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

We focus on developing a method for isolating and adoptively transferring tumor-infiltrating lymphocytes, TILs, in a pancreatic cancer model. We are trying to answer whether the subsets of TILs can effectively target and kill pancreatic tumors in vivo and how T cells can be optimized for future clinical trials. Recent development in adoptive cell therapy, including enhancing TIL isolation and expression techniques, gently modifying TILs to improve their targeting and and the combining TIL therapy with other immunotherapies like checkpoint inhibitors or targeted therapies for enhanced antitumor efficacy.

We have established a robust method for isolating and expanding tumor-reactive TILs from pancreatic cancer mouse model. We demonstrated enhanced anti-tumor efficacy of adoptively transferred tumor-reactive T cells. Our protocol offers a more targeted approach to adoptive cell therapy by enabling the isolation and the selection of tumor-specific TILs compared to other less specific methods.

It also uses by bioluminescent imaging for noninvasive tumor monitoring. Our findings will advance the field by providing a standardized preclinical model for evaluating TIL therapy efficacy and optimizing treatment parameters of T-cell subsets for pancreatic cancer. To prepare a KPC-Luciferase cell suspension for inducing an orthotopic pancreatic tumor in mice, trypsinize the KPC-Luciferase cell line for one minute at 37 degrees Celsius.

After trypsinization, add PBS, transfer the cells, and then centrifuge the cell suspension. Count the cells and resuspend them at a concentration of 1 times 10 to the power of 6 cells per milliliter in PBS containing 30%basement membrane matrix. Place the anesthetized mouse on an operating platform.

After making a 0.5 to 1 centimeter left abdominal incision, hold the spleen using forceps and expose the pancreas. After preparing the KPC-Luciferase cells and basement membrane matrix mix, inject 50 microliters of the cell suspension containing 50, 000 cells into the pancreas using a 29 gauge needle. Return the pancreas into the abdominal cavity.

Sequentially suture the peritoneum and skin. Allow the mouse to recover on a warm pad until fully awake. After seven days, intraperitoneally inject the mouse with 60 microliters of D-Luciferin potassium salt solution for live imaging.

Following 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 naturally from anesthesia. To begin, place the donor euthanized mouse on an operating platform.

Sterilize the entire body with 75%ethanol. In a sterile hood, use scissors and forceps to open the abdominal cavity. Remove the tumor and place it in PBS on ice.

Measure the tumor size with a Vernier caliber. Then, calculate the tumor volume using the onscreen formula. After preparing the 1X digestion solution using collagenase, Dispase II, and DNAse I in RPMI 1640, add one to two milliliters of the solution per well in a 12-well plate.

Mince the tumors into small pieces using scissors. Transfer the tumor pieces into each well of a 12-well plate and digest them at 37 degrees Celsius while shaking at 70 RPM for 30 minutes. To terminate the digestion, add one to two milliliters of 10%fetal bovine serum and transfer the cell suspension to a collection tube.

Wash the wells of a 12-well plate twice with PBS to collect any remaining cells. Filter the cell suspension through a 70 micrometer cell strainer, mince and crush the tissue fragments to release the single cells using the flat end of a syringe plunger. Centrifuge the filtrate at 400 G for five minutes at four degrees Celsius, and resuspend the pellet in 5%bovine serum albumin for flow cytometry staining.

To begin, place the cells isolated from the tumor-bearing mouse along with the spleen or peripheral blood mononuclear cells from the non-tumor-bearing mouse on a working platform. Stain cells with FC blockers and flow antibodies at a 1 to 100 dilution. Incubate the tubes on ice in the dark for 30 minutes.

After incubation, wash the cells twice in PBS by centrifugation before resuspending them in 1%bovine serum albumin in PBS. On a flow cytometer, to sort CD45+CD3+cells, gate live cells based on FSC-A and SSC-A to create gate P1.Then, gate single cells from P1 based on FSC-A and FSC-H to create gate P2.Sort CD45+CD3+cells from P2 using CD45 and CD3 gating. Collect the sorted cells into collection tubes.

Centrifuge sorted CD45+CD3+cells at 400 G for five minutes at four degrees Celsius. Resuspend the cells in mouse serum at a concentration of 1 times 10 to the power of 6 cells per milliliter, and place them on ice. Place the tumor-induced recipient mouse on an operating platform.

Using a 29 gauge 1/2-inch insulin syringe, intraperitoneally inject the sorted immune cells. Then, administer interleukin-2 intraperitoneally for three consecutive days.