Abstract B017: NINJA PDAC: A robust murine pancreatic cancer organoid transplant model with inducible neoantigens for studying tumor microenvironment interactions with anti-tumor T-cell immunity
Authors: Jacox Jeremy B. , Foster Gena G. , Shah Dhruvi , Joshi Nikhil S. , Muzumdar Mandar D. 
Published: January 2024

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is highly lethal due to early metastasis and poor therapeutic response including to anti-PD1/CTLA4-based immunotherapy, characteristics attributed to a desmoplastic tumor microenvironment (TME) and low immunogenicity. However, long-term human PDAC survivors exhibit higher neoantigen expression with active T-cell immunogenicity, and most human PDAC express predicted neoepitopes, arguing that T-cell mediated anti-tumor immunity shapes PDAC progression. By contrast, tumors in classical pre-clinical (KRASLSL-G12D; TRP53F/F; pancreas-specific Cre, or KPC) murine models exhibit low neoantigen burden, are non-immunogenic, with growth kinetics independent of T cells, making them poorly suited to study T-cell immunity. Combined chemoimmunotherapy regimens for human PDAC might be developed more efficiently via preclinical studies in murine models with physiologic stroma, defined neoantigens that can be induced in established tumors, and measurable neoantigen-specific T-cell responses. We developed NINJA PDAC, a novel, robust transplantable murine pancreatic cancer organoid model, to study neoantigen-specific anti-tumor T-cell responses and how they are influenced by the TME. The NINJA (Damo et al., 2021) system allows for spatially (Cre) and temporally (doxycycline+tamoxifen) controlled inducible expression of LCMV-derived neoantigens with low-to-moderate MHCI/II-binding affinity (GP33 and GP66, respectively) comparable to human PDAC, in Kras-mutated, p53-deficient PDAC organoids. Transplanted NINJA PDAC organoids engraft robustly both subcutaneously and orthotopically in syngeneic mice, with reproducible kinetics. Unlike 2D cell line transplants, these organoid-derived tumors have a complex ductal architecture, immune islands, and fibrotic stroma more representative of human PDAC. T-cell depletion studies demonstrated that full rejection of tumors with constitutive neoantigen expression (NINJA ‘ON’) requires both CD4+ and CD8+ neoantigen-specific T-cells, allowing the study of the roles of both helper and cytotoxic T-cells in anti-PDAC immunity. ‘Poised’ NINJA PDAC organoids do not express neoantigen until induction, and we confirmed high tumor neoantigen expression can be induced with doxycycline/tamoxifen in vivo (38%±18% neoantigen-expressing tumor cells). Early induction in vivo leads to intratumoral CD3+ T-cell infiltration, with increased neoantigen-specific (H2Db/gp33-41-tetramer positive) T-cells in tumors and tumor-draining lymph nodes. Late induction in vivo leads to neoantigen induction, but more muted T-cell infiltration and minimally attenuated tumor growth, suggesting a role of an established TME in suppressing T-cell responses. NINJA PDAC is a promising model that affords a high-fidelity toolset to study how murine neoantigen-specific anti-tumor T-cell responses are perturbed by the TME and further altered by host variables (such as obesity) that modulate the TME with the potential to expedite the development of new combination immunotherapies.
Citation Format: Jeremy B. Jacox, Gena G. Foster, Dhruvi Shah, Nikhil S. Joshi, Mandar D. Muzumdar. NINJA PDAC: A robust murine pancreatic cancer organoid transplant model with inducible neoantigens for studying tumor microenvironment interactions with anti-tumor T-cell immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B017.