Abstract A056: Pancreatic ductal adenocarcinoma derived cancer-associated fibroblasts suppress tumor infiltrating lymphocytes in the tumor microenvironment
Authors: Bailey Charles , Mcdonald Hannah , Reagan Anna , Cavnar Michael , Barry-Hundeyin Mautin , Pandalai Prakash , Kim Joseph 
Published: September 2024

Abstract
Patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis due to late presentation of disease and frequent resistance to therapy. Immunotherapy regimens have uniformly failed in PDAC, which has long been considered an immunologically cold tumor. It is believed that immunotherapy has been unsuccessful in PDAC due to its dense, fibrotic, and immunosuppressive microenvironment. Cancer-associated fibrosis may account for up to 90% of the PDAC microenvironment, thus further study of this immunosuppressive component is urgently needed. Methods Human PDAC surgical specimens were procured, sectioned, and allocated for histology, flow cytometry, tumor-infiltrating lymphocyte (TIL) expansion, and fibroblast culture. For histology, tumor slices were saved in 10% formalin and wax embedded. Histological PDAC slides were stained against fibroblasts markers alpha smooth muscle actin, fibroblast activation protein; and T-cell markers CD3, CD69, CD25, FOXP3. First, tumor sections were digested using mechanical and chemical techniques. For flow cytometry, digested tumor cells were stained with fluorescent conjugated antibodies targeting CD45, CD3, CD4, CD8, CD25, CD69, PD-1 and ICAM. For TIL isolation and expansion, resultant single-cells were bead-isolated for CD3 positive cells, cultured and expanded with 2000 IU interleukin-2 and 1.5 mg/mL anti-CD3/CD28 antibodies. For fibroblast culture, fibroblasts were bead isolated and sub-cultured in RPMI supplemented with FGF. TILs were co-cultured with patient-derived fibroblasts/fibroblast media with the PD-L1 inhibitor Atezolizumab. TILs were then analyzed by flowcytometry, and media from co-culture was analyzed via ELISA for gamma interferon release. Results TILs isolated from PDAC (N=5) co-cultured with patient-matched fibroblasts or media were phenotypically compared using flow cytometry and measure for gamma interferon release. Interestingly, TILs co-cultured with fibroblasts or fibroblast media showed improved expansion over peripheral blood derived control T-cells, but both groups showed highly impaired gamma interferon release. Co-cultured TILs had high levels of PD-1 and low levels of CD69, compared to control TILs, indicating a suppressed phenotype. TILs co-cultured with fibroblasts and Atezolizumab resulted in significantly increased CD69 expression and gamma interferon release (P&gt;0.05), indicating an activated phenotype. Histological PDAC slides showed abundant T-cell infiltrates with minimal CD69 expression. CD25+ T-cells were identified throughout fibrotic areas of the TME. Conclusion Here, we challenge the long-held belief that PDAC is immunologically cold as our tumor cross-sections (N=5) were rich in T-cells. More importantly, our results show that tumor-derived fibroblasts appear to play a role in suppression of TIL function, which is partially reversible with immunomodulatory techniques (e.g., anti-PDL1therapy). Our results warrant further investigation into targeting CAFs in PDAC microenvironment to improve patient response to immunotherapy.
Citation Format: Charles Bailey, Hannah Mcdonald, Anna Reagan, Michael Cavnar, Mautin Barry-Hundeyin, Prakash Pandalai, Joseph Kim. Pancreatic ductal adenocarcinoma derived cancer-associated fibroblasts suppress tumor infiltrating lymphocytes in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A056.