Background
Pancreatic cancer (PDAC) is considered an immunologically cold cancer type. This perception is due to its rapid progression, strong presence of immunosuppression and lack of response to current immunotherapies. However, we and others have shown that a significant proportion of PDAC patients do contain robust lymphocyte infiltration, often aggregated in the form of tertiary lymphoid structures (TLS). The presence of TLS in primary PDAC tumors is prognostic for long-term survival and in other cancer types, predictive of response to immunotherapy. Despite the clinical benefit to generating TLS in tumors, why they form in some PDAC patients but not others, remains an unanswered question. Reticular fibroblast subsets are critical to regulating lymphocyte aggregation and follicular retention in lymph nodes, but cancer associated fibroblasts (CAF) in PDAC tumors are predominantly TGF-beta induced myofibroblasts (myCAF) phenotypes with immunosuppressive and pro-tumorigenic properties. To that end, we hypothesized that repolarizing CAF programming may improve lymphocyte recruitment and TLS formation
Methods
We tested mouse fibroblast cultures for their response to various inflammatory modulators. We treated fibroblasts with TGFb1 and TGFbR1 inhibitor prior to treatment with pro-inflammatory ligands. Gene expression for select chemokines and myCAF genes were assayed with RT-PCR. Protein expression was assayed by flow cytometry and ELISA. Using cell lines derived from KPC mice, we induced orthotopic tumors followed by treatments with agonist LTBR antibody, TGFbR1 inhibitor and chemotherapy. At study end-point, tumors and peripheral blood were harvested for flow cytometry and immunohistochemistry to characterize the CAF phenotype and immune infiltrate.
Results
Treatment of fibroblasts with TNFa and anti-LTBR agonist resulted in upregulation of VCAM1, CXCL13, and CCL19 and increase B and T cell migration to activated fibroblasts. TGFb treatment repressed upregulation of VCAM1, CXCL13 and CCL19 while upregulating myCAF programming. PDAC tumor-bearing mice treated with TGFbR1 inhibitor, LTBR agonists, and chemotherapy increased T and B cell infiltration resulting in TLS-like lymphocyte aggregates and tumor stasis. This associated with a repolarization of myCAF phenotypes to chemokine-expressing reticular CAF subsets.
Conclusions
CAF comprise a disproportionate fraction of PDAC tumor cellularity resulting in fibrosis and immunosuppression driven by TGFb. Here we demonstrate that relieving TGFbR1 signaling in CAF allows synergy between the pro-inflammatory mediators TNFa and LTBR to upregulate lymphocyte recruiting chemoattractants thereby enhancing T and B cell recruitment, TLS formation, and greater tumor control. Future studies will seek to understand the molecular crosstalk between these signaling pathways and characterize novel mediators of this response.
Acknowledgements
This work was supported by NCI R01CA286017-01 and the Pelotonia Institute of Immuno-Oncology at the Ohio State Comprehensive Cancer Center.