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 cell....

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 ce.......

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 them advantageous for treating solid tumors

Materials

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