Abstract 4051: Microfluidic incubation of patient derived tumor and immune cells boosts lymphocyte cytotoxic phenotype
Authors: Hutchins Damian C. , Schaaf Cecilia R. , Edenhoffer Nicholas P. , Kooshki Mitra , Greissenger Robyn , Forsythe Steven D. , Hall Adam R. , Miller Lance D. , Soker Shay , Triozzi Pierre L. , Votanopoulos Konstantinos 
Published: April 2023

Abstract
Introduction: Tumor infiltrating lymphocytes (TILs), hold promise in advancing adoptive cell therapy for patients with otherwise limited treatment options. However, many tumors do not attract TILs, or the TILs themselves are exhausted which limits treatment availability and efficacy. Consequently, there is a need for alternative sources of tumor reactive T cells. Here, we address this need through the ex vivo 3D cell culture biofabrication of immune competent patient tumor organoids (iPTOs). These constructs, composed of patient-matched cancer cells, antigen presenting cells (APCs), and stromal cells encapsulated in extracellular matrix (ECM)-like hydrogel, closely mimic the in vivo tumor microenvironment. We demonstrate that constant circulation of peripheral blood mononuclear cells (PBMCs) through a microfluidic device housing iPTOs can emulate lymph node activation of immune cells to the tumor, yielding organoid interacting lymphocytes (OILs) that possess increased markers of activation, cytotoxicity, proliferation, homing markers, and inflammatory cytokine production relative to uncirculated PBMCs.
Methods: Tumor tissue, APCs (from lymph nodes or spleen), and peripheral blood were collected from 8 patients with mesothelioma (3), melanoma (2), and appendiceal cancer (3). Chips were built from a glass slide and laser cut Polymethyl methacrylate. IPTOs were made such that a concentration of 3 APCs for every 1 tumor cell were added to a collagen and hyaluronic acid mixture that was then photocrosslinked in the chip chamber. PBMCs were circulated through chips housing the photocrosslinked iPTOs for 7 days and then expanded. Following expansion, a subpopulation of these cells were cocultured with tumor only organoids for a 7 day period. Single and secondary tumor exposure populations of effector, memory, and reactive T cells were analyzed via flow cytometry, immunohistochemistry, and cell culture medium proteomic analysis, and compared with both uncirculated PBMCs and patient TILs.
Results: Data collected from patients with appendiceal, melanoma, and mesothelioma tumors shows generation of OILS with increase in cytotoxic T lymphocyte, effector memory, and central memory phenotypes when compared to uncirculated PBMCs, greater than or comparable to TILs and with similar effector cytokine and enzyme expression. OILs were also found to express less immunosuppressive signals (i.e., Tim3, PD1, and IL 10). Critically, chip-based T cell activation resulted in 10x more viable OILs than were typically observed with TILs following parallel growth period of 14 days.
Conclusions: This data suggests that microfluidic incubation of lymphocytes with lymph node and tumor cells can generate large amounts of viable tumor-specific T cells on-demand with characteristics similar to TILs but with increased viability, cell expansion propensity, and cytotoxic responsiveness.
Citation Format: Damian C. Hutchins, Cecilia R. Schaaf, Nicholas P. Edenhoffer, Mitra Kooshki, Robyn Greissenger, Steven D. Forsythe, Adam R. Hall, Lance D. Miller, Shay Soker, Pierre L. Triozzi, Konstantinos Votanopoulos. Microfluidic incubation of patient derived tumor and immune cells boosts lymphocyte cytotoxic phenotype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4051.