Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

Podsumowanie

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

Streszczenie

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

Wprowadzenie

The development of effective and safe anticancer agents requires appropriate preclinical models that can also provide insight into mechanisms of action that can facilitate the identification of predictive and pharmacodynamic biomarkers. Inter- and intratumor heterogeneity^1,2,3,4,5 and the TME^{6,7,8,9,10,11,12} are known to influence anticancer drug responses, and many existing preclinical cancer models such as cell lines, organoids, and mouse models are not able to fully accommodate these crucial features. An "ideal" model is one that can recapitulate the complex spatial interactions of malignant with non-malignant cells within tumors as well as reflect the regional differences within tumors. This article focuses on PDEs as an emerging platform that can fulfil many of these requirements¹³.

The first example of the use of human PDEs, also known as histocultures, dates back to the late 1980s when Hoffman et al. generated slices of freshly resected human tumors and cultured them in a collagen matrix^14,15. This involved establishing a 3D culture system that preserved tissue architecture, ensuring the maintenance of stromal components and cell interactions within the TME. Without deconstructing the original tumor, Hoffman et al.¹⁶ heralded a new approach of translational research, and since this time, many groups have optimized different explant methods with the aim of preserving the tissue integrity and generating accurate drug response data^{17,18,19,20,21,22,23,24}, although some differences between protocols are evident. Butler et al. cultured explants in gelatin sponges to help the diffusion of nutrients and drugs through the specimen^20,21,25, whereas Majumder et al. created a tumor ecosystem by culturing explants on top of a matrix composed of tumor and stromal proteins in the presence of autologous serum derived from the same patient^22,23.

More recently, our group set up a protocol whereby explants are generated by fragmentation of tumors into 2 - 3 mm³-sized pieces that are then placed without additional components on permeable membranes at the air-liquid interface of a culture system²⁴. Taken together, these numerous studies have demonstrated that PDEs allow the culture of intact, live fragments of human tumors that retain the spatial architecture and regional heterogeneity of the original tumors. In original experiments, explants or histocultures were usually subjected to homogenization following drug treatment, after which various viability assays were applied to the homogenized samples such as the histoculture drug response assay^20,21, the MTT (3-(6)-2,5-diphenyltetrazolium bromide) assay, the lactate dehydrogenase assay, or the resazurin-based assay^26,27,28. Recent progress in endpoint analysis techniques, particularly digital pathology, have now expanded the repertoire of endpoint tests and assays that can be performed on explants^29,30. To apply these new technologies, instead of homogenization, explants are fixed in formalin, embedded in paraffin (FFPE) and then analyzed using immunostaining techniques, allowing spatial profiling. Examples of this approach have been documented for non-small cell lung cancer (NSCLC), breast cancer, colorectal cancer, and mesothelioma explants whereby immunohistochemical staining for the proliferation marker, Ki67, and the apoptotic marker, cleaved poly-ADP ribose polymerase (cPARP), was used to monitor changes in cell proliferation and cell death^{24,31,32,33,34}.

Multiplexed immunofluorescence is particularly amenable for spatial profiling of drug responses in explants at endpoint³⁵. For example, it is possible to measure the relocalization and spatial distribution of specific classes of immune cells, such as macrophages or T cells, within the TME upon drug treatment^13,36,37,38, and investigate whether a therapeutic agent can favor the transition from "cold tumor" to "hot tumor"³⁹. In recent years, this group has focused on the derivation of PDEs from different tumor types (NSCLC, renal cancer, breast cancer, colorectal cancer, melanoma) and the testing of a range of anticancer agents including chemotherapies, small-molecule inhibitors, and immune checkpoint inhibitors (ICIs). Endpoint analysis methods have been optimized to include multiplexed immunofluorescence to allow spatial profiling of biomarkers for viability as well as biomarkers for different constituents of the TME.

Protokół

1. Tissue collection

1. After surgery, transfer freshly resected human tumor specimens into a tube containing 25 mL of fresh culture medium (Dulbecco’s modified Eagle medium supplemented with 4.5 g/L glucose and L-glutamine + 1% (v/v) fetal calf serum + 1% penicillin–streptomycin) and stored on ice. Process the explant within 2 h of surgery in a sterile class II hood.

2. Explant preparation

1. Clean all surgical equipment (graft blades/dental wax surface/tweezers) with 70% industrial methylated spirit (IMS) solution.
2. Fill a 10 cm culture dish (see the Table of Materials) with ~25 mL of fresh medium, and place it on top of a bed of ice.
3. Using tweezers, transfer the specimen onto a dental wax surface (see the Table of Materials), and slice the tissue into fragments of approximately 2–3 mm³ using two skin graft blades.
   NOTE: For best performance, start slicing the tissue to obtain an individual strip of 2–3 mm of thickness, and then cut the strip along the length to obtain the final explants. Repeat the process until the whole specimen is chopped.
4. Transfer the explants promptly into the ice-cold culture medium in the 10 cm dish. Take a proportion of the explants (6–9 pieces), place them in a 1 mL tube containing 10% of non-buffered formalin solution, and leave at room temperature (RT) for 24 h.
   NOTE: These fragments will represent the control explants for the “uncultured” condition.
5. Fill the desired number of wells of 6-well plates with 1.5 mL of fresh medium per well, and place an organotypic culture insert disc (see the Table of Materials) in each well so that it floats on top of the medium, carefully avoiding air bubbles at the media-insert interface.
6. Select explants randomly, and place them one at a time on top of the insert disc. To be consistent with the “uncultured” condition, use 6–9 explants per well. Place the multiwell plate in a CO₂ incubator at 37 °C and 5% CO₂, and allow the explants to recover for 16 h.
   NOTE: To avoid crushing the tissue, gentle handling of the tweezers is highly recommended.

3. Drug treatment

1. After 16 h, take new 6-well plates, add 1.5 mL of fresh medium to each well, add the relevant drugs at the desired concentrations, and include a well for the vehicle control. Using the tweezers, move each insert containing the explants to the newly prepared 6-well plates containing the drugs. Place the plates in the CO₂ incubator at 37 °C and 5% CO₂ for 24–48 h.
2. Transfer the uncultured explants previously fixed in formalin into a histology cassette (see section 4 below), and store in 70% IMS until the end of the experiment.

4. Histological processing

1. Tissue fixation
   1. After drug treatment, transfer the insert discs containing the explants into new 6-well plates containing 10% formalin, and add a few drops of 10% formalin onto the top of the explants to ensure complete coverage with the fixative.
      CAUTION: Formalin is hazardous. Avoid any contact with skin, and handle it inside a fume food to prevent inhalation.
   2. Allow the explants to remain overnight (20–24 h) in the 10% formalin.
   3. On the next day, soak the relevant number of small histology sponges in 70% IMS, and place them inside histology cassettes. Gently transfer all the explants from a given drug treatment condition onto a single sponge (maintain the orientation of the explants so that the sides of the explants in contact with the insert discs, and therefore the drug-treated areas, are placed in contact with the sponge). Place another presoaked sponge on top of the explants, and secure within a histology cassette (one cassette per well/treatment).
   4. Submerge the cassettes in 70% IMS, and leave at RT for 24 h.
2. Paraffin embedding and tissue sectioning
   1. On the following day, transfer the histology cassettes containing the explants into the sample basket of the tissue processor (see the Table of Materials), and secure the lid. Place the basket in the retort and close gently. Select the desired program, and start the processor. Drain the retort, open it to remove the basket, and transfer the basket into the embedding center wax bath.
   2. After embedding, transfer the metal cassette lids into the basket, and return to the retort of the tissue processor for the cleaning program. Wipe the sensor with a dry tissue, remove any excess wax from the retort lid, and proceed with the cleaning program.
   3. Position a paraffin block in the rotary microtome, cut a few sections at 4 µm, and return the block to ice. Reposition the block, and cut more 4 µm sections. Transfer the sections with a small paint brush and forceps onto a water bath to remove creases and bubbles.
   4. Position the sections centrally onto microscope slides, drain the slides for few minutes, and place them into a slide rack to dry overnight in an incubator at 37 °C. When the sections are dry, store them at RT in a slide folder or box to protect from dust.

5. Hematoxylin and eosin (H&E) staining

1. Fill the stainer (see the Table of Materials) with reagents, and set the required program for H&E staining: hematoxylin incubation time: 5 min; eosin incubation time: 3 min.
2. Attach the slide rack to the carrier, place it into the first container with xylene, and start the program. Remove the slide carrier from the rack, and take the container with the slide rack to the mounting area.
3. Mount the slides with dibutyl phthalate xylene (DPX) under the extraction hood. Place DPX onto each coverslip, and lower the slide onto the coverslip with the section downwards, allowing the DPX to spread out.
   NOTE: DPX is hazardous. Avoid any contact with skin and use in a designated fume hood.

6. Immunostaining

NOTE: The following steps should be carried out at RT unless stated otherwise.

1. Deparaffinization and rehydration
   1. Place the slides in a slide rack, and heat at 65 °C for 10 min before deparaffinizing the sections in xylene for 3 min (2x). Minimize the amount of carry-over solution.
      NOTE: Xylene is flammable, hazardous, and irritant. Handle inside the fume hood whilst using double-layered gloves. Avoid skin and eye contact. Dispose of waste within a sealed container.
   2. Rehydrate the slides in an alcohol gradient as follows: 99% IMS for 1 min (2x) and 95% IMS for 1 min. Minimize the amount of carry-over solution.
      NOTE: IMS is highly flammable, volatile, and irritant. Handle inside the fume hood, and avoid skin and eye contact.
   3. Completely submerge the slide rack in ultrapure (UP) water for 5 min.
2. Antigen retrieval
   1. Prepare antigen retrieval buffer according to the manufacturer’s guidelines for the particular antibody used.
      NOTE: Citrate buffer 0.2 M, pH 6, or tris(hydroxymethyl)aminomethane (Tris)-ethylenediamine tetraacetic acid (EDTA) 0.1 M, pH 9, are the common buffers used for acidic or alkaline conditions, respectively. Citric acid monohydrate and Tris are irritant agents. Prepare all stock solutions in the fume hood. Avoid contact with skin and eyes.
   2. Submerge the rack containing the slides into antigen retrieval buffer, and microwave (see the Table of Materials) at full power (800 W) for 20 min.
      NOTE: Keep the slides fully submerged in the buffer during the entire process. To avoid excessive reduction of the buffer volume, place a lid on top of the container used.
3. Multiplex immunofluorescence (mIF)
   NOTE: This process takes 1–2 days.
   1. Fill a clean container with 250 mL of UP water, and completely submerge the slide rack for 2 min (2x). Proceed with assembling each slide into a slide coverplate (see the Table of Materials).
      1. To assemble a slide onto a coverplate, prepare a glass trough with water. Submerge both the coverplate and slide in the water, placing the tissue section side facing the coverplate.
      2. Position the bottom edge of the slide on top of the protuberances at the bottom of the coverplate, and finally align the slide to the coverplate, allowing the water to fill the gap in between with no air trapped. Hold the coverplate and slide together, and place them in a coverplate rack.
   2. Fill the coverplate with phosphate-buffered saline (PBS), and wait for the buffer to wash the slides, flowing out by gravity (2x). Pipet 110 μL of blocking buffer (see the Table of Materials) onto each coverplate and incubate for 10 min.
   3. Prepare the desired primary antibody dilution in PBS or blocking buffer according to the manufacturer’s recommendations. Incubate the slides with primary antibody (110 μL each slide) for 30 min. Wash the slides with 5 mL of PBS (2x), as described in 6.3.2.
   4. Incubate the slides with 110 μL of horseradish peroxidase polymer for 30 min. Wash the slides with 5 mL of PBS (2x), as described in 6.3.2.
   5. Prepare fluorophore dilution in amplification diluent 1:100 (v/v), and incubate the slides with fluorophore (110 μL each slide) for 10 min. Wash the slides with 5 mL of PBS (2x), as described in 6.3.2.
      NOTE: For the use of fluorophore 780, follow the manufacturer’s guidelines. The experiment may be paused here if the slides are incubated in the final wash of PBS and stored at 4 °C overnight.
   6. Unclip the slides from the coverplates, and go back to the antigen retrieval step to start the staining with a different antibody. Repeat the staining for the desired number of antibodies to be tested.
   7. Following step 6.3.5 for the last fluorophore in use, keep the slides on the coverplates, prepare 6 μM solution of 4’-6-diamidino-2-phenylindole (DAPI) dilactate with UP water, and counterstain the slides for 5 min (110 μL each slide). Wash the slides with UP water (2x), and remove excess water by drying the edges of the slides. Assemble the coverslips on the slides using mounting medium, and store at RT in the dark.

7. Scanning

NOTE: Slide scanning was performed using a multispectral automated imaging system (see the Table of Materials).

1. Assemble the slides into the carrier, and load them into the scanner in the desired order.
   NOTE: It is recommended that a control slide be included in which no fluorophore or DAPI is used during immunohistochemical staining. This slide will be used as the control for intrinsic florescence (autofluorescence (AF)) of the tissue.
2. After launching the slide scanner software, select Edit Protocol from the Home page. Create a new protocol addressing the name, imaging mode, the type of multispectral scan to be performed (4–7 channels), and study (directory).
   NOTE: To modify the default settings of bands analyzed, select/deselect the desired filters using the function Select Scan Bands
3. Proceed with Scan Exposure and Load carrier, and finally select the carrier for the protocol setup. Select Take Overview to detect the tissue on the slide. Choose a slide displayed on the carrier, and select a specific area of the tissue with the red cursor to bring it into live view on the left window of the screen.
4. Select the DAPI filter, and click on Autofocus or manually adjust the Stage Height slider to focus the field of interest. Click on Autoexpose to allow the system to find the best exposure for that filter/band.
   NOTE: The exposure time for each filter is set at 12 ms. After autoexposing the system, refine the exposure time to a better estimate. It is also possible to override the autoexposure value by typing a value manually into the highlighted cell.
5. Repeat the above steps for all filters in the protocol. If necessary, change the locations and/or slides to find the best signals for setting the exposures.
   NOTE: If the tissue is highlighted in red in live view, the fluorescent signal of the filter analyzed is saturated, and autoexposure must be repeated.
6. Once the exposure settings have been established, click on the Back button, and Save the protocol. From the software home page (see the Table of Materials), select Scan Slides.
7. Stack all the carriers to be scanned into the Slide Carrier Hotel, and take note of the order of the slides to assign their ID into the system.
   NOTE: On the software screen, each carrier will be related to a slot containing 4 slides.
8. To configure multiple slides with the same parameters, click on Configure Tasks, and select one or more Slots with the option Using the same rules for the same slides. Once the Edit Slides opens, configure: Tasks, Study, and Protocol, and click on OK.
9. Label each slide by clicking on the Slot icon, and type a name in the Slide ID cell. Click on Scan to start scanning.
   NOTE: Once the Slot icon turns blue and the slides are marked with a blue arrow, the carrier has all the rules and is ready to be scanned. It is possible to save the slide IDs, studies, protocols, and tasks by clicking on the Save Setup button.

8. Analysis

NOTE: The protocol below illustrates the method for phenotype analysis.

1. Image preparation
   1. To prepare the images for analysis, open the viewer software (see the Table of Materials), and select Load slides. Select the desired scans for the training project on the right-hand side of the screen.
   2. For each scan, click on Stamp, and choose Project in the box Select for: Define a stamp with size 1 x 1 in the image field to be used later as a template for the training analysis. Observe that this stamp will be marked as a red square.
      NOTE: The number of stamp annotations for the Project is arbitrary. It is recommended to generate one for the intrinsic fluorescence scan and a few more that are generally representative of the signal distribution within the tissue.
   3. Finally, open all the scans of the experiment. For each one, click on Stamp, and this time, choose the option Batch in the box Select for: Place a stamp circumscribing each explant.
      NOTE: This time, the stamps will be marked as green squares and will be required once the batch analysis is launched. Choose the appropriate size for the stamps (1 x 1, 2 x 2, 3 x 3) and avoid overlapping of the stamps, which would generate duplicated data in the exported file.
2. Training analysis
3. Launch the software for analysis (see the Table of Materials), and create a new project: Select File | New | Project.
4. Type a name in the Project Name box, and configure the type of project.
   NOTE: The experiments were performed using the following specifications: trainable tissue segmentation; adaptive cell segmentation; phenotyping.
5. Load the scans containing the stamps for training: File | Open | Image. In single mode view, navigate through the images and select the scans with intrinsic fluorescence.
6. Click on the Autofluorescence (AF) button, and with the Autofluorescence picker, draw on an unstained portion of tissue. Click on Prepare images to allow the software to subtract the intensity of intrinsic fluorescence from all the images uploaded for training.
7. Click on the Edit Markers and Colors button, and enter the marker names in the box associated with their fluorophore. Click on the Segment Tissue step on top of the screen to open the Tissue Segmentation Training window.
8. In the Tissue Categories window, type in the name of the tissue categories to segment the images into (e.g., Tumor, Stroma, Background, Necrosis).
9. In the Tissue Segmentation Training window, click on the Draw button and draw regions around groups of cells to define a tissue category of interest. For example, to define a Tumor area, draw a region around a group of tumor cells. Switch to the next category and repeat the drawing process.
   NOTE: It is recommended to draw regions in most of the images uploaded for training. It is also possible to refine the training by editing the Pattern Scale and the Segmentation Resolution whose details are better described in the user manual.
10. Having defined the training regions, proceed with Train the Tissue Segmenter.
    NOTE: During the process of training, the software displays the percentage of accuracy of tissue segmentation. For optimal results, it is recommended that tissue segmentation be at least 90% accurate. Once the Segmenter has stabilized, click on the Done button.
11. To see the Tissue Category mask on all images, click on the Segment All button.
12. After verifying the quality of tissue segmentation, re-draw the training regions around the areas that were incorrectly classified, and re-train the Tissue Segmenter until the process is successful.
13. Click on the Cell Segmentation step in the Step Bar at the top of the window to proceed with the analysis.
14. Choose the cellular compartments to segment: Nuclei, Cytoplasm, and/or Membrane.
    NOTE: As adaptive cell segmentation can only detect cells with nuclear signal, do not de-select Nuclei. Although it is also possible to segment cytoplasm and/or membrane, it is recommended to choose the strongest, most abundant nuclear component first (e.g., DAPI).
15. Configure the nuclear component using the Typical Intensity slider to adjust the threshold used to detect nuclear pixels. Take note of the preview window that opens and provides live feedback as the threshold is adjusted.
    NOTE: This threshold is adaptive and is measured relative to the surrounding background. Increase this value if too much background is detected as nuclear. Lower this value if faint nuclei are being missed. Use the Show/Hide Regions button to blink the segmentation masks on and off, and check whether the correct pixels are being detected as nuclear.
16. To split clusters of nuclear pixels, use the Nuclear Component Splitting panel. Choose the button that best describes the staining quality of the nuclei. Then, use the slider bar to adjust the Splitting Sensitivity, keeping in mind that lower values will result in more splitting.
17. Click on the Segment All button to segment all the images. If the result is not accurate, modify the settings and re-train the software until a satisfying segmentation is achieved.
18. Proceed to the last step of the analysis: Phenotyping. In the Phenotypes panel, add the list of phenotypes to be detected, and type the name in the corresponding text box. Click on the Edit Phenotypes button, select a particular cell to bring up a dropdown menu, and choose the corresponding phenotype.
    NOTE: At least five cells for each phenotype are necessary to train the classifier. Turning off the visualization of the nuclei allows better visualization of the cell phenotype that needs to be assigned.
19. Once the selection for training is complete, click on the Train Classifier button.
    NOTE: The software will classify every single cell in the image, and whenever errors in classification occur, it is possible to edit the phenotype of the cells and repeat the training of the classifier. Save the project before proceeding to batch analysis, and click on the Export in the tool bar on the top of the screen.
20. Select the Bath Analysis tab on the left menu, and load the project in the Batch Algorithm or Project box.
21. Click on Export Options to create separate directories for each image, and with the Browse button, navigate to find the location to export the data. Click on all the options of the Images and Tables to export.
22. On the right side of the screen, click on Add Slides and load all the images containing stamps for previously prepared batches (step 7.3). Click on the Run button to start the batch analysis and process one stamp at a time, exporting the corresponding data.

Wyniki

Multispectral imaging of mIF-stained histological sections permits identification and phenotyping of individual cell populations and identification of tumor and stromal components in the explant TME (Figure 2). Multispectral imaging is particularly useful for the analysis of tissues with high intrinsic autofluorescence, such as tissue with a high collagen content, as it allows the autofluorescence signal to be deconvoluted from other signals and excluded from subsequent analysis. Subsequent ...

Dyskusje

This paper describes the methods for generation, drug treatment, and analysis of PDEs and highlights the advantages of the platform as a preclinical model system. Ex vivo culturing of a freshly resected tumor, which does not involve its deconstruction, allows for the retention of the tumor architecture^13,24 and thus, the spatial interactions of cellular components in the TME as well as intratumoral heterogeneity. This method demonstrates how, by using a tumo...

Materiały

Name	Company	Catalog Number	Comments
Acetic acid	Sigma	320099	Staining reagent
Antibody Diluent / Block, 1x	Perkin Elmer	ARD1001EA	Antibody diluent/blocking buffer
Barnstead NANOpure Diamond	Barnstead		Ultra Pure (UP) H2O machine
Citric Acid Monohydrate	Sigma-Aldrich	C7129	Reagent for citrate buffer
Costar Multiple Well Cell Culture Plates	Corning Incorporated	3516	6 multiwell plate
DAPI Dilactate	Life Technologies	D3571
100 x 17 mm Dish, Nunclon Delta	ThermoFisher Scientific	150350	100 mm diameter dish for tissue culture
DMEM (1x) Dubelcco's Modified Eagle Medium + 4.5 g/L D-Glucose + 110 mg/mL Sodium Pyruvate	Gibco (Life Technologies)	10569-010	Tissue culture medium (500 mL)
DPX mountant	VWR	360294H	Mounting medium
DPX mountant	Merck	6522	Mounting medium
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	3609	Reagent for TE buffer
Eosin	CellPath	RBC-0100-00A	Staining reagent
Foetal Bovine Serum	Gibco	10500-064	For use in tissue culture medium
37% Formaldehyde	Fisher (Acros)	119690010	10% Formalin
iGenix, microwave oven IG2095	iGenix	IG2095	Microwave used for antigen retreival
Industrial methylated spirit (IMS)	Genta Medical	199050	99% Industrial Denatured Alcohol (IDA)
InForm Advanced Image Analysis Software	Akoya Biosciences	InForm
Leica ASP3000 Tissue Processor	Leica Biosystems		Automated Vacuum Tissue Processor
Leica Arcadia H and C	Leica Biosystems		Embedding wax bath
Leica RM2125RT	Leica Biosystems		Rotary microtome
Leica ST4040 Linear Stainer	Leica Biosystems		H&E stainer
Mayer's Haematoxylin	Sigma	GHS132-1L	Staining reagent
Millicell Cell Culture Inserts, 30 mm, hydrophilic PTFE, 0.4 µm	Merck Milipore	PICMORG50	Organotypic culture insert disc
Novolink Polymer Detection System	Leica Biosystems	RE7150-K	DAB staining kit
OPAL 480	Akoya Biosciences	FP1500001KT	Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 520	Akoya Biosciences	FP1487001KT	Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 570	Akoya Biosciences	FP1488001KT	Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 620	Akoya Biosciences	FP1495001KT	Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 650	Akoya Biosciences	FP1496001KT	Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 690	Akoya Biosciences	FP1497001KT	Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 780 / OPAL TSA-DIG Reagent	Akoya Biosciences	FP1501001KT	Fluorophore with Dimethyl Sulfoxide (DMSO) diluent and TSA-DIG reagent
Opal Polymer HRP Ms Plus Rb, 1x	Perkin Elmer	ARH1001EA	HRP polymer
Penicillin/streptomycin solution	Fisher Scientific	11548876	For use in tissue culture medium
PhenoChart Whole Slide Contextual Viewer	Akoya Biosciences	PhenoChart	Viewer software for scanned images
Phosphate Buffered Saline Tablets	Thermo Scientific Oxoid	BR0014G	PBS
1x Plus Amplification Diluent	Perkin Elmer	FP1498	Fluorophore diluent
Prolong Diamond Antifade Mountant	Invitrogen	P36961	Mounting medium
Slide Carrier	Perkin Elmer		To load slides into Slide Carrier Hotel for scanning with Vectra Polaris
Sodium Chloride	Fisher Scientific	S/3160/63	10% Formalin
Sodium Hydroxide pellets	Fisher Scientific	S/4920/53	Reagent for citrate buffer
Tenatex Toughened Wax - Pink (500 g)	KEMDENT	1-601	Dental wax surface
Thermo Scientific Shandon Sequenza Slide Rack for Immunostaining Center	Fisher Scientific	10098889	Holder for slides and slide clips
Thermo Scientific Shandon Plastic Coverplates	Fisher Scientific	11927774	Slide clips
Tris(hydroxymethyl)aminomethane (Tris)	Sigma-Aldrich	252859	Reagent for TE buffer
VectaShield	Vecta Laboratories	H-1000-10	Mounting medium
Vectra Polaris Slide Scanner	Perkin Elmer	Vectra Polaris	Slide scanner
Xylene	Genta Medical	XYL050	De-waxing agent