Abstract 19: Patient derived tumor-on-a-chip platform potentiates adaptive immune response against primary tumor cells
Authors: Schaaf Cecilia R. , Liu Tiefu , Hutchins Damian , Kooshki Mitra , Wagner Calvin , Edenhoffer Nicholas , Forsythe Steven , Greissinger Robyn , Miller Lance , Triozzi Pierre , Levine Edward , Soker Shay , Hall Adam R. , Votanopoulos Konstantinos I. 
Published: March 2024

Abstract
Background: Adoptive cellular therapy (ACT) is a promising antitumor immunotherapy showing success in select trials/patients. However, major challenges limit ACT in all tumors including insufficient cell numbers and failure to target heterogeneous tumors. To improve ACT, we developed a novel biomimetic device where patient-derived tumor organoids, enriched with antigen presenting cells, are cocultured with autologous peripheral blood mononuclear cells (PBMCs) in a microfluidic tumor-on-a-chip system (TOC). As PBMCs circulate through the device, they are exposed to dispersed tumor polyclonal neoantigens, leading to formation of patient- and tumor-specific organoid interacting lymphocytes (OILs).
Objectives: Demonstrate that a 3D tumor-on-a-chip (TOC) platform generates organoid interacting lymphocytes (OILs) with enhanced antitumor reactivity.
Methods: Specimens were collected from 15 surgical patients: melanoma (2), mesothelioma (6), and metastatic appendiceal adenocarcinoma (9). Tumor cells were combined with autologous antigen presenting cells (healthy spleen/lymph node) in a 3D matrix to generate immune-enhanced tumor organoids in the TOC system. Autologous PBMCs circulated through the system to produce OILs (7 days). Uncirculated PBMCs and tumor infiltrating lymphocytes (TILs) were cultured as controls. Resultant CD8+ controls and OILs were examined by single cell proteomics to compare cytokine secretion polyfunctionality. To assess anti-tumor reactivity, OILs and controls were reintroduced to tumor cells via coculture. Secreted bulk cytokine profiles were compared by Isoplexis CodePlex and confirmed by ELISA. NanoString Geomx spatial proteomic analysis of coculture PTOs identified key lymphocyte activity and phenotypes during anti-tumor reactivity. Coculture cytotoxic lymphocyte activation and tumor cell death was confirmed by flow cytometry. Data were analyzed using student t-test and differential expression from linear mixed-effect model.
Results: Compared to controls, CD8+ OILs showed increased polyfunctionality within single cells immediately after training, particularly in effector T-cell associated cytokine profiles (INF-γ, Granzyme B, Perforin). Upon re-introduction to PTOs, OILs excreted significantly increased inflammatory-associated proteins IFN-γ, MCP-1, and Granzyme A (p&lt;0.05). Furthermore, OIL re-exposure to tumor led to cytotoxic lymphocyte activation (CD8+CD69+) and significantly increased tumor cell death compared to control (p&lt;0.05).
Conclusion: Compared to controls, OILs produced in the TOC system show enhanced cytotoxic lymphocyte polyfunctionality and induce tumor cytotoxicity, independent of tumor type. With further validation of the OILs’ increased tumor-targeting efficacy, we propose a new potential ACT modality to target a broad array of tumors previously untreatable by current therapies.
Citation Format: Cecilia R. Schaaf, Tiefu Liu, Damian Hutchins, Mitra Kooshki, Calvin Wagner, Nicholas Edenhoffer, Steven Forsythe, Robyn Greissinger, Lance Miller, Pierre Triozzi, Edward Levine, Shay Soker, Adam R. Hall, Konstantinos I. Votanopoulos. Patient derived tumor-on-a-chip platform potentiates adaptive immune response against primary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 19.