Ex Vivo T Cell Stimulation in the Pancreatic Tumor: A Method to Increase Cytokine Production in T Cells

Protocolo

1. Digestion of pancreatic tumors

1. Place the dissected pancreatic tumor, metastatic site tumors, or healthy pancreatic tissue in ice-cold PBS, and store on ice.
   1. Use forceps to transfer the tumor onto a Petri dish.
   2. Add 5.0 mL of digestion medium (2 mg/mL Collagenase, 0.025 mg/mL DNase RPMI) into a 50 mL tube; store on ice to prevent enzyme activity commencing.
      NOTE: This protocol uses Collagenase Type V, which has an activity of ≥1 units/mg FALGPA and > 125 collagen digestion units (CDU)/mg solid. Collagenase and DNase aliquots can be stored at -20 °C and thawed on ice before use. When both are completely solubilized in sterile RPMI, they can be passed through a 0.2 µm filter to remove contaminants. Collagenase must be completely solubilized before filtering to avoid loss of material.
   3. Take a small aliquot of this solution to cover the tumor on the Petri dish.
   4. Use sterile scalpel and forceps to cut the tumor into small pieces, roughly less than 3 mm in length.
   5. Scrape the tumor pieces into the tube and gently invert the tube until all the pieces are submerged in digestion media. Store on ice if other tumor samples need to be prepared in a batch.
   6. Transfer onto a shaking device for 20 min at 37 °C. Make sure all pieces of tumor are submerged and not stuck to the edge of the tube. If shaking is not possible, then vortex the sample every 5 min to aid digestion.

2. Preparation of single-cell suspension from digested tumor

1. Immediately after the digestion step, place the tube on ice to slow enzyme activity.
   1. Add EDTA to achieve a final concentration of 20 mM and briefly vortex sample to mix. This will further slow enzyme activity.
   2. Open the tube and rinse any tumor digest off the lid of the tube with fresh RPMI medium.
   3. Prepare a 70 µm strainer (the µm size of the strainer can be altered as desired) on a 50 mL open tube, on ice.
   4. Pre-wet the filter with medium.
   5. Resuspend the digested cells and wash the sides of the tube using a 25 mL stripette, or larger. The wider opening of the stripette is important to allow the thick digest to pass easily.
   6. Transfer all of the digest, using the 25 mL stripette, onto the strainer.
   7. Mash the tumor on top of the filter using a 1 mL syringe plunger. Mash only directly up and down to minimize shear stress to cells.
   8. Continuously wash cells through the strainer with RPMI. Make sure to wash with enough force to push cells through.
   9. If there is still material to mash, but the RPMI stops flushing through, the strainer will be saturated. Therefore, transfer the sample into a new filter and continue.
      NOTE: Eventually only extracellular matrix components will remain in the filter, all single cells should have passed through.
2. Centrifuge the tube for 5 min at 300 x g and 4 °C.
   1. Carefully resuspend the cell pellet in complete RPMI and pass directly through another filter to remove any extracellular matrix or large cell clumps that cannot be adequately resuspended.
   2. At this point, if no stimulation is required, immediately stain the isolated cells for flow cytometry analysis by skipping to centrifuge the plate for 5 min at 300 x g and 4 °C. Remove the supernatant by flicking the plate upside down in one sharp movement. Alternatively, resuspend them in freezing medium (10% DMSO in FBS) and store at -80 °C followed by long-term storage in liquid nitrogen.
      NOTE: The freezing step can allow purification of immune cells at a later date; however, the quantification of immune cell subsets may require optimization to confirm that cell numbers and phenotype is not affected by the freeze/thaw process. The ex vivo T cell stimulation is best performed on fresh tumor samples. At this point the sample can be further purified by bead-based dead-cell removal or immune cell enrichment assays if required.

3. Preparing cells for ex vivo stimulation

1. Count the cells to achieve a concentration of 2 x 10⁶/ 100 µL in complete medium (RPMI 10 % FBS, 1X penicillin and 1X streptomycin).
   NOTE: The high number of total cells plated ensures that there will be adequate T cells within this sample to analyze. However, the number can be scaled up or down depending on sample availability and the rare nature of T-cell subsets of interest.
   1. Plate 100 µL of cells in a U-bottomed 96-well plate.
   2. Add 100 µL of complete medium containing a 2x preparation of PMA/ ionomycin (to achieve a final concentration 0.081 µM and 1.34 µM, respectively, as recommended by the manufacturer).
      NOTE: If measuring degranulation/exocytosis, also include here a fluorescently conjugated anti-mouse CD107a in the media. A control sample that does not contain CD107a must also be performed.
   3. Place in 37 °C incubator with 5% CO₂ for 1 h.
   4. Add 20 µL of a 10x preparation of brefeldin A and monensin (to achieve a final concentration 1.06 µM and 2.0 µM, respectively (as recommended by the manufacturer) in complete media.
      NOTE: Brefeldin A and monensin are protein transport inhibitors and thus block extracellular release of cytokines, etc. permitting their detection by flow cytometry. If measuring cytokine release into the supernatant by ELISA or similar methods – then this step can be skipped.
   5. Place the plate in a 37 °C incubator with 5% CO₂ for further 4 h.

Materiais

Name	Company	Catalog Number	Comments
70 μm pore-size cell strainer	Fisher Scientific	11597522
anti-CD107a PE (clone 1D4B)	Biolegend	121612	1:100 to culture media
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4503
Cell Stimulation Cocktail (500x) (phorbol 12-myristate 13-acetate (PMA) and ionomycin)	eBioscience	00-4970-03	Final concentration PMA 0.081 μM, ionomycin 1.34 μM
THP1 Human Monocytic Leukemia Cells	ATCC	TIB-202
Collagenase Type V from Clostridium histolyticum	Sigma-Aldrich	C9263	2 mg/mL in media
Dimethyl sulphoxide (DMSO)	Sigma-Aldrich	D2650-100mL
DNase (Deoxyribonuclease I from bovine pancreas Type II-S) stock 10 mg/mL in 0.15 M NaCl	Sigma-Aldrich	D4513	Final concentration in digestion media 0.025 mg/mL
Foetal calf-serum (FCS)	GE Healthcare	A15-104	10% in RPMI
Hamilton syringe 700 series, 25 μL volume, 22s gauge needle bevel tip	Fisher Scientific	10100332
Penicillin/streptomycin	PAA	15140122	100 units/mL Penicillin, 100 μg/mL Streptomycin
Protein transport inhibitor cocktail (500x) (brefeldin A and monesin)	eBioscience	00-4980-03	1x Final concentration Brefeldin A 10.6 μM, monensin 2 μM
RPMI-1640 (containing 0.3 g/L Glutamine)	Sigma-Aldrich	R8758
Surgical Scalpel Blade No.10	Swann-Morton	501
Trypsin-EDTA Solution 10x	Sigma-Aldrich	594-18C	Trypsin (0.1%) EDTA (0.4%) final concentration
U-bottomed 96 microwell plate	VWR	734-2080