Abstract B52: Effects of tissue site and antigenicity on KPC-derived pancreatic tumor growth and response to combination immunotherapy
Authors: Ager Casey R. , Curran Michael A. 
Published: February 2019

Abstract
Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to checkpoint blockade therapy is commonly attributed to its status as an immunologically cold tumor. However, it remains to be determined whether the lack of endogenous immunity to PDAC is predominantly due to a paucity of targetable neoantigens or to the unique tolerogenic environment of malignant pancreatic tissue. Using an implantable model derived from the Kras+/LSL-G12DTrp53+/LSL-R172HPdx1-Cre (KPC) mouse, we investigated how tissue implantation site and exogenous antigen presentation influenced immune surveillance of KPC tumors and response to combination immunotherapy.
Methods: The KPC organoid-derived cell line mT4-2D was stably transduced with various expression constructs including luciferase (mT4-Luc), ovalbumin (mT4-OVA), or mCherry (mT4-Cherry). Each line was implanted subcutaneously (2.5x105 cells) or orthotopically at indicated doses to assess effects of exogenous antigen expression on engraftment efficiency. To evaluate how tissue environment governs response to therapy, mice were co-implanted with mT4-Luc at both orthotopic and subcutaneous sites and were exposed to combination therapy consisting of intratumoral STING agonist ML-RR-CDA (10ug/mouse) and checkpoint antagonists αCTLA-4 and αPD-1 beginning on day 14 post-implantation. Tumor growth at both sites was monitored weekly by IVIS bioluminescent imaging and mice were followed for survival, or tumors were harvested on day 25 for analysis of immune infiltrates by flow cytometry.
Results: mT4 tumors grew aggressively at both subcutaneous and orthotopic sites, with doses as low as 3.5x104 causing mortality within 3 weeks post-implantation. However, expression of either OVA or mCherry resulted in spontaneous clearance in 100% of mice, regardless of implantation site. Luciferase expression yielded progressing tumors, and ex vivo peptide restimulation failed to identify Luciferase-specific T cells within mT4-Luc challenged mice; suggesting that Luciferase is a minimally immunogenic protein in C57BL/6 mice. Despite the poor immunogenicity of the mT4 model, we found that intratumoral injections of ML-RR-CDA delayed growth of subcutaneously-implanted mT4, and that concomitant administration of αCTLA-4/αPD-1 induced complete clearance in ~40% of mice. In contrast, intratumoral delivery of ML-RR-CDA was ineffective against orthotopic mT4 tumors, and combination therapy with αCTLA-4/αPD-1 achieved only modest delays in tumor progression. Flow cytometric analysis of tumor infiltrating immune populations in mice with co-implanted orthotopic and subcutaneous lesions revealed dichotomies in the density of CD8 T cells, CD4 effector T cells, and dendritic cell populations between tissue sites in response to local therapy.
Conclusions: Although implantable cell lines isolated from KPC tumors are highly aggressive and resistant models of PDAC, we found that engraftment and growth of mT4-2D was completely negated upon forced expression of a single foreign protein antigen. This suggests that the lack of targetable mutanome products may explain the resistance of KPC and potentially human PDAC tumors to immunotherapy. However, when implanted subcutaneously, mT4-2D was sensitive to in situ STING activation in combination with checkpoint blockade, which indicates that immune responses against tissue-specific antigens are potentially sufficient to control growth of KPC tumors when removed from the suppressive pancreatic environment. Flow analysis revealed a greater capacity of CD8 T cells, CD4 T cells, and dendritic cells to infiltrate subcutaneous vs orthotopic tumors following intratumoral therapy, reinforcing the theory that therapies which enhance infiltration of select immune populations are needed in order to sensitize PDAC tumors to therapy.
Citation Format: Casey R. Ager, Michael A. Curran. Effects of tissue site and antigenicity on KPC-derived pancreatic tumor growth and response to combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B52.