Abstract 5850: Development and characterization of patient-derived pancreatic fibroblasts to study CAF heterogeneity
Authors: Dwivedi Nidhi V. , Gautam Shailendra K. , Rachagani Satya , Venkata Ramakanth , Dalal Vipin , Jain Maneesh , Batra Surinder K. 
Published: April 2023

Pancreatic ductal adenocarcinoma (PDAC) has complex tumor microenvironment (TME), characterized by phenotypically and functionally heterogenous cancer-associated fibroblasts (CAFs). Different CAF subtypes have been identified that evidently contribute to desmoplasia, immunosuppression, and therapy response variably. The lack of models to study their plasticity and heterogeneity reproducibly poses a major limitation. We, thus, describe the development and characterization of a panel of immortalized patient-derived pancreatic fibroblasts (iPDPFs), studying their impact on PDAC cell lines in vitro and in vivo.

Method: We developed six iPDPF lines using human telomerase reverse transcriptase (hTERT) from cancer-adjacent normal pancreas (9-26-NP), chronic pancreatitis (CPP1), and PDAC CAFs (9-17-P, 10-15-P, 10-03-P, & 10-32-P). Immunoblotting, bulk-RNAseq, and qPCR were performed for their molecular characterization. Functional characterization was done in vitro using collagen contraction assay and co-culture studies with COLO357. The impact of iPDPFs on the tumor cell behavior in vivo was studied by their co-implantation with COLO357 in athymic nude mice.

Results: The iPDPFs exhibited variable expression of activation, inflammatory, and ECM markers indicative of heterogeneity across the panel. While CAFs expressed high levels of αSMA (alpha-smooth muscle actin) and fibroblast-specific protein-1 (FSP1), the expression of these markers was low in 9-26 NP & CPP-1. Also, the iPDPFs variably expressed TGFβ, PDGFRβ, VDR, COL1A, and small GTPases. High expression of inflammatory markers was observed in 9-26 NP, CPP-1, & 10-32-P suggesting their iCAF type while 9-17-P, 10-03-P, and 10-15-P were of myCAF type. These observations were recapitulated in the transcriptomic analysis. Interestingly, the marker expression changed with passage suggesting existence of plasticity in the iPDPFs. 9-17-P induced highest collagen contraction (47.9%) while 10-15-P induced the least contraction (25.89%). Co-culture of iPDPFs with COLO357 enhanced the expression of ROCK-1. In vivo, iPDPFs resulted in stroma-rich tumors; however, the extent of stroma and growth rate were variable across iPDPF lines and implantation sites. 9-17-P xenograft tumors exhibited the highest percent αSMA (14.5%), and FSP1 (30.7%) positive cells. Limited analysis showed that iPDPF co-implanted tumors exhibited variable vascularity and ECM composition suggesting that the heterogeneity observed in vitro was manifested in vivo.

Conclusion: Overall, the iPDPFs recapitulate pancreatic fibroblasts heterogeneity, reflecting the overlapping nature of CAF subtypes. Also, the iPDPFs are functionally variable and plastic in vitro. Importantly, these fibroblasts exert variable effects on the tumor cells in vivo. The iPDPFs can thus serve as a useful model to reproducibly study CAF heterogeneity and plasticity in PDAC.

Citation Format: Nidhi V. Dwivedi, Shailendra K. Gautam, Satya Rachagani, Ramakanth Venkata, Vipin Dalal, Maneesh Jain, Surinder K. Batra. Development and characterization of patient-derived pancreatic fibroblasts to study CAF heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5850.