Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma.

Abstract Introduction: Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12) axis is a key immunosuppressive pathway where CXCL12 released by cancer-associated fibroblasts (CAF) binds to its receptor CXCR4 expressed by cytotoxic CD8+ T cells (CTL) which results in their decreased abundance within tumors. Encouraging preclinical and clinical data from mouse PDAC models and human PDAC tumor sections have shown T cell tumor accumulation after co-inhibition of CXCR4 and PD1/PDL1, however number of tumor regressions were limited. Results: To further explore the mechanism by which inhibition of the CXCR4/CXCL12 pathway modulates the immune TME in triggering an anti-tumor immune response in human PDAC, we generated an autologous co-culture system with patient-derived tumor organoids (PDTO) and peripheral blood mononuclear cells (PBMC). PDAC tumor tissue from surgical resection was used to generate lines of PDTO that recapitulate the histological and genetic characteristics of the original tumor. We demonstrate that tumor-specific reactive T cells can be obtained in PDAC by co-culture with autologous PDTO, with up to 5% of CD8+ T cells producing IFNγ after tumor organoid stimulation. No T cell reactivity was observed against normal pancreatic organoids, and blocking tumor MHC I/MHC II decreased the proportion of activated T cells. Ex vivo migration assays were established to better understand the role of CXCL12 in PDAC tumor T cell exclusion. Tumor organoids attracted autologous PBMC and addition of recombinant CXCL12 decreased PBMC migration, while treatment of PBMC with the CXCR4 inhibitor AMD3100, rescued the number of PBMCs that were able to migrate towards the tumor organoids. PBMC infiltrated PDTO-containing but not empty Matrigel domes. Addition of AMD3100 increased PBMC infiltration within the Matrigel dome containing PDTOs. Finally, we found that pre-treatment of PBMC with AMD3100 increased T cell reactivity against PDTO, and that T cell activation was further increased when stimulated with PDTO pre-treated with chemotherapy. Conclusions: We found that CXCL12 modulates the migration of T cells towards tumor organoids generated from pancreatic cancer tissue, and that inhibition of CXCR4 not only increases the migration potential, but also increases tumor-specific T cell activation. The use of ex vivo autologous co-culture and migration assays provide a unique strategy to identify modulators that may enhance T cell mediated neoplastic cell death to guide therapeutic intervention in PDAC. Citation Format: Emily Alouani, Ilenia Pellicciotta, Winston Wong, Alexander S. Thomas, Michael D. Kluger, Anna M. Chiarella, Anil K. Rustgi, Gulam A. Manji. Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-001.