Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Summary

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

Abstract

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient's own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration. Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

Introduction

Pancreatic cancer immunotherapy is in its nascent stages, with ongoing preclinical exploration and validation primarily in mouse models¹. Current treatments for pancreatic cancer include surgery, chemotherapy, radiotherapy, and targeted therapies. Unfortunately, these methods often fail to completely eradicate tumor lesions, leading to recurrence and progression. Traditional treatments focus on tumor cells but often overlook the tumor microenvironment (TME), which includes both tumor cells and associated stroma composed of tumor-infiltrating lymphocytes (TILs), fibroblasts, and extracellular matrix². TILs are immune cells within the TME, including cytotoxic T cells, helper T cells, regulatory T cells, B cells, NK cells, macrophages, and myeloid-derived suppressor cells³. These cells participate in immune responses that influence tumor growth and therapeutic outcomes⁴.

Adoptive cell therapy (ACT) involves collecting a patient's immune cells, modifying and expanding them in vitro, and then reintroducing them to target and kill tumor cells. TIL therapy, a type of ACT, is being researched for treating various cancers, including melanoma, lung cancer, and cervical cancer. This process involves isolating lymphocytes from the tumor, culturing them with IL-2 in vitro, and reinfusing them into the patient, often after lymphodepletion with chemotherapy or radiotherapy⁵.

Since Rosenberg's 1986 study demonstrated the efficacy of TILs in mice⁶, adoptive cell transfer immunotherapy has become a research hotspot and has shown promise in cancer treatment^7,8,9,10. We aim to extract TILs from mice, sort them for specific T cells via flow cytometry, and validate their tumor-killing effects through adoptive transfer.

In this protocol, we describe a method to utilize TILs from mice through flow cytometry for adoptive cell transfer. The goal is to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer. This method includes implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, isolating lymphocytes from primary tumors via flow cytometry sorting, or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells into tumor-bearing mice. Subsequent administration of interleukin-2 and bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, particularly evaluating tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer, as well as the optimization of cell processing parameters and combination treatment regimens.

Protocol

The mouse colony is housed in an AAALAC International-accredited facility, adhering to all relevant USDA, HHS, and NIH regulations and standards. The Tianjin Medical University Cancer Institute and Hospital Animal Care and Use Committee reviewed and approved all animal procedures, including the rationale for strain selection, experimental group assignments, and the statistical justification for animal numbers and randomization.

1. Tumor Induction

1. Use the KPC-Luc cell line¹¹ to induce orthotopic pancreatic tumors in mice.
2. Prepare a cell suspension by trypsinizing the KPC-Luc cells, washing them twice with PBS, and resuspending them at a concentration of 1 x 10⁶ cells/mL in PBS with a 30% basement membrane matrix (BMM).
3. Anesthetize the mouse with isoflurane at an induction dose of 4% and then switch to a maintenance dose of 2%. Provide thermal support using a heating pad and monitor as necessary. Apply ophthalmic ointment to both eyes to prevent dryness.
4. Disinfect the surgical area multiple times in a circular motion using both an iodine-based scrub and alcohol. Administer a subcutaneous injection of carprofen 5 mg/kg as analgesia prior to the surgery. Using a sterile scalpel blade, perform a small left abdominal incision (0.5-1 cm) under the left rib of the mouse to expose the pancreas.
5. Use forceps to hold the mouse spleen and expose the mouse pancreas, mix cells and inject them into the pancreas of the mouse with 50,000 cells per mouse. Inject 50 µL of the cell suspension directly into the pancreas using a 29 G needle.
6. Return the mouse pancreas and sequentially suture the peritoneum and skin with sutures.
7. Allow the mouse to recover on a warm pad until fully awake. Administer 5 mg/kg of carprofen subcutaneously as analgesia after surgery daily for 3 days.
8. Perform live imaging 7 days post-injection to monitor tumor growth.
   NOTE: Treatments may be administered at this step.
   1. Intraperitoneally inject the mouse with D-Luciferin potassium salt (15 mg/mL in PBS, 60 µL per mouse).
   2. Following isoflurane anesthesia, position the mouse in the in vivo imaging system for bioluminescence detection. After imaging, remove the mouse from the system and allow it to recover from anesthesia naturally.

2. Harvest tumors

1. Euthanize the donor mice using a CO₂ chamber. Sterilize the whole body with 75% ethanol.
2. Place the mouse in a sterile hood. Use sterile scissors and forceps to open the abdominal cavity.
3. Remove the tumors and place them in PBS on ice. Measure the tumor size with a vernier caliper and calculate the tumor volume using the formula (π x length × width × width)/6.

3. Tumor digestion

1. Prepare the digestion solution by making 0.125 mg/L collagenase, 0.125 mg/L Dispase II, and 10 μg/mL DNase I in RPMI 1640 as 1x concentration. Add 1-2 mL of digestion solution per well in a 12-well plate.
2. Mince the tumors into small pieces using sterile scissors. Digest the tumor pieces at 37°C with shaking at 70 rpm for 30 minutes.

4. Cell collection

1. Terminate the digestion by adding 2-4 mL of 10% fetal bovine serum. Transfer all cells to a collection tube.
2. Wash the wells with PBS twice to collect any remaining cells. Filter the cell suspension through a 40 µm or 70 µm cell strainer.
3. Collect the cells by centrifugation at 400 x g for 5 min at 4 °C. Resuspend the cells in 5% BSA for flow cytometry staining.

5. Flow cytometry preparation

1. Prepare spleen or peripheral blood mononuclear cells (PBMCs) from non-tumor-bearing mice as controls.
   NOTE: Optional: PBMCs from tumor-bearing mice can also be used as controls.
2. Stain cells with Fc blockers (<1 μg per million cells) and flow antibodies (CD3-APC, CD45-APC/Fire750 at a 1:100 dilution).
3. Incubate on ice in the dark for 30 min. Wash twice with cold PBS and resuspend the cells in 1% BSA in PBS.

6. Cell sorting

1. Sort CD45+CD3+ cells using a flow cytometer. Gate live cells based on FSC-A and SSC-A (P1). Then, gate single cells from P1 based on FSC-A and FSC-H (P2).
2. Finally, sort CD45+CD3+ from P2 using CD45 and CD3 gating and collect them into the collecting tubes. Ensure proper resuspension and maintenance of cell viability during sorting.

7. Cell preparation for transfer

1. Centrifuge sorted cells at 400 x g for 5 min at 4 °C. Resuspend the cells in mouse serum at a final concentration of 1 x 10⁶ cells/mL. Keep the cells on ice.
   NOTE: Ex vivo culture or activation of the sorted cells may be performed.

8. Adoptive transfer

1. Perform tumor induction for the recipient mouse 7 days before the transfer day.
2. Inject 2 x 10⁵ cells per mouse intraperitoneally into recipient mice using 29 G x 1/2" insulin syringe. Administer IL-2 (50 IU per mouse) intraperitoneally for three consecutive days.

9. Tumor monitoring

1. Perform live imaging on day 4 and day 7 post-transfer to assess tumor growth, with additional live imaging weekly with the same procedure described in step 1.8.
2. Follow up on the survival of the recipient mice. At the end of the experiment, euthanize the mouse as described above. Use ophthalmic scissors and forceps to remove the tumors, and then place them in PBS on ice for a quick measurement of tumor size and may continue the subsequent experiments as needed.

Results

The recipient mice are divided into seven groups to thoroughly evaluate the efficacy of the TIL transfer: (a) Saline control (blank control): Mice receiving saline injections to serve as a baseline. (b) IL-2 only (blank control): Mice receiving IL-2 injections to account for any effects of the cytokine alone. (c) The same single treatment as to the treated donor mice (positive control): Mice receiving the same treatment as the donor mice to compare direct treatment vs. TIL transfer. (d) T...

Discussion

Potential immunotherapy for pancreatic cancer may enhance the immune system to target and eliminate tumor cells, involving adoptive immunotherapy, immune checkpoint inhibitors, and tumor vaccines¹². TIL therapy extracts and expands lymphocytes from tumors, which are then reinfused into patients. Compared to other adoptive cell therapies, TIL therapy has diverse TCR clonality, superior tumor homing ability, and low off-target toxicity, making it advantageous for treating solid tumors

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bioluminescent imaging system	PerkinElmer	IVIS Spectrum	For monitoring tumor growth
Bovine Serum Albumin V	Solarbio	A8020	For cell resuspension
Cell strainer (40 μm or 70 μm)	Jet	CSS013040	For filtering cell suspensions
Centrifuge	Eppendorf	5810R	For cell collection and preparation
CO2 chamber	Tianjin Liliang	N/A	For euthanizing mice
Collagenase	Sigma-Aldrich	C5138	For digestion solution
CD3-APC antibody	Biolegend	100236	For flow cytometry staining
CD45-APC/Fire750 antibody	Biolegend	100154	For flow cytometry staining
C57BL/6 albino mice	Jackson Laboratory	58	Donor and recipient mice
Dispase II	Gibco	17105-041	For digestion solution
DNase I	SparkJade	AC1711	For digestion solution
D-Luciferin potassium salt	Beyotime	ST198-10g	For live imaging
Ethanol (75%)		N/A	For sterilization
Fc blockers	BD Biosciences	553142	For flow cytometry staining
Fetal Bovine Serum	Gibco	A5669701	For cell culture
Flow cytometer	BD Biosciences	FACSAria III	For cell sorting
IL-2 (Interleukin-2)	PeproTech	200-02	For administration post-transfer
Isoflurane	Shandong Ante	N/A	For narcotism
KPC-Luc cell line	PI lab generated	N/A	For inducing orthotopic pancreatic tumors
Matrigel Matrix	Corning	356234	For orthotopic implantation
PBS (Phosphate-Buffered Saline)	Servicebio	G4207-500ML	For washing and resuspending cells
RPMI 1640 medium	Gibco	11875093	For preparing digestion solution
Sterile hood	ESCO	N/A	For sterile tumor extraction and processing
Sterile scissors and forceps		N/A	For tumor extraction
Suture (4-0, PGA absorbable suture)	Jinhuan Medical	R413	For wound closure
Syringe	Jiangxi Fenglin	1 mL 0.45 × 16RWLB	For injection
Tissue culture plate 12 well	Jet	TCP001012	For tumor digestion