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1. (WO2016146616) LAMES DE CONTRÔLE POUR IMMUNOHISTOCHIMIE GÉNÉRÉES À PARTIR DE CULTURES DE LIGNÉES DE CELLULES TRIDIMENSIONNELLES
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CONTROL SLIDES FOR IMMUNOHISTOCHEMISTRY GENERATED FROM THREE-DIMENSIONAL CELL LINE CULTURES

CROSS-REFERENCE TO RELATED APPLICATIONS

The benefit of United States Provisional Patent Application No. 62/133,885, filed March 16, 2015, is hereby claimed, the content of which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

This disclosure relates to control slides for immunohistochemistry, namely, slides generated using three-dimensional cultures of cell lines.

Description of Related Art

One challenge in developing commercial histochemical assays is the selection of control slides. The ideal control slide provides consistent staining between slides and ready supply, while also providing an accurate representation of the typical tissue microenvironment. Unfortunately, no good solutions currently exist.

Traditionally, tissues expected to display typical staining patterns for the analyte of interest are used as controls. However, 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. Additionally, 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. However, xenografts can be expensive to generate, as they require live animals. Moreover, 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 reminiscent 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.

Three dimensional cell cultures have been used to immunostaining studies.

Pinto et al. analyzed three-dimensional cultures of mixed cell colonies microscopically. Pinto's method relies on a MATRIGEL™ matrix, which is a matrix based on a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. 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.

Additionally, because the matrix is of mammalian origin, there is a potential for cross-reaction between the matrix and the antibodies. Moreover, 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. However, 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. However, the inclusion of human components to the slide introduces a potential source of non-specific binding. Moreover, 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.

Godugu et al. developed a 3D Lung Tumor Model using an ALGIMATRIX 3D culture system. Spheroids were cultured in the ALGIMATRIX matrix and, after reaching a sufficient size, the matrix was removed and the cells were deposited onto a microscope slide using a cytospin method. However, this procedure is incompatible with large-scale manufacture, due to the required cytospin technique.

Although the aforementioned methods have been useful for isolated studies, the procedures are not compatible with mass produced control slides. The procedures are too complex and time- and labor-intensive and/or do not result in sufficient density of spheroids on the slide to provide a reliable control for routine pathological IHC screening.

SUMMARY OF THE INVENTION

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.

In an embodiment, a method of manufacturing a histological matrix block embedded with a control cell line is provided, the method 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. In certain embodiments, the animal origin-free bioscaffold is an alginate matrix having a pore size of from about 50 μιη to about 200 μιη. In some embodiments, the spheroids are removed from the bioscaffold when they have a diameter approximately equal to the pore size of the bioscaffold. In other embodiments, 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. In other embodiments, 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. In some embodiments, the porous embedding material in which the spheroids are deposited is an agarose-based matrix. In other embodiments, the porous embedding material is embedded in a paraffin block.

In another embodiment, a cell block obtained by the methods disclosed herein is provided. In some embodiments, 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.

In another embodiment, a method of manufacturing a control slide for a histochemical assay is provided, the method 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. In an embodiment, at least 500 slides are manufactured from a single block.

In another embodiment, a control slide is provided, the control slide obtained by the methods as disclosed herein.

In another embodiment, a kit for performing a histochemical assay is provided, the kit 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. In an embodiment, 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. In some embodiments, 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. In some embodiments, 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. In other embodiments, 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. In other embodiments, the kit is useful for calibrating an automated IHC/ISH slide stainer.

In another embodiment, methods of histochemically analyzing a primary tissue sample using the control slides obtained by a method as disclosed herein are

disclosed. In an embodiment, an immunohistochemical method of assaying a primary tissue sample for an antigen indicative of a disease or tissue state is provided, the method 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.

In another embodiment, an in situ hybridization method of assaying a primary tissue sample for a disease or tissue state is provided, the method 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.

In another embodiment, the staining is performed on an automated platform.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION OF THE INVENTION

I. Development background

3D cell culture techniques have previously been used to produce slides for immunohistochemical assays. However, 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. We therefore sought to develop an improved three-dimensional cell culture process for generating cell blocks on a commercial scale.

II. Cell blocks and methods of generating the same

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.

A. Spheroid generation

As used herein, 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. As can be seen, 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. For example, 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. One specific example of a commercially available algal bioscaffold is the ALGIMATRIX 3D culture system (Thermo Fisher Scientific, Inc.), which uses an alginate sponge having a pore size of about 50 μιη to 200 μιη. Examples of 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

US 2013-0344036 Al to Yliperttula et al, and Modulevsy et al). Examples of 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). As other examples, the bioscaffold may comprise or consist of 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. In an embodiment, the animal origin-free bioscaffold has an average pore size in the range of 30 um to 300 μιη, 30 μιη to 275 μιη, 30 μιη to 250 μιη, 35 μιη to 300 μιη, 35 μιη to 275 μιη, 35 μιη to 250 μιη, 40 μιη to 300 μιη, 40 μιη to 275 μιη, 40 μιη to 250 μιη, 45 μιη to 300 μιη, 45 μιη to 275 μιη, 45 μιη to 250 μιη, 50 μιη to 300 μιη, 50 μιη to 275 μιη, or 50 μιη to

250 μιη. In another embodiment, the animal origin- free bioscaffold has an average pore size of from about 50 μιη to about 250 μιη. In another embodiment, the animal origin-free bioscaffold is an alginate matrix having an average pore size of from about 50 μιη to about 250 μιη. Pore sizes referenced herein are average pore sizes as determined by scanning electron microscopy

In some embodiments, the cell lines used to generate the cell blocks are selected to be relatively fast growing and relatively small. As used herein, 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

20 to 35 hours, from 20 to 30 hours, from 25 to 40 hours, from 25 to 35 hour, from 25 to 30 hours, and subranges thereof. The "doubling times" referred to herein refer to doubling times of the cell line when cultured in a standard 2-dimensional culture at the exponential growth phase. As used herein, a "relatively small cell line" shall refer to a cell line having an average cell size about 15 μιη or smaller. In certain examples, the cell line is a cell line that has an average cell size such that from 5 to 15 μΜ, 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

100%, from 60% to 99%, from 60% to 98% from 60% to 97%, from 60% to 96% from 60% to 95%, from 60% to 94% from 60% to 93%, from 60% to 92%, from 60% to 91%, from 60% to 90%, 70% to 100%, from 70% to 99%, from 70% to 98% from 70% to 97%, from 70% to 96% from 70% to 95%, from 70% to 94% from70% to 93%, from 70% to 92%, from 70% to 91%, from 70% to90%, from

80% to 100%, from 80% to 99%, from 80% to 98% from 80% to 97%, from 80% to 96% from 80% to 95%, from 80% to 94% from 80% to 93%, from 80% to 92%, from 80% to 91%, from 80% to90%, from 85% to 100%, from 85% to 99%, from 85% to 98% from 85% to 97%, from85% to 96% from 85% to 95%, from 85% to 94% from85% to 93%, from 85% to 92%, from 85% to 91%, from 85% to 90%, from 90% to 100%, from 90% to 99%, from 90% to 98%, from 90% to 97%, from 90%) to 96%o, from 90%> to 95%, and subranges thereof. In an embodiment, 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. In another embodiment, the spheroids have an average diameter in the range of 50 μιη to 250 μιη within 12 to

14 days of seeding. In an embodiment, cells having an average diameter in the range of 5 to 15 μΜ are seeded in an alginate bioscaffold at a cell density in the range of 2 x 104 to 2 x 106 cells per well of a standard 6 well plate, and grown under conditions that spheroids having an average diameter in the range of 50 μιη to 250 μιη are obtained within 12 to 14 days of seeding.

B. Spheroid concentration

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. In an embodiment, the average spheroid size of collection is selected such that at least 90% of cells per spheroid are viable. In an embodiment, 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. In some embodiments, 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.

C. Spheroid fixation

The concentrated spheroids are fixed according to any method consistent with the end analysis for which the resulting cell blocks are intended. In an embodiment, 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. 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. In a specific embodiment, the spheroids are fixed in

10% NBF for 24 hours at room temperature.

In certain embodiments, the fixative is an aldehyde-based cross-linking fixative, such as glutaraldehyde- and/or formalin-based solutions. Examples of aldehydes frequently used for immersion fixation include:

• formaldehyde (standard working concentration of 5-10% formalin for most tissues, although concentrations as high as 20%> formalin have been used for certain tissues);

• glyoxal (standard working concentration 17 to 86 mM);

• glutaraldehyde (standard working concentration of 200 mM).

In one embodiment, the fixative comprises a standard concentration of formaldehyde, glyoxal, or glutaraldehyde. In one exemplary embodiment, 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 :

Solution Standard Composition

Neutral Buffered Formalin 5-20% formalin + phosphate buffer

Formal Calcium 10% formalin + 10 g/L calcium chloride

Formal Saline 10% formalin + 9 g/L sodium chloride

Zinc Formalin 10% formalin + 1 g/L zinc sulphate

50 mL 100% formalin + 1 L aqueous solution

Helly's Fixative containing 25 g/L potassium dichromate + 10 g/L sodium sulfate + 50 g/L mercuric chloride

2 mL 100% formalin + 20 mL aqueous solution

B-5 Fixative containing 6 g/L mercuric chloride + 12.5 g/L sodium acetate (anhydrous)

100 mL 100% formalin + 15 mL Acetic acid + 1L

Hollande's Solution aqueous solution comprising 25 g copper acetate and 40g picric acid

250 mL 100% formalin + 750 mL saturated

Bouin's Solution

aqueous picric acid + 50 mL glacial acetic acid

Table 1

In certain embodiments, the fixative solution is selected from Table 1.

In the context of concentrations of components of the aldehyde-based fixatives, 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).

In an embodiment, the cells are fixed using a two-temperature fixation process. As used herein, the term "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. In an embodiment, the cold temperature is in the range of 0 °C to 10 °C, and the warm temperature is in the range of 15 °C to 50 °C. In an embodiment, 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. 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. Applicant has found that low melting point agarose, i.e. agarose having a melting point in the range of about 55 to 65° C, works particularly well as a porous embedding media. An exemplary porous embedding material is the HISTOGEL™ 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.

E. Histological matrix

After the pellet is formed, the pellet is subjected to post-fixation processing. As used herein, the phrase "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. Other post-fixation processing steps would be well-known to a person having ordinary skill in the art.

In one embodiment, post-fixation processing comprises wax-embedding. In the typical example, 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. After the last alcohol treatment step the pellet is then immersed into another organic solvent, commonly referred to as a clearing solution. 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. In other examples, the sample may be embedded in resin blocks (such as epoxy or acrylic resins) instead of wax blocks. Exemplary resins include methyl methacrylate, glycol methacrylate, araldite, and epon. Each requires specialized post-fixation processing steps, which are well known in the art. Typically, before analysis, 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.

III. Kits

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. As used herein, the term "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. For example, a biomarker may be a molecule or group of molecules whose presence, absence, or relative abundance is:

• characteristic of a particular disease state;

• indicative of the severity of a disease or the likelihood or disease progression or regression; and/or

• predictive that a pathological condition will respond to a particular treatment.

As another example, 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. As used herein, 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. In another embodiment, the cell line is derived from the same tissue type as a tissue sample or cytological sample of interest. As used herein, the term "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. As used herein, the term "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. Examples of cytological samples include tissue scrapings (such as a cervical scraping), fine needle aspirates, samples obtained by lavage of a subject, et cetera. In an embodiment, the tissue or cytological sample of interest is a specific tumor and the cell line is derived from the same type of tumor. In other examples, 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.

In an embodiment, the kit comprises a control slide and a specific binding entity capable of specifically binding to a biomarker of interest. As used herein, 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. For example, 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. In one embodiment, 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). In certain embodiments, a binding entity that specifically binds to a target has a dissociation constant (Kd) of <1 μΜ, <100 nM, <10 nM, <1 nM, or <0.1 nM. In another embodiment, specific binding can include, but does not require exclusive binding. As used herein, 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. 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. aureus; Affibody AB, Solna, Sweden), AVIMERs (scaffold based on domain A/LDL receptor; Amgen, Thousand Oaks, CA), dAbs (scaffold based on VH or VL antibody domain; Glaxo SmithKline PLC, Cambridge, UK), DARPins (scaffold based on Ankyrin repeat proteins; Molecular Partners AG, Zurich, CH), ANTICALINs

(scaffold based on lipocalins; Pieris AG, Freising, DE), NANOBODYs (scaffold based on VHH (camelid Ig); Ablynx N/V, Ghent, BE), TRANS-BODYs (scaffold based on Transferrin; Pfizer Inc., New York, NY), SMIPs (Emergent Biosolutions, Inc., Rockville, MD), and TETRANECTINS (scaffold based on C-type lectin domain (CTLD), tetranectin; Borean Pharma A/S, Aarhus, DK). Descriptions of such engineered specific binding structures are reviewed by Wurch et al, Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the content of which is incorporated by reference. In some embodiments, 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. In other embodiments, 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.

In some cases, 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.

IV. 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

DISCOVERY series automated platforms), Dako (such as the COVERSTAINER, OMNIS, AUTOSTAINER, and ARTISAN series automated slide stainer), and the LEICA BOND and ST series stainers. Exemplary systems are disclosed in U.S. Pat. No. 6,352,861, U.S. Pat. No. 5,654,200, U.S. Pat. No. 6,582,962, U.S. Pat. No. 6,296,809, and U.S. Pat. No. 5,595,707, all of which are incorporated herein by reference. Additional information concerning automated systems and methods also can be found in PCT/US2009/067042, which is incorporated herein by reference.

In an embodiment, the control slides are used as controls for detecting specific analytes using immunohistochemistry (IHC). In the typical IHC protocol, 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. In exemplary embodiments, 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). This enzyme-antibody conjugate is referred to as an

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). In illustrative embodiments, the approach uses non-endogenous haptens (e.g. not biotin, see U.S. application Ser. No. 12/660,017 which is hereby incorporated by reference in its entirety for disclosure related to detection chemistries). In illustrative embodiments, 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. If 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). If the enzyme is horseradish peroxidase, then one suitable substrate is diaminobenzidine (DAB). 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. In some embodiments, 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.

In some embodiments, the fixed tissue samples are analyzed by immunohistochemistry for the presence of post-translationally modified proteins. In such a case, 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. In the typical process, 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. In an embodiment, 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. One could determine whether a given post-translational modification is susceptible to residual enzyme activity by treating a sample with an entity that leads to increased presence of the post-translational modification. 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.

Thus, in an embodiment, 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. In an embodiment, the post-translational modification is a diagnostic or prognostic marker for a disease state of the tissue sample. In an embodiment, the post-translational modification is a predictive marker for an effect of a therapy on a disease state of the tissue. In an embodiment, the post-translational modification is a phosphorylation.

In some embodiments, the fixed tissue samples are analyzed by in situ hybridization for the presence of specific nucleic acids. In the typical process, 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. In an embodiment, 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. One could determine whether a given nucleic acid is susceptible to residual enzyme activity by treating a sample with an entity that leads to increased presence of the nucleic acid. 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.

Thus, in an embodiment, 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. In an embodiment, the analyte nucleic acid is a diagnostic or prognostic marker for a disease state of the tissue sample. In an embodiment, the analyte nucleic acid is a predictive marker for an effect of a therapy on a disease state of the tissue. In an embodiment, the analyte nucleic acid is an RNA molecule, such as mRNA or miRNA.

V. Examples

A. Growth rates and viability

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. For the 3D method, HT-29 cells were seeded at 1.25 x 105 (low density) or 5.3 x 105 (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

37 °C. Cell count and viability of the collected spheroids were measured using Trypan Blue and TC20 cell counter at days 4, 6, 8, and 10 for the low density wells and at days 3, 4, 6, 8, and 10 for high density wells. The cell counts and viability counts are recorded at Table 2 and plotted at Fig. 1. The HT-29 cells in spheroids grow slower in 3D culture than in 2D, with a doubling time of -40 hours versus 23 hours reported by ATCC for 2D cultures. As illustrated at Fig. 2, exponential growth of low-density plates can be observed, whereas the cell population is approximately steady-state for high-density plate. Exponential growth is desirable, as cell cultures entering sub-exponential growth phases tend to become senescent, which changes the expression profile.

B. Live/Dead Assay

Additionally, 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 (EthD-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.

Digital images of the spheroids were used to provide estimates of spheroid size and number of cells in each spheroid using NIH ImageJ software. 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. For low density cultures, spheroid sizes varied from a minimum of 36 μιη diameter to a maximum 152 μιη, with a median size of 73 μιη. For high density cultures, spheroid sizes varied from a minimum of 34 μιη diameter to a maximum 214 μιη, with a median size of 103 μιη. Overall, viability of cells in spheroids was about 90%.


Table 2

Area Volume Number Cells

Radius (μπι) Diameter

Spehroid (Square (μιη3) (Typical cell

0.65*(Area/7i)1/2 (μπι)

Pixels) (4/3) Tir3 radius = 7 μιη)

16 8798 34 69 1.70E+05 1 19

17 18953 50 101 5.39E+05 375

Table 3

Area Volume Number Cells

Radius (μπι) Diameter

Spehroid (Square (μιη3) (Typical cell

0.65*(Area/7i)1/2 (μπι)

Pixels) (4/3) Tir3 radius = 7 μιη)

4 53066 84 169 2.53E+06 1758

5 85260 107 214 5.14E+06 3580

Table 4

Spheroid Size Measurements

Low Density Well 1 Spheroids

Area Volume Number Cells

Radius (μπι) Diameter

Spehroid (Square (μιη3) (Typical cell

0.65*(Area/7i)1/2 (μπι)

Pixels) (4/3) Tir3 radius = 7 μιη)

1 24098 57 1 14 7.73E+05 538

2 24525 57 1 15 7.93E+05 552

3 1431 1 44 88 3.54E+05 246

4 9295 35 71 1.85E+05 129

5 18250 50 99 5.09E+05 354

6 8365 34 67 1.58E+05 1 10

7 9666 36 72 1.96E+05 137

8 9513 36 72 1.92E+05 133

9 6834 30 61 1.17E+06 81

10 29567 63 126 1.05E+05 731

1 1 16854 48 95 4.52E+05 315

12 10331 37 75 2.17E+05 151

13 9838 36 73 2.02E+05 140

14 6312 29 58 1.04E+05 72

15 8319 33 67 1.57E+05 109

16 8798 34 69 1.70E+05 1 19

17 18953 50 101 5.39E+05 375

18 1 1586 39 79 2.58E+05 179

19 8595 34 68 1.65E+05 1 15

Spheroid Size Measurements

Low Density Well 1 Spheroids

Area Volume Number Cells

Radius (μπι) Diameter

Spehroid (Square (μιη3) (Typical cell

0.65*(Area/7i)1/2 (μπι)

Pixels) (4/3) Tir3 radius = 7 μπι)

20 181 13 49 99 5.04E+05 351

21 29201 63 125 1.03E+06 717

22 1 1243 39 78 2.46E+05 171

23 5852 28 56 9.25E+04 64

24 20814 53 106 6.20E+05 432

25 6209 29 58 1.01E+05 70

26 13717 43 86 3.32E+05 231

27 10830 38 76 2.33E+05 162

28 9739 36 72 1.99E+05 138

29 6746 30 60 1.14E+05 80

30 10612 38 76 2.26E+05 157

31 12264 41 81 2.81E+05 195

Table 5

Spheroid Size Measurements

Low Density Well 2 Spheroids

Area Volume Number Cells

Radius (μπι) Diameter

Spehroid (Square (μιη3) (Typical cell

0.65*(Area/7i)1/2 (μπι)

Pixels) (4/3) Tir3 radius = 7 μηι)

1 21894 54 109 6.69E+05 466

2 2474 18 36 2.54E+04 18

3 2758 19 39 2.99E+04 21

4 20628 53 105 6.12E+05 426

5 9443 36 71 1.90E+05 132

6 17329 48 97 4.71E+05 328

7 29662 63 126 1.06E+06 735

8 17241 48 96 4.68E+05 326

9 18041 49 99 5.01E+06 348

10 7383 32 63 1.31E+05 91

1 1 9302 35 71 1.85E+05 129

12 42796 76 152 1.83E+06 1273

13 42877 76 152 1.83E+06 1277

14 18620 50 100 5.25E+05 365

15 3596 22 44 4.45E+04 31

16 6353 29 58 1.05E+05 73

Spheroid Size Measurements

Low Density Well 2 Spheroids


Table 6

Spheroid Size Measurements

High Density Well 1 Spheroids

Area Volume Number Cells

Radius (μπι) Diameter

Spehroid (Square (μιη3) (Typical cell

0.65*(Area/7i)1/2 (μπι)

Pixels) (4/3) Tir3 radius = 7 μηι)

1 45149 78 156 1.98E+06 1379

2 74368 100 200 4.19E+06 2916

3 40438 74 147 1.68E+06 1 169

4 53066 84 169 2.53E+06 1758

5 85260 107 214 5.14E+06 3580

6 22326 55 1 10 6.89E+05 480

7 18227 50 99 5.08E+05 354

8 21858 54 108 6.68E+05 465

9 20815 53 106 6.20E+05 432

10 50860 83 165 2.37E+06 1649

1 1 21539 54 108 6.53E+05 455

12 20746 53 106 6.17E+05 430

13 37670 71 142 1.51E+06 1051

14 62155 91 183 3.20E+06 2228

15 34238 68 136 1.31E+06 91 1

16 24525 57 1 15 7.93E+05 552

17 19792 52 103 5.75E+05 400

18 13882 43 86 3.38E+05 235

19 12245 41 81 2.80E+05 195

20 73873 100 199 4.15E+06 2887

21 16082 68 137 1.34E+06 931

22 23969 20 41 3.51E+04 24

Spheroid Size Measurements

High Density Well 1 Spheroids

Area Volume Number Cells

Radius (μπι) Diameter

Spehroid (Square (μιη3) (Typical cell

0.65*(Area/7i)1/2 (μπι)

Pixels) (4/3) Tir3 radius = 7 μπι)

23 14106 44 87 3.46E+05 241

24 15391 45 91 3.94E+05 275

25 16994 48 96 4.58E+05 319

26 19576 51 103 5.66E+05 394

27 10716 38 76 2.29E+05 160

28 19381 51 102 5.57E+05 388

29 22056 54 109 6.77E+05 471

Table 7


Table 8

C. Immunohistochemical assays

To test whether the present culture methods are useful for histochemical assays, 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 105 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.

For 2D cultures, 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.

Spheroids and 2D cultures were centrifuged after fixation, the supernatant discarded, and the cell pellet suspended in an 0.7%> agarose solution or HISTOGEL

(in deionized distilled water) at 45 °C. 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.

Slides for the 2D and 3D cultures, the xenografts, and the colon adenocarcinoma were cut and stained using anti-c-Myc, anti-beta-catenin, anti-BRAF V600E, and anti-PTEN antibodies and the OptiView DAB kit on a VENT ANA BENCHMARK automated staining platform. Data are shown at Fig. 5.

As seen at Fig. 5, staining pattern and IHC intensity results using various antibodies on 3D cell cultures prepared with the ALGIMATRIX system as performed on the VENTANA BENCHMARK automated staining platform were consistent with results obtained with the same antibodies in 2D cell culture as well as in xenografts and tumor tissue. However, the cell morphology was improved with the 3D culture method compared to the 2D culture method. The developed protocols and methods demonstrated that a 3D culture system could be the preferred technique especially in IHC applications to evaluate the cell-cell adhesion environment on automated staining platforms. Also, the control slides made from 3D culture system can be an alternative to use of tumor tissue and xenografts. Moreover, the resulting slides were compatible with standard automated IHC staining procedures without modification of protocols.

D. Labile biomarkers

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).

REFERENCES

Sompuram et al., A novel quality control slide for quantitative immunohistochemistry testing, J Histochem Cytochem. Vol. 50, Issue 11, pp. 1425- 34 (Nov. 2002).

Xiao et al., Cell Lines as Candidate Reference Materials for Quality Control of ERBB2 Amplification and Expression Assays in Breast Cancer, Clinical Chemistry, Vol. 55, Issue 7, pp. 1307-15 (2009).

Pinto et al, An Immunohistochemical Method to Study Breast Cancer Cell

Subpopulations and Their Growth Regulation by Hormones in Three-Dimensional Cultures, Frontiers in Endocrinology, Vol. 2, Issue 15, web publication (2011).

Kunz-Schughart et al., A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation, Exp. Cell Res. Vol. 266, Issue 1, pp. 74-86 (2001).

Graham et al., Hormone-responsive model of primary human breast epithelium, J. Mammary Gland Biol. Neoplasia Vol. 14, Issue 4, pp. 367-379 (2009).

Fogh et al., One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice, Journal of the National Cancer Institute 59: 221-226 (1977).

Modulevsy et al., Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture, PLoS One 9(5): e97835 (May 19-2014).

Tibbitt & Anseth, Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture, Biotechnol Bioeng. 103(4): 655-663 (Jul. 1, 2009).

Rossouw et al. Thermo-responsive non-woven scaffolds for "smart" 3D cell culture, Biotechnol Bioeng. 109(8):2147-58 (Aug. 2012).

Lei & Schaffer, A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation, Proceedings of the Nat'l Academy of Sci., 110(52), E5039-E5048 (Dec. 24, 2013).

Smith et al., An Initial Evaluation of Gellan Gum as a Material for Tissue Engineering Applications, J. Biomaterials Applications 22(3), pp241-54 (Dec. 2007).

Mendes et al., Encapsulation and Survival of a Chondrocyte Cell Line within Xanthan Gum Derivative, Macromolecular Bioscience 12(3), pp. 350-59 (2012).