Interrogating the Tumor Microenvironment by Deep Tissue Immunoprofiling

Abstract

With the modern success of immunotherapy from chimeric antigen receptor T-cell therapy to checkpoint blockade, an understanding of the tumor microenvironment has been more essential than ever. Knowing the key immune players in the tumor can provide the basis for studying cancer host immunity and interactions governing tumor tolerance. This endeavor can be approached in a step like manner—from a broad overview with immunohistochemistry to genomic expression analysis, followed by detailed functional analysis of each cell with multiparameter flow cytometry.

1. Introduction

Many tools are available to assess the tumor microenvironment. The simple process of immunohistochemistry (IHC) can provide a comprehensive survey of the tumor landscape from formalin-fixed, paraffin embedded (FFPE) tissue [1]. A method pioneered in 1941 by Coons, Creech, and Jones [2], IHC takes advantage of specific antigen expression from different tissues and binds antigen with complementary antibody that is conjugated to a chromogen, allowing for gross visualization of antigens. In the 1990s, techniques for antigen retrieval were optimized, allowing for better exposure of antigens that were masked from the formalin fixation process [3]. Now, with the creation of custom antibodies, this method can detect a variety of immune biomarkers that are unique to individual cellular populations and identify them in situ within heterogeneous tumor tissue.

IHC also provides a blueprint of the tumor cellular composite that can be utilized for more in-depth interrogations of the tumor microenvironment such as gene expression with tools like Laser Capture Microdissection (LCM). The combination of laser technology with microscopy visualization in LCM provides a method to isolate specific cell populations and areas of interest from the diverse tumor landscape. FFPE tissue is cut onto PEN (polyethylene naphthalate)-coated glass slides (Fig. 1). The PEN membranes help collect larger pieces of tissue, avoid need for dehydration, and detach completely from the glass slide. These isolated pieces of tissue can then be extracted for RNA or DNA, paving way for gene expression investigations.

Fig. 1.

Scheme of laser capture microdissection methodology

Real-time polymerase chain reaction (RT-PCR) employs standard PCR techniques to provide gene expression data. Extracted RNA from tumor tissue is converted to complementary DNA (cDNA), which is then amplified with attached fluorescent probes that can quantify the amount of product produced, translating this to gene expression data.

Aside from FFPE tissue, fresh tumor tissue is even more valuable and can yield live immune cells. Through an enzymatic digestion process, tumor infiltrated lymphocytes can be isolated from fresh tumor tissue. The tumor isolated lymphocytes (TILs) can be stained with antibodies, conjugated to a specific fluorochrome, which will bind a particular antigen of interest. Through analysis of the light scatter pattern emitted by the attached fluorochrome, each single lymphocyte can then be phenotypically characterized by multiparameter flow cytometry. This tool is not limited to cell surface characterization, but also can assess intracellular cytokine production and transcription factors.

These multitude of tools will build the essential foundation for exploring the tumor microenvironment. Identifying different cells in the tumor microenvironment, characterizing their function, and decoding their genomic signature will provide clues to underlying cellular interactions in the cancer milieu [4, 5].

2. Materials

2.1. Immunohistochemistry

2.1.1. Reagents

1. Xylene.

2. Ethanol.

3. Distilled water.

4. Hydrogen peroxide 30%: dilute with methanol.

5. PBS.

6. Citrate Buffer Solution: 10 mM sodium citrate, 0.05% TWEEN 20, pH 6.0×.

7. Histostain-Plus third Gen IHC Detection Kit (Invitrogen Cat. No. 85-9073) or similar IHC staining kit.

8. Primary antibody of your choice titrated to effect. Dilute antibodies in 5% bovine serum albumin (BSA), 2.5% Triton X-100 solution, Triton X-100, and BSA should be diluted in PBS.

9. Hematoxylin.

10. Resin or mounting solution such as Histomount Mounting Solution Consumables.

11. Slide cover slips.

2.1.2. Equipment

1. Vegetable cooker or pressure cooker.

2. Humidifying chamber.

2.2. Laser Capture Microdissection and RNA Isolation

2.2.1. Reagents

1. RNase Away.

2. Hematoxylin.

3. Eosin.

4. RNA isolation kit.

2.2.2. Consumables

1. PEN membrane slides.

2. RNA-free 0.5-ml Eppendorf tubes.

2.2.3. Equipment

1. Leica LMD 700 Laser Microdissection.

2.3. Real-Time PCR

2.3.1. Reagents

1. High-Capacity RNA to cDNA kit (Applied Biosystems, Cat. No. 4387406).

2. Customized TaqMan Array FAST Plates with Custom TaqMan PreAmp Pool.

3. TaqMan PreAmp Master Mix 2× (Applied Biosystems, Cat. No. 4384266).

4. TaqMan Fast Advanced Master Mix (Applied Biosystems, Cat. No. 4444557).

2.3.2. Consumables

1. PCR tubes.

2.3.3. Equipment

1. Nanodrop to measure concentration of RNA.

2. Thermal Cycler.

3. StepOnePlus Real-Time PCR System (Applied Biosystems, Cat. No. 4376600).

2.4. Tumor Tissue Processing for Lymphocytes

2.4.1. Reagents

1. RPMI 1640.

2. Fetal bovine serum (FBS).

3. DNase 1×.

4. Liberase 1×.

5. EDTA.

6. 1× PBS.

7. 10× PBS.

8. Percoll.

9. ACK lysis buffer.

10. DMSO.

2.4.2. Consumables

1. 50-ml Falcon conical tubes.

2. 15-ml Falcon conical tubes.

3. Pasteur pipette attached to vacuum.

4. 2-ml Cryovials.

5. 70-μM cell strainers.

6. 100-μM cell strainers.

7. Petri dish.

8. Surgical scalpel blades.

2.4.3. Equipment

1. Mr. Frosty or CoolCell Freezing Containers.

2. Hemocytometer.

3. Sterile forceps.

4. 150-ml Erlenmeyer flask.

5. Magnetic stir bars.

6. 250-ml beakers.

7. Pipettes μl and ml.

8. Hot plate with stir function.

9. Centrifuge.

2.5. Intracellular and Extracellular Staining for Multi-parameter Flow Cytometry Analysis

2.5.1. Reagents

1. RPMI 1640.

2. Fetal bovine serum (FBS).

3. PBS 1×.

4. IMDM (Iscove’s Modified Dulbecco’s Medium).

5. Cell stimulation cocktail with PMA and ionomycin plus protein transport inhibitors (eBioscience Cat. No. 00-4975-93).

6. Live/dead viability dye of your choice.

7. Antibodies of your choice.

8. Foxp3/transcription factor fixation/permeabilization concentrate and diluent (eBioscience Cat. No. 00-5521-00).

9. BD Perm/Wash (BD Biosciences Cat. No 554723).

10. Distilled water.

2.5.2. Consumables

1. 5-ml round bottom polystyrene tubes with caps.

2. Foil.

2.5.3. Equipment

1. Hemocytometer.

2. Centrifuge.

3. Vortex mixer.

4. 37 °C incubator.

5. Flow cytometry machine (we use Attune NxT Flow Cytometer and BD LSRFortessa).

3. Methods

3.1. Immunohistochemistry

3.1.1. Deparaffinization

Working with FFPE tissue cut onto plus charged slides.

1. Deparaffinize slides with xylene bath, and then rehydrate through series of graded ethanol solutions. Wash with distilled water. Do not let tissue dry, slide with tissue should be kept moist from this step on. Keep slides in humidifying chamber during incubations (see Note 1).

2. To eliminate endogenous peroxidase activity, submerge slides in 30% hydrogen peroxide (diluted with methanol). Leave for 10 min (see Note 2).

3. Wash slides with PBS for 2 min. Repeat for total of three times.

3.1.2. Antigen Retrieval

This heating process reveals epitopes that were masked during the formalin fixation process.

1. Heat citrate buffer solution (10 mM sodium citrate, 0.05% TWEEN 20, pH 6.0×) with vegetable cooker or pressure cooker (see Note 3).

   1. With vegetable cooker, heat solution to 100 °C. Immerse slides in heated citrate buffer solution and incubate for 20 min.
   2. With pressure cooker, once solution starts boiling, place slides in pressure cooker for 3 min.

2. Remove slides and place in cold tap water for 10 min.

3. Wash slides with PBS for 1 min. Repeat for total of three times.

At this step we use a kit such as Histostain-Plus kit. This portion adapted from the kit:

1. Add serum blocking solution (10% non-immune serum—goat) to tissue, about 100 μl.

2. Leave for 10 min. Then drain blocking solution from slides.

3. Place ~100 μl of primary antibody or enough to cover the tissue completely. Leave overnight in 4 °C humidification chamber.

4. Wash slides with PBS for 2 min. Repeat for total of three times.

5. Add ~100 μl biotinylated secondary antibody. Leave for 10 min.

6. Wash slides with PBS for 2 min. Repeat for total of three times.

7. Add ~100 μl streptavidin-peroxidase conjugate. Leave for 10 min.

8. Wash slides with PBS for 2 min. Repeat for total of three times.

9. Make DAB Chromogen solution—add 1 drop of DAB chromogen to 1 ml DAB substrate buffer (20×). Mix and keep covered in foil away from light. Use within 2 h.

10. Add ~100 μl DAB Chromogen solution to tissue on slide. Leave for 5 min.

11. Wash slides with distilled water for 2 min. Repeat for total of three times.

12. Counterstain with ~100 μl of hematoxylin. Leave for 2–3 min. Wash in tap water until blue. Rinse with distilled water.

13. Mount slides with resin or mounting solution, covered with cover slip, then dry in 60 °C oven for 20–30 min or until dried.

3.2. Laser Capture Microdissection and RNA Isolation

3.2.1. Preparing Slides

Every step must be done with RNA precautions. We use Roche High Pure RNA Paraffin Kit, but any other brand can be used (see Note 4, 5, 6 and 7).

1. Cut FFPE tissue onto PEN membrane slides. Cut to 10 μm thickness.

2. Stain all cut PEN membrane slides with hematoxylin and eosin with RNA precautions (we use a tissue service to have this performed, but this can be done in lab). The purpose of the H&E stain is to show tissue structures and help navigate where to cut. Once stained, the PEN membrane slides are ready for laser capture microscopy.

3. Using the Leica LMD 700 Laser Microdissection System. Load slides face down onto the slide holder. Load a 0.5-ml tube onto the collection holder.

   1. The baseline settings we start at 10× magnification are below. You can increase or decrease power depending on thickness of tissue and how deep you wish the tissue to be cut (Fig. 2).

      Power	29
      Aperture	15
      Speed	20
      Specimen balance	15
      Head current	100
      Pulse frequency	120

Fig. 2.

Microscopy photo of tissue pre and post-microdissection

• 4.Laser areas of interest. Flakes of tissue should fall into cap of 0.5-ml tube. Put 20 μl of Tissue Lysis Buffer onto the cap to prevent flakes from flying awake. Carefully close cap and centrifuge, so that flakes sink to bottom of tube.
• 5.Add 80 μl of Tissue Lysis Buffer, so that final volume is 100 μl and proceed to step 1 of Subheading 3.2.2. RNA should be isolated right after LCM. There is no need to deparaffinize as this should have been done during the H&E staining process.

3.2.2. RNA Isolation and RT-PCR: Adapted from Applied Biosystems Protocols

Conversion of RNA to cDNA with applied biosystems kit (see Notes 8, 9, 10 and 11)

1. Would recommend 2 μg of total RNA per 20 μl reaction. If there is not enough RNA, can use minimum of 1 μg RNA.

2. Calculate volumes needed of each component based on number of samples.

   The total volume for each sample is 20 μl and is composed of the following:

   1. 2× RT buffer, 10 μl
   2. 20× Enzyme Mix, 1 μl
   3. RNA sample, up to 9 μl
   4. Nuclease-free water, 9 μl. Volume of RNA sample (so anywhere from 0 and up)

3. Make the master mix. Add together the appropriate volume of RT buffer, enzyme mix, and nuclease-free water. Would make an extra sample to account for pipetting loss.

4. Using RNAase-free PCR tubes, first aliquot master mix to each tube.

5. Then add appropriate amount of RNA to each PCR tube, pipette up and down to mix with master mix.

6. Seal tubes and spin down to ensure all drops come down and no air bubble exists.

7. Place on thermal cycler for 60 min at 37 °C followed by 5 min at 95 °C to stop the reaction. Hold at 4 °C.

8. The product will be cDNA which can be stored in −15 °C to −25 °C.

Preamplification of cDNA

1. The recommended concentration of cDNA to use is 0.2 ng to 100 ng for amplification.

2. Depending on the size of the custom panel, you will use different volumes of the TaqMan PreAmp pool, TaqMan preAmp Master Mix, and different quantities of cDNA. We commonly use a custom 48 gene panel for which we use these volumes.

   Total volume is 30 μl for each sample and composed of:

   1. Custom TaqMan PreAmp Pool: 7.5 μl.
   2. TaqMan PreAmp Master Mix (2×): 15 μl.
   3. cDNA 0.2–100 ng: max volume of 7.5 μl.
   4. Nuclease-free water: 7.5 μl—volume of cDNA (will anywhere from 0 μl and up, depending on volume of cDNA used).

3. If you are running many samples, a master mix can be created with Custom TaqMan PreAmp Pool, TaqMan PreAmp Master Mix, and Nuclease-free water. Add 20% extra to account for pipetting loss.

4. Using 0.2 ml RNAase-free PCR tubes, aliquot master mix to each tube.

5. Then add appropriate amount of RNA to each PCR tube, pipette up and down to mix with master mix.

6. Seal tubes and spin down to ensure all drops come down and no air bubble exists.

7. Proceed to thermal cycler with these settings:

   Volume of 50 μl (to heat entire tube):

   1. Start with 95 °C for 10 min.
   2. 14 cycles of: 95 °C for 15 s (denature) followed by 60 °C for 4 min (anneal/extend).
   3. Keep at 99.9 °C for 10 min.
   4. Hold at 4 ^°C.

8. Proceed to PCR amplification or store at −20 °C.

PCR Amplification with TaqMan Array Plate

1. The volumes written here may change depending on size of custom plate. Applied Biosystems offers a Standard TaqMan Array Plate or a Fast TaqMan Array Plate. We designed a 48 gene panel on Fast TaqMan Array Plate which allows two samples per a 98-well plate. The final volume per each well is 10 μl.

2. Make the following master mix with preamplified product:

   This is based on a 48 gene format on the Fast TaqMan Array Plate. The final volume will be 576 μl:

   1. 2× Master Mix: 288 μl.
   2. Nuclease-free water: 276 μl.
   3. Preamplified product: 12 μl.

3. Aliquot 10 μl of master mix solution with preamplified product to each 48 well.

4. Seal plate well with sealing tape, ensuring each well is covered.

5. Spin down well to ensure all drops are settled. Plate is ready to be run.

6. We use Applied Biosystems Real-Time PCR system, specifically the StepOnePlus system. The thermal-cycling profile for this system is:

   1. UNG Incubation: 50 °C for 2 min.
   2. Polymerase activation: 95 °C for 20 s.
   3. PCR 40 cycles of: 95 °C for 1 s (denature), followed by 60 °C for 20 s (anneal/extend).

3.3. Tissue Collection and Storage

Collect fresh tissue and store in 50-ml Falcon tubes in RPMI +5% FBS. Tissue should not be left to dry out. If processing next day, can store tissue overnight in 50-ml Falcon tubes placed on rotating rocker in 4 °C fridge or cold room.

3.4. Weigh Tissue for Immune Cell Density

Prior to processing the tissue, the fresh specimen should be weighed. This will determine the amount of digestion enzymes to use. Use a covered petri dish to transport the tissue. First zero scale with covered petri dish. Then transport tissue in same covered petri dish and weigh. This weight will also be important for calculation of immune cell density once TILs are isolated.

3.5. Tissue Processing

Preparation: Use 250 ml beakers filled to ~150 ml on a hot plate to create 37 °C water baths.

1. Working in a hooded space, finely mince tumor tissue in petri dish with scalpel and forceps. Use a small amount of RPMI to aid with mincing and prevent tissue from drying out (see Notes 12 and 13).

2. Transport minced tissue into a 150-ml Erlenmeyer flask with magnetic stir bar. Add RPMI +5% FBS to minced tissue to make a suspension, so that the tissue can be transported via a 25-ml pipette. Depending on size of tissue the amount of RPMI +5% FBS to use will be different.

   1. Tissue weight ≤ 0.5 g use 10 ml of RPMI +5% FBS.
   2. Tissue weight 0.5–1.0 g use 20 ml of RPMI +5% FBS.
   3. Tissue weight ≤ 1.5 g use 30 ml of RPMI +5% FBS.

   Use 5 ml at a time, so the remaining volume can be used to rinse the petri dish to collect any remaining tissue.

3. Add digestion enzymes DNASE and Liberase to the Erlenmeyer Flask with minced tissue suspension. Amount will depend on tissue weight:

   1. Tissue weight ≤ 0.5 g add 2 ml DNASE and 200 μl Liberase. This should all be in 10 ml of RPMI +5% FBS.
   2. Tissue weight 0.5–1.0 g add 4 ml DNASE and 400 μl Liberase. This should all be in 20 ml of RPMI +5% FBS.
   3. Tissue weight ≤ 1.5 g, add 6 ml DNASE and 600 μl Liberase. This should all be in 30 ml of RPMI +5% FBS.

4. Place weighted rings on Erlenmeyer flask and then place in the 250-ml beaker heated water bath. Turn stirrer on and let tissue digest for 45 min. Check intermittently to ensure water temperature remains at 37 °C and tissue is not completely digested. If at the 45 min mark there are still large chunks of tissue, leave for another 15 min, checking intermittently to ensure tissue is not completely digested.

5. Stop the reaction by adding 100 mM EDTA (can remove flask from water bath and add in hooded space) and incubate for another 10 min on 37 °C water bath with stirrer on.

6. Working back in a hooded area, filter the digested tissue. Using a 50-ml Falcon tube, place the 100 μm filter on top. Use a 25-ml pipette to transport the digested tissue into the filter to strain the sample. If filter clogs, replace with new filter. Rinse the Erlenmeyer flask with 10 ml of RPMI +5%FBS and 2 mM EDTA and strain through the filter.

7. Strain filtered tissue suspension into a new 50-ml Falcon tube with 70 μm filter on top.

8. Centrifuge filtered tissue suspension at 1500 rpm for 10 min at 4 °C. Aspirate off the supernatant keeping the pellet and move on to Percoll gradient.

3.6. Percoll Gradient

1. Will need to prepare four gradients: 100%, 80%, 40%, 20%. Prepare in 50-ml Falcon tubes.

   1. 100% Percoll: Take 45 ml of pure Percoll and add 5 ml of 10 × PBS. Mix well.
   2. 80% Percoll: Take 40 ml of 100% Percoll and add 10 ml of 1 × PBS. Mix well.
   3. 40% Percoll: Take 20 ml of 80% Percoll and add 20 ml of 1× PBS. Mix well.
   4. 20% Percoll: Take 4 ml of 100% Percoll and add 16 ml of RPMI. Mix well.
2. The Percoll gradients will be made in 15-ml Falcon tubes (Fig. 3). The initial tissue weight will determine the number of gradients (see Notes 14 and 15).

   1. For tissue ≥2 g, use 6 gradients. Can use more gradients if necessary. If tissue sample is smaller, can use 4 or 2 gradients.

3. Take 80% Percoll and resuspend the pellet from tissue processing step. Depending on the number of gradients, the amount of Percoll to add will be different.

   Add 4 ml of Percoll per a gradient:

   1. 6 tubes = 24 ml of 80% Percoll.
   2. 4 tubes = 16 ml of 80% Percoll.
   3. 2 tubes = 12 ml of 80% Percoll.

4. Mix the pellet well with the Percoll, pipetting up and down.

5. Aliquot 4 ml into each 15-ml Falcon tube.

6. Mix 40% Percoll and very gently add 4 ml to each gradient by dripping it down the sides of the Falcon tube. Be careful not to break the interphase between the different Percoll concentrations.

7. Mix 20% Percoll and add 3 ml to each gradient very gently, laying it on top of the 40% Percoll. Be careful not to disturb the interphase.

8. Centrifuge gradients at 3000 rpm for 30 min at 25 °C with no breaks.

9. You may be able to visualize a layer of cloudy beige cells at the interphase of 40% and 80%, if not, it is still present. Remove the gradient with a Pasteur Pipette attached to vacuum, moving in a circular manner, removing gradient form the sides. Stop at about 1 in. above the 40% interphase.

10. Harvest the mononuclear layer (interphase of 40% and 80%) with a 5-ml pipette, again moving in circular motion and removing cells from the sides. Collect to about the 2 ml mark from the red blood cells, which are located at the bottom.

11. Place all harvested mononuclear cells into a 50-ml Falcon tube. Add RPMI +5% FBS and 2 mM EDTA, bringing up the volume to 50 ml. If the sample appears thick, split to two 50-ml Falcon tubes, and add RPMI +5% FBS and 2 mM EDTA to both, bringing up the volume to 50 ml. This process is to wash the cells.

12. Mix well and centrifuge at 1500 rpm at 4°C for 10 min with breaks on.

13. Remove supernatant. If the pellet has red blood cells present (appears red), can add 3 ml ACK lysis buffer, resuspend the pellet and let sit for 5 min.

14. Wash cells again by adding 1× PBS and bringing up the volume to 50 ml. Spin at 1500 rpm for 10 min at 4 °C.

15. Remove supernatant and resuspend pellet in 10 ml of 1× PBS. Count the cells with a hemocytometer.

Fig. 3.

Diagram of tubes post-digestion to obtain single cell suspensions

3.7. Freezing Cells

1. Prepare cryo-freezing container—can use Mr. Frosty or CoolCell Freezing containers, basically any container that cools slowly at −1 °C/min. Place these in −20 °C for 15–20 min.

2. Spin down cells at 1500 rpm for 10 min at 4 °C. Remove the supernatant. Will add solution of FBS and DSMO for freezing.

3. Number of cryovials will depend on cell amount. Do not freeze more than ten million cells per a vial, this will affect viability. Aim for 5–10 million/vial. Each cryovial should be filled half way to 1 ml. Once number of cryovials calculated determine total volume of cell suspension at 1 ml per a cryovial. From there 90% of the volume will be FBS and 10% will be DSMO. For example if there is only one cryovial, final volume of cell suspension will be 1 ml, so add 900 μl (90%) of FBS to the pellet followed by 100 μl (10%) of DSMO. If there are four cryovials, final volume of cell suspension will be 4 ml, so add 3.6 ml (90%) of FBS and 400 μl (10%) of DSMO.

4. Add FBS first, pipetting up and down to resuspend the cell pellet. Then add DSMO and pipette up and down to mix. DSMO is cytotoxic, so once added, move quickly. Aliquot 1 ml of FBS + DSMO cell suspension to each cryovial and place cryovials in cryo-freezing containers. Store in −80 °C for 4–48 h and transfer to −180 °C for long-term storage.

3.8. Thawing for Frozen Cells

1. If using cryo-frozen cells, thaw tubes by place in 37 °C water bath for quick defrost. When the tube is half thawed, remove from water bath and let rest defrost at room temperature (see Note 16).

2. Wash cells of cryo-freezing solution: transfer cells to a 15-ml Falcon tube with thawing buffer (RPMI +20% FBS).

3. Spin at 1500 rpm for 5–10 min. Dispose supernatant and repeat wash with thawing buffer and spin again at 1500 rpm for 5–10 min. Dispose supernatant. Resuspend in PBS and count cells with hemocytometer.

3.9. Counting Cells and Making Single-Cell Suspension

1. For each sample, aim for 0.5–2 million cells. If performing intracellular staining, would aim for at least one million cells a sample (the more cells the better as you will lose cells with intracellular wash steps). Use 5-ml round bottom tubes per a sample—these will be the tubes used for staining.

2. If performing surface stain only, suspend cell pellet in 100 μl of 1× PBS. There should be 0.5–2 million cells/tube. The final volume will be 200 μl/tube after 100 μl of antibody solution is added in the below staining step.

3. If performing intracellular stain, suspend cell pellet in 100 μl of 1× IMDM. There should be 1–2 million cells/tube. The final volume will be 200 μl/tube after 100 μl of stimulation cocktail is added in the below intracellular staining step below.

3.10. Surface Stain with Viability Dye

1. Recommend use of viability dye first to label dead cells. Prepare viability dye, dilute 1:1000. Add 200 μl to single stain control and tumor sample, vortex. Add 200 μl PBS to rest of samples for control (see Notes 17, 18 and 19).

2. Cover samples with foil and leave for 30 min at room temperature.

3. Add 2 ml PBS to each sample, vortex. Centrifuge at 1300 rpm for 5–10 min.

4. After centrifuging, the cells should be pelleted to the bottom of the tube. The pellet may not be visualized but is present. In one swift motion, pour off the supernatant. There will drops of PBS left over, but that is ok. Do not shake or use forceful motion, as this will dislodge cells that are adherent to the tube.

5. Prepare antibody solution: add proper amount of antibody to 1× PBS—final volume will be 100 μl.

6. Add antibody solution to each sample of single cell suspension, vortex. For any unstained samples, add 100 μl of 1× PBS for control.

7. Cover samples with foil to keep in dark. Incubate at room temp for 30 min.

8. Add 2 ml of 1× PBS to each tube to wash cells, vortex. Centrifuge at 1300 rpm for 5–10 min. In one swift motion, pour off the supernatant.

9. Add 500 μl of 1× PBS, vortex. Now cells are ready to be run on the flow cytometer. If there are chunks of cells present, run through a filter prior to prevent clogging of flow cytometer.

3.11. Intracellular Stimulation

1. From the single-cell suspension step, each sample should be in 100 μl IMDM. Will add 100 μl of stimulation cocktail for final volume of 200 μl.

   1. Cell Stimulation Cocktail with PMA and ionomycin comes in 500×. Some include protein transport inhibitors. If not, protein transport inhibitor will need to be added.

2. Dilute Cell Stimulation Cocktail from 500× to 2× with IMDM (after adding 100 μl of Stimulation Cocktail to 100 μl of single-cell suspension, final volume will be 200 μl, diluting Cell Stimulation Cocktail to 1×).

3. Add 100 μl of 2× Cell Stimulation Cocktail to 100 μl single-cell suspension, vortex and incubate for 3 h at 37 °C.

4. After incubation, wash cells: add 1 ml 1× PBS, vortex. Centrifuge 1300 rpm for 5–10 min. Pour off supernatant and proceed surface stain.

5. Follow steps 1–8 of Subheading 3.10.

3.12. Fixation and Permeabilization

1. Prepare fixation/permeabilizaiton solutions by making one to four dilution with concentrate and diluent. For example, for 2 ml solution, use 500 μl of concentration and add to 1500 μl of diluent.

2. Add 100 μl of Fix/Perm solution to spin down cells from step 5 of Subheading 3.11. Vortex, cover in foil and let set for 1 h at room temperature.

3. Prepare permeabilization wash buffer. Solution comes in 10×, dilute to 1×. For 50 ml, add 5 ml of 10× solution, and add 45 ml of distilled water.

4. After the 1 h incubation, add 1–2 ml of 1× permeabilization wash buffer, vortex. Centrifuge at 1300 rpm for 5–10 min.

5. Pour off supernatant and add 500 μl of PBS. Vortex and store cells in 4 °C fridge overnight and proceed to intracellular stain next day.

3.12.1. Intracellular Stain

1. Remove cells from fridge and centrifuge at 1300 rpm for 5–10 min. Pour off supernatant.

2. Add 200 μl of permeabilizaiton wash buffer to each sample, vortex, and let sit covered with foil for 15–20 min. Centrifuge at 1300 rpm for 5–10 min and pour off supernatant.

3. For intracellular stain use permeabilization wash buffer as base solution. Prepare antibody solution: add proper amount of antibody to 1× permeabilization wash buffer—final volume will be 100 μl.

4. Add antibody solution to each sample of single cell suspension, vortex. For any unstained samples, add 100 μl of 1× permeabilization wash buffer for control.

5. Cover samples with foil to keep in dark. Incubate at room temperature for 30 min.

6. Add 1 ml of 1× permeabilization wash buffer to each tube to wash cells, vortex. Centrifuge at 1300 rpm for 5–10 min. In one swift motion, pour off the supernatant.

7. Add 500 μl of 1× PBS, vortex. Now cells are ready to be run on the flow cytometer. If there are chunks of cells present, run through a filter prior to prevent clogging of flow cytometer.

4. Notes

Immunohistochemistry

1. Once deparaffinized, it is important to keep tissue moist till completion of stain.

2. If staining Collagen Type II, incubate with 0.35% hyaluronidase after eliminating endogenous peroxidase activity.

3. During the Antigen Retrieval Process, a 100 °C water bath can also be used if a vegetable cooker or pressure cooker is not available. Microwave is not recommended given heterogeneity of heating and formation of cold pockets.

LCM and RNA isolation

• 4.Remember that all supplies used for this process should be done with RNA precautions. Use RNAase Away spray to clean your workbench, all pipettes, and the slide loader. The 0.5-ml tubes need to be RNA free.
• 5.To improve RNA yield, this entire protocol—from cutting of PEN membranes to RNA isolation—should be done in 1 day.
• 6.When collecting the tissue from 0.5-ml Eppendorf tubes, be very careful as the tissue flakes can fly off during the unloading process.
• 7.During the LCM process, part of the PEN membrane will be lasered off with the tissue and will need to be removed during the RNA isolation portion. Remove PEN membranes after overnight Proteinase K incubation and adding of Binding Buffer, but before ethanol precipitation. After binding buffer is added, spin down tubes. You will see a small pellet of PEN membranes. The supernatant will have RNA. Transfer the supernatant to a new tube, and then proceed with ethanol precipitation.

RT-PCR

• 8.Use RNase Away to ensure work bench and all pipettes are RNA free.
• 9.This process does not need to be completed in one day. RNA is unstable, so should be freeze/thawed as infrequently as possible. However, cDNA is more stable and once RNA is converted to cDNA, it can be stored in −20 °C for a week or so.
• 10.Reagents are fragile, so do not vortex, instead flip tubes upside down, flick tubes, and pipette up and down to mix.
• 11.With the 98-well plates used during PCR amplification, remember that the volumes are very small, so all drops count. Ensure all wells appear to have same volume when all reagents are added and spin down plate.

Tissue processing for TILs

• 12.During the tissue procurement process, keep tissue moist and bathed in RPMI. If tissue dries, cell viability will be low. For optimal cell viability, tissue should be processed within 24 h of collection.
• 13.When mincing the tissue, the finer you can mince, the better. This allows for a more effective tissue digestion process.
• 14.Use low setting on your pipette when creating the Percoll gradient. Let Percoll slowly drip down from side of tube.
• 15.If you accidentally break the Percoll gradient while laying down the different layers, you can still save the cells. Add RPMI and centrifuge with breaks to spin down the cells. Remove the Percoll supernatant, leaving only the cell pellet. From there you can resuspend the pellet in 80% Percoll and restart the gradient.

Staining for flow cytometry

• 16.If using frozen cells, the defrosting process should be quick. Keep cells frozen, transport with dry ice, until ready to defrost in 37 °C water bath.
• 17.Cell staining can also be done in 96-well plates. However, with thick tumor tissue, we prefer staining in 5-ml round bottom tubes.
• 18.Attempt to perform staining process away from direct light. Keep antibody solutions on ice during use.
• 19.Pouring of the supernatant from 5-ml tubes should be done in one swift motion. This applies to all steps requiring removal of supernatant.