hours followed by at 37 °C for 2 hours protocol;
  • B 3-dimensional culture of HT-29 cells fixed using at 4 °C for 2 hours followed by at 37 °C for 2 hours protocol;
  • C 3-dimensional culture of HT-29 cells fixed using at 4 °C for 26 hours;
  • D 3-dimensional culture of HT-29 cells fixed using a "2+2" fixation protocol;
  • E 3-dimensional culture of HT-29 cells fixed at 4°C for 24 hours;
  • F HT-29 xenograft fixed using at room temperature for 24 hours; and
  • G Calu-3 xenograft fixed using a "2+2" fixation protocol. All images were photographed at 10X magnification.
  • Fig. 7 illustrates an exemplary in situ hybridization assay for HER2 using various fixation conditions.
  • A 2-dimensional culture of HT-29 cells fixed at 4 °C for 2 hours;
  • B 3-dimensional culture of HT-29 cells fixed using a "2+2" fixation protocol;
  • C HT-29 xenograft fixed at room temperature for 24 hours.
  • 3D cell culture techniques have previously been used to produce slides for immunohistochemical assays.
  • the utility of such processes has been limited to research uses, as these techniques typically rely on process steps that are impractical for commercial slide production.
  • Cell blocks are made using essentially a four-step method: (1) culturing the cell line in a 3D bioscaffold; (2) concentrating the resulting spheroids once they have reached an adequate size; (3) fixing the concentrated spheroids; (4) suspending the fixed spheroids in a porous embedding material to form a spheroid pellet, and embedding the spheroid pellet in a histological matrix.
  • the term "spheroid" shall refer to clumps of cells resulting from a 3-dimensional (3D) culture of a cell line.
  • 3D culture techniques can generally be divided into (1) scaffold-based techniques and (2) non-scaffo ld-based techniques. Scaffold-based techniques use a porous matrix onto which the cells adhere (referred to herein as a "bioscaffold”) to guide spheroid formation, whereas scaffold-free techniques rely on self-adhesive properties of the cell lines to drive spheroid formation, and thus lack a bioscaffold.
  • the cell blocks of the present invention are generated from spheroids cultured using a scaffold-based technique.
  • a family tree of various 3D cell culture methods is displayed at Fig. 1.
  • bioscaffolds can generally be divided into hydrogel-based bioscaffolds and inert material bioscaffolds.
  • Hydrogel-based bioscaffolds can be further divided into animal-derived hydrogels, non-animal cellular material-derived hydrogels, and synthetic hydrogels.
  • the cell blocks of the present invention are generated using bioscaffolds that are substantially free of animal-origin hydrogels.
  • the bioscaffold may comprise or consist of a non-animal cellular material-derived hydrogel, a synthetic hydrogel, a porous inert material, or combinations thereof.
  • Exemplary non-animal cellular material-derived hydrogels include hydrogels derived from algae -based materials, microbial-based materials, and plant-based materials. Examples of algae - based materials include carrageenans, agarose, alginates.
  • algal bioscaffold is the ALGIMATRIX 3D culture system (Thermo Fisher Scientific, Inc.), which uses an alginate sponge having a pore size of about 50 ⁇ to 200 ⁇ .
  • plant-based hydrogels include galactomannan gum-based matrices (such as those disclosed by WO 2008-112170 Al to Corning Inc. et al.) and cellulose-based matrices (such as those disclosed by
  • microbial-based hydrogels include gellan gum-based matrices (such as those evaluated by Smith et al.) and xanthan gum-based matrices (such as those disclosed by Mendes et al.).
  • exemplary synthetic hydrogels include, for example: poly(ethylene glycol), poly(vinyl alcohol), and poly(2-hydroxy ethyl methacrylate)-based matrices (such as those reviewed by Tibbitt & Anseth); and poly(N-isopropylacrylamide)-based matrices (such as those disclosed by Rossouw et al. and Lei & Schaffer).
  • the bioscaffold may comprise or consist of a porous inert material, such as ceramic or polystyrene -based bioscaffolds.
  • a porous inert material such as ceramic or polystyrene -based bioscaffolds.
  • Many other bioscaffolds that are substantially free of animal-origin hydrogels are known in the art.
  • the animal origin-free bioscaffold has an average pore size in the range of 30 um to 300 ⁇ , 30 ⁇ to 275 ⁇ , 30 ⁇ to 250 ⁇ , 35 ⁇ to 300 ⁇ , 35 ⁇ to 275 ⁇ , 35 ⁇ to 250 ⁇ , 40 ⁇ to 300 ⁇ , 40 ⁇ to 275 ⁇ , 40 ⁇ to 250 ⁇ , 45 ⁇ to 300 ⁇ , 45 ⁇ to 275 ⁇ , 45 ⁇ to 250 ⁇ , 50 ⁇ to 300 ⁇ , 50 ⁇ to 275 ⁇ , or 50 ⁇ to
  • the animal origin-free bioscaffold has an average pore size of from about 50 ⁇ to about 250 ⁇ .
  • the animal origin-free bioscaffold is an alginate matrix having an average pore size of from about 50 ⁇ to about 250 ⁇ . Pore sizes referenced herein are average pore sizes as determined by scanning electron microscopy.
  • the cell lines used to generate the cell blocks are selected to be relatively fast growing and relatively small.
  • a "relatively fast growing cell line” is a cell line that has a doubling time of 40 hours or less, explicitly including, for example, cell lines having doubling times of 35 hours or less, 30 hours or less, 25 hours or less, 20 hours or less, from 20 to 40 hours, from
  • doubling times refer to doubling times of the cell line when cultured in a standard 2-dimensional culture at the exponential growth phase.
  • a “relatively small cell line” shall refer to a cell line having an average cell size about 15 ⁇ or smaller. In certain examples, the cell line is a cell line that has an average cell size such that from 5 to 15 ⁇ , and subranges thereof.
  • the cells are seeded at a cell density such that the cells are in an exponential growth phase when they reach an average spehroid size of not less than 60% of the average pore size of the animal origin- free bioscaffold, for example from 60% to
  • the cells are seeded at a cell density such that the cells reach the aforementioned average spheroid size within 12 to 14 days of seeding.
  • the spheroids have an average diameter in the range of 50 ⁇ to 250 ⁇ within 12 to
  • cells having an average diameter in the range of 5 to 15 ⁇ are seeded in an alginate bioscaffold at a cell density in the range of 2 x 10 4 to 2 x 10 6 cells per well of a standard 6 well plate, and grown under conditions that spheroids having an average diameter in the range of 50 ⁇ to 250 ⁇ are obtained within 12 to 14 days of seeding.
  • the cells are collected from the animal origin-free bioscaffold after they have reached the aforementioned average spheroid size, but while they are still in the exponential growth phase.
  • the average spheroid size of collection is selected such that at least 90% of cells per spheroid are viable.
  • viability is determined using Trypan Blue with manual counting and/or calcein AM and ethidium homodimer-1 (EthD-1) incorporation to count live/dead cells.
  • the manner in which the spheroids are separated from the bioscaffold depends on the precise identity of the bioscaffold.
  • the bioscaffold can be dissolved (such as for alginate-based bioscaffolds) or melted. The spheroids can then be separated from the bioscaffold material and the cells concentrated, for example, by centrifugation and removal of the supernatant.
  • the fixation process is a chemical fixation process.
  • Chemical fixation involves immersing a tissue sample in a volume of chemical fixative, typically at least 20 times the volume of the tissue to be fixed. The fixative diffuses through the tissue sample and preserves structures (both chemically and structurally) as close to that of living tissue as possible.
  • Cross-linking fixatives typically aldehydes, create covalent chemical bonds between endogenous biological molecules, such as proteins and nucleic acids, present in the tissue sample. Formaldehyde is the most commonly used fixative in histology.
  • Formaldehyde may be used in various concentrations for fixation, but it primarily is used as 10% neutral buffered formalin (NBF), which is about 3.7% formaldehyde in an aqueous phosphate buffered saline solution.
  • NBF neutral buffered formalin
  • Paraformaldehyde is a polymerized form of formaldehyde, which depolymerizes to provide formalin when heated.
  • Glutaraldehyde operates in similar manner as formaldehyde, but is a larger molecule having a slower rate of diffusion across membranes.
  • Glutaraldehyde fixation provides a more rigid or tightly linked fixed product, causes rapid and irreversible changes, fixes quickly and well at 4 °C, provides good overall cytoplasmic and nuclear detail, but is not ideal for immunohistochemistry staining.
  • Some fixation protocols use a combination of formaldehyde and glutaraldehyde. Glyoxal and acrolein are less commonly used aldehydes.
  • the spheroids are fixed in
  • the fixative is an aldehyde-based cross-linking fixative, such as glutaraldehyde- and/or formalin-based solutions.
  • aldehydes frequently used for immersion fixation include:
  • the fixative comprises a standard concentration of formaldehyde, glyoxal, or glutaraldehyde.
  • the aldehyde -based fixative solution is about 5% to about 20% formalin.
  • Aldehydes are often used in combination with one another. Standard aldehyde combinations include 10% formalin + 1% (w/v) Glutaraldehyde. Atypical aldehydes have been used in certain specialized fixation applications, including: fumaraldehyde, 12.5% hydroxyadipaldehyde (pH 7.5), 10%> crotonaldehyde (pH 7.4), 5% pyruvic aldehyde (pH 5.5), 10% acetaldehyde (pH 7.5), 10% acrolein (pH 7.6), and 5% methacrolein (pH 7.6). Other specific examples of aldehyde- based fixative solutions used for immunohistochemistry are set forth in Table 1 :
  • Hollande's Solution aqueous solution comprising 25 g copper acetate and 40g picric acid
  • the fixative solution is selected from Table 1.
  • the term "about” shall be understood to encompass all concentrations outside of the recited range that do not result in a statistically significant difference in diffusion rate in the same type of tissue having the same size and shape as measured by Bauer et al. , Dynamic Subnano second Time-of-Flight Detection for Ultra-precise Diffusion Monitoring and Optimization of Biomarker Preservation, Proceedings of SPIE, Vol. 9040, 90400B-1 (2014-Mar-20).
  • the cells are fixed using a two-temperature fixation process.
  • two-temperature fixation refers to a fixation protocol using an aldehyde-based fixative in which the sample is first immersed in the aldehyde- based fixative at a temperature sufficiently cold to retard reaction of the fixative with the sample for a sufficient period of time to allow the fixative to diffuse throughout the sample, and then subjecting the sample to a warmer temperature for a sufficient period of time to allow the aldehyde-based fixative to fix the cellular sample.
  • the cold temperature is in the range of 0 °C to 10 °C
  • the warm temperature is in the range of 15 °C to 50 °C.
  • the cold temperature is from 0 °C to 7 °C for not more than at least 1 hour.
  • Two- temperature fixation protocols are particularly useful when the downstream analysis is conducted on a labile biomarker, such as a phosphorylated protein or an R A molecule.
  • a labile biomarker such as a phosphorylated protein or an R A molecule.
  • One particular fixation protocol, termed "2+2" involves a 2 hour immersion in 10% NBF at 4 °C followed by a 2 hour immersion in 10% NBF at 45 °C.
  • D Porous embedding material
  • the concentrated and fixed spheroids are aggregated to one another by embedding them in a porous embedding material.
  • the porous embedding media should be a material which is normally solid at room temperature and at tissue processing temperatures, and should have a melting point or liquidus point which is below the temperature at which the spheroids would become denatured or otherwise changed by heat.
  • the porous embedding media also is a material that is porous to treating solutions commonly used in processing and fixing tissues, e.g. acetic acid, acetone, chromic or picric acid, alcohols, aldehydes, mercuric chloride, osmium tetroxide, potassium dichloride, xylene, etc.
  • low melting point agarose i.e. agarose having a melting point in the range of about 55 to 65° C
  • An exemplary porous embedding material is the HISTOGELTM brand of hydroxyethyl agarose.
  • the fixed and concentrated spheroids are suspended in the porous embedding material at a temperature at which the porous embedding material is liquid, placed into a mold, and held in the mold until at least the porous embedding material has solidified.
  • post-fixation processing shall encompass any process following aldehyde fixation that is used to prepare the fixed sample for storage and/or analysis. Many such processes are well-known and would be well understood by a person of ordinary skill in the art. For example, protocols for using zinc formalin, Helly's fixative and Hollande's require a water wash after fixation to remove various contaminates. Some protocols for Bouin's and B-5 suggest storing the fixed samples in 70% ethanol before processing. Additionally, some specimens may be difficult to cut on a microtome because of calcium carbonate or phosphate deposits, and thus may require decalcification.
  • post-fixation processing comprises wax-embedding.
  • the pellet is subjected to a series of alcohol immersions to dehydrate the sample, typically using increasing alcohol concentrations ranging from about 70% to about 100%.
  • the alcohol generally is an alkanol, particularly methanol and/or ethanol.
  • the clearing solution (1) removes residual alcohol, and (2) renders the sample more hydrophobic for a subsequent waxing step.
  • the clearing solvent typically is an aromatic organic solvent, such as xylene.
  • Wax blocks are formed by applying a wax, typically a paraffin wax, to the pellet.
  • the sample may be embedded in resin blocks (such as epoxy or acrylic resins) instead of wax blocks.
  • resin blocks include methyl methacrylate, glycol methacrylate, araldite, and epon. Each requires specialized post-fixation processing steps, which are well known in the art.
  • the blocks are sliced into thin sections using a microtome. The thin sections may then be mounted on a slide and stored for later analysis and/or subjected to post-processing analysis.
  • the slides generated from the present methods are particularly useful as control slides for histochemical assays, including immunohistochemical assays and in situ hybridization assays.
  • the cells used for such control slides are typically picked for expressing a biomarker or group of biomarkers of interest.
  • biomarker shall refer to any molecule or group of molecules found in a biological sample that can be used to characterize the biological sample or a subject from which the biological sample is obtained.
  • a biomarker may be a molecule or group of molecules whose presence, absence, or relative abundance is:
  • the biomarker may be an infectious agent (such as a bacterium, fungus, virus, or other microorganism), or a substituent molecule or group of molecules thereof.
  • infectious agent such as a bacterium, fungus, virus, or other microorganism
  • the term "biological sample” shall refer to any material obtained from a subject capable of being tested for the presence or absence of a biomarker.
  • the cell line is derived from the same tissue type as a tissue sample or cytological sample of interest.
  • tissue sample shall refer to a sample that preserves the cross-sectional spatial relationship between the cells as they existed within the subject from which the sample was obtained.
  • cytological sample refers to a cellular sample in which the cells of the sample have been partially or completely disaggregated, such that the sample no longer reflects the spatial relationship of the cells as they existed in the subject from which the cellular sample was obtained.
  • cytological samples include tissue scrapings (such as a cervical scraping), fine needle aspirates, samples obtained by lavage of a subject, et cetera.
  • tissue or cytological sample of interest is a specific tumor and the cell line is derived from the same type of tumor.
  • the tissue sample of interest is a specific tumor and the cell line is a model system for normal cells of the same tissue types.
  • the kit comprises a control slide and a specific binding entity capable of specifically binding to a biomarker of interest.
  • a specific binding entity capable of specifically binding to a biomarker of interest.
  • the phrase “specific binding”, “specifically binds to,” or “specific for” refers to measurable and reproducible interactions such as binding between a target and a specific binding agent, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • a binding entity that specifically binds to a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets.
  • the extent of binding of a binding entity to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).
  • a binding entity that specifically binds to a target has a dissociation constant (Kd) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • Kd dissociation constant
  • specific binding can include, but does not require exclusive binding.
  • the term "specific binding agent" shall refer to any compound or composition that is capable of specifically binding to a biomarker or a specific structure within that biomarker.
  • nucleic acid probes specific for particular nucleotide sequences examples include nucleic acid probes specific for particular nucleotide sequences; antibodies and antigen binding fragments thereof; and engineered specific binding structures, including ADNECTINs (scaffold based on 10th FN3 fibronectin; Bristol-Myers-Squibb Co.), AFFIBODYs (scaffold based on Z domain of protein A from S.
  • ADNECTINs scaffold based on 10th FN3 fibronectin; Bristol-Myers-Squibb Co.
  • AFFIBODYs scaffold based on Z domain of protein A from S.
  • the specific binding entity is an antibody and the biomarker is characteristic of a disease or tissue state of which the cell line is representative.
  • the specific binding entity is a nucleic acid probe and the biomarker is a nucleic acid characteristic of a disease or tissue state of which the cell line is representative.
  • a positive control slide and a negative control slide is provided.
  • the positive control slide is generated using a cell line that is expected to express the biomarker or biomarkers of interest, or is a cell line derived from the same diseased tissue type as the tissue or cytological sample of interest.
  • the negative control slide is a cell line derived or representative of a normal tissue of the tissue or cytological sample of interest.
  • Post-processing analysis Control slides obtained by the processes and compositions disclosed herein can be used together with any staining systems and protocol known in the art of histochemistry, as well as affinity histochemistry, immunohistochemistry and in situ hybridization.
  • the present invention can also be used together with various automated staining systems, including those marketed by Ventana Medical Systems, Inc. (such as the VENTANA HE600, SYMPHONY, BENCHMARK, and
  • control slides are used as controls for detecting specific analytes using immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • a tissue sample is contacted first with an analyte-specific antibody under conditions sufficient to permit specific binding of the analyte-specific antibody to the analyte.
  • detection of specific analytes is realized through antibodies capable of specific binding to the analyte (or antibody fragments thereof) conjugated with multiple enzymes (e.g. horse radish peroxidase (HRP), alkaline phosphatase (AP).
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • HRP or AP multimer in light of the multiplicity of enzymes conjugated to each antibody.
  • Multimer technologies are described in U.S. Patent No. 8,686,122, which is hereby incorporated by reference in its entirety.
  • This type of detection chemistry technology is currently marketed by Ventana Medical Systems Inc., as ultra View Universal DAB detection kit (P7N 760-500), ultraView Universal AP Red detection kit (P/N 760-501), ultraView Red ISH DIG detection kit (P/N 760-505), and ultraView SISH DNP detection kit (P/N 760-098).
  • the approach uses non-endogenous haptens (e.g. not biotin, see U.S. application Ser. No.
  • a tyramide signal amplification may be used with this approach to further increase the sensitivity and dynamic range of the detection (See PCT/US2011/042849 which is hereby incorporated by reference in its entirety for disclosure related to detection chemistries).
  • Any suitable enzyme/enzyme substrate system can be used for the disclosed analysis/detection method.
  • Working embodiments typically used alkaline phosphatase and horseradish peroxidase.
  • the enzyme is alkaline phosphatase
  • one suitable substrate is nitro blue tetrazolium chloride/(5-bromo-4-chloro-lH-indol-3- yl)dihydrogen phosphate (NBT/BCIP).
  • NBT/BCIP nitro blue tetrazolium chloride/(5-bromo-4-chloro-lH-indol-3- yl)dihydrogen phosphate
  • DAB diaminobenzidine
  • Numerous other enzyme- substrate combinations are known to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149, and 4,318,980.
  • the enzyme is a peroxidase, such as horseradish peroxidase or glutathione peroxidase or an oxidoreductase.
  • U.S. Patent Publication 2008/0102006 the entire disclosure of which is incorporated herein by reference, describes robotic fluid dispensers that are operated and controlled by microprocessors.
  • U.S. Patent Publication 2011/0311123 the entire disclosure of which is incorporated herein by reference, describes methods and systems for automated detection of IHC patterns. The automated detection systems disclosed in these patent applications can be used to detect analytes in the fixed tissue samples of the present invention.
  • the fixed tissue samples are analyzed by immunohistochemistry for the presence of post-translationally modified proteins.
  • the cell line for use in manufacturing the control slide is selected for endogenous presence or absence of the post-translational modification of the target protein, or may be treated (for example with receptor ligands, phosphatase or kinase inhibitors, and/or other excitatory or inhibitory molecules), or may be recombinantly engineered to express and/or produce the post-translationally modified protein.
  • the slides are contacted with an analyte- binding entity capable of specifically binding to the post-translationally modified protein under conditions sufficient to effect binding of the analyte -binding entity to the post-translationally modified protein; and binding of the analyte-binding entity to the post-translationally modified protein is detected.
  • the precise conditions for effective IHC generally need to be worked on an individual basis, depending upon, for example, the precise antibody used, the type of sample used, sample size, further processing steps, et cetera.
  • the post-translational modification is one that is susceptible to loss during a standard aldehyde fixation process due to residual enzyme activity within the tissue sample.
  • the sample could then be fixed using a standard technique (such as 24 hour fixation in room temperature NBF) and a fixation process as disclosed herein and the amount of signal detectable in each of the samples can be compared. If signal is absent or significantly lower in the sample fixed according to standard techniques, then one can assume that the post- translational modification is susceptible to degradation by residual enzyme activity.
  • the post-translational modification is a post-translational modification that has a lower level of detection in a tissue fixed for 24 hours in room temperature NBF without a cold temperature pre-treatment than in a substantially identical tissue sample that has been fixed using a two-temperature fixation as described above.
  • the post-translational modification is a diagnostic or prognostic marker for a disease state of the tissue sample.
  • the post-translational modification is a predictive marker for an effect of a therapy on a disease state of the tissue.
  • the post- translational modification is a phosphorylation.
  • the fixed tissue samples are analyzed by in situ hybridization for the presence of specific nucleic acids.
  • the fixed tissue sample is contacted with a nucleic acid probe complementary to the analyte nucleic acid under conditions sufficient to effect specific hybridization of the probe to the analyte nucleic acid; and binding of the nucleic acid probe to the analyte nucleic acid is detected.
  • the precise conditions for effective ISH generally need to be worked on an individual basis, depending upon, for example, the precise nucleic acid probe used, the type of sample used, sample size, further processing steps, et cetera.
  • the analyte nucleic acid is one that is susceptible to loss during a standard aldehyde fixation process due to residual enzyme activity within the tissue sample.
  • the sample could then be fixed using a standard technique (such as 24 hour fixation in room temperature NBF) and a fixation process as disclosed herein and the amount of signal detectable in each of the samples can be compared. If signal is absent or significantly lower in the sample fixed according to standard techniques, then one can assume that the analyte nucleic acid is susceptible to degradation by residual enzyme activity.
  • the analyte nucleic acid has a lower level of detection in a tissue fixed for 24 hours in room temperature NBF without a cold temperature pre- treatment than in a substantially identical tissue sample that has been fixed using a two-temperature fixation as described above.
  • the analyte nucleic acid is a diagnostic or prognostic marker for a disease state of the tissue sample.
  • the analyte nucleic acid is a predictive marker for an effect of a therapy on a disease state of the tissue.
  • the analyte nucleic acid is an RNA molecule, such as mRNA or miRNA.
  • a colorectal adenocarcinoma cell line - HT-29 - was cultured in McCoy's 5A media, using a 3D cell culture method.
  • the HT-29 cell line is described at Fogh et al.
  • HT-29 cells were seeded at 1.25 x 10 5 (low density) or 5.3 x 10 5 (high density) cells per plate in 24-well plates of the ALGIMATRIX 3D cell culture system (Life Technologies Inc., Rockville, MD). Media were changed daily or every other day as required based on observed cell growth.
  • Spheroids were allowed to form for 3, 4, 6, 8, or 10 days, after which they were extracted from the ALGIMATRIX matrix by dissolving the matrix using ALGIMATRIX dissolving buffer which releases the spheroids.
  • the spheroids are washed and spun down for fixation with 10% NBF for 2 hours at 4 °C followed by 10% NBF for 2 hours at
  • spheroids were seeded at either low or high density as described above and grown for 10 days. Spheroids were collected and stained with Live/Dead Viability/Cytotoxicity Assay (Life Technologies Inc., Rockville, MD) in order to determine viability.
  • the Live/Dead ® Viability/Cytotoxicity Assay Kit is a two-color fluorescence cell viability assay used to produce immunofluorescent images of 3D Spheroids as shown below. Live cells contain intracellular esterase activity which can be detected by the enzymatic conversion of nonfluorescent cell- permeable calcein AM to intensely fluorescent calcein, shown as the green color in the images.
  • Ethidium homodimer-1 enters cells with damaged membranes and undergoes fluoresence upon binding to nucleic acids, thereby producing a bright red fluoresence in dead cells. EthD-1 is excluded by the intact plasma membrane of live cells.
  • Fig. 2 is an exemplary image of spheroids plated at low density, and Table 3 provides the associated data.
  • Fig. 3 is an exemplary image of spheroids from cells seeded at high density, and Table 4 provides the associated data. Visible red cells (i.e. dead) are shown in Figs. 2 and 3 by the white hatched arrows. As may be seen in the images at Figs. 2 and 3, the majority of the cells in the spheroids are alive.
  • Aggregated data is displayed at Tables 5-8.
  • spheroid sizes varied from a minimum of 36 ⁇ diameter to a maximum 152 ⁇ , with a median size of 73 ⁇ .
  • spheroid sizes varied from a minimum of 34 ⁇ diameter to a maximum 214 ⁇ , with a median size of 103 ⁇ .
  • viability of cells in spheroids was about 90%.
  • HT-29 cells cultured according to the present 3D culture methods were compared to HT-29 cells cultured with traditional 2D culture methods.
  • HT-29 xenografts and primary colon adenocarcinoma FFPE tissue sections were used as positive controls.
  • To generate the 3D cultures HT-29 cells were seeded at 2.0 x 10 5 per well in 6-well culture plates and cultured as described above in an ALGIMATRIX matrix. Spheroids were collected after approximately two weeks in culture. The matrix was dissolved from the collected spheroids using ALGIMATRIX Dissolving Buffer as described in the commercially available kit.
  • Spheroids were then pooled, washed once with IX Hank's balanced salt solution (HBSS), and then fixed in 10% neutral buffered formalin (NBF) for 2 hours at 4 °C followed by 2 hours at 37 °C.
  • HBSS IX Hank's balanced salt solution
  • NVF neutral buffered formalin
  • the HT-29 cells are seeded in traditional 2D plates or flasks and cultured. Cells are then harvested using enzymatic or non-enzymatic dissociation buffer and counted. Approximately 20- to 30-million cells (based on the cell counts) are collected, washed in HBSS, and then fixed in 10% neutral buffered formalin (NBF) for 2 hours at 4 °C followed by 2 hours at 37 °C.
  • NPF neutral buffered formalin
  • the suspended cells were then pulled into a transfer pipette (approximately 2-3 mm diameter stem) and held in the stem of the pipette for 20-60 minutes at room temperature to allow the agarose to solidify.
  • the resulting pellets were then wrapped in lens paper, placed in a cassette, and dipped in 10% NBF for an additional 2 hours at room temperature.
  • the resulting pellets were then processed in a tissue processor and then embedded in paraffin blocks following standard cell line fixation procedures.
  • HT-29 xenografts tissue were produced by inoculating HT-29 cells subcutaneously in mice using standard techniques, and the resulting xenografts were fixed and processed for paraffin embedding using standard techniques.
  • Colon adenocarcinoma samples were obtained and fixed and processed for paraffin embedding using standard techniques.
  • 2D- and 3D cultures and xenografts of HT-29 cells were obtained as described above and fixed using a 2+2 fixation protocol.
  • the resulting fixed cultures were processed, embedded in HISTOGEL or agarose, and embedded in paraffin as described above.
  • the resulting slides were immunohistochemically stained for phospho-AKT or labeled for HER2 nucleic acid via an ISH protocol on a VENTANA BENCHMARK automated slide stainer using standard protocols on the device. Data is shown at Figs. 6 (p-AKT) and 7 (HER2 ISH).

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Abstract

La présente invention concerne des matériaux et des procédés pour générer des blocs de matrice histochimique intégrée de lignée cellulaire. Les lignées cellulaires sont cultivées dans une culture tridimensionnelle comprenant un échafaudage biologique sans origine animale et mis en croissance sur des sphéroïdes. Les sphéroïdes qui en résultent sont ensuite concentrés, fixés et incorporés dans un matériau d'enrobage poreux (tel que des matrices à base d'agarose), et les granulés résultants sont incorporés dans une matrice histologique, ce qui génère les blocs. Les blocs peuvent être utilisés, par exemple, pour fabriquer des lames de contrôle pour des dosages histochimiques, notamment des dosages immunohistochimiques et d'hybridation in situ.
PCT/EP2016/055514 2015-03-16 2016-03-15 Lames de contrôle pour immunohistochimie générées à partir de cultures de lignées de cellules tridimensionnelles WO2016146616A1 (fr)

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IT202000014863A1 (it) * 2020-06-22 2021-12-22 Hospitex Int S R L Kit per la preparazione di citoinclusi

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT202000014863A1 (it) * 2020-06-22 2021-12-22 Hospitex Int S R L Kit per la preparazione di citoinclusi
EP3929559A1 (fr) * 2020-06-22 2021-12-29 Hospitex International S.r.l. Méthode et kit pour la production de blocs cellulaires

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