Abstract
Abstract Introduction (or Objective): Pancreatic ductal adenocarcinoma (PDAC) is characterized by having an extensive desmoplastic stroma that contributes to its resistance to therapeutic treatments resulting in a poor prognosis. Cancer associated fibroblasts (CAFs) are the most prevalent stromal cells within the pancreatic tumor microenvironment (TME), making up roughly 70-80% of the tumor volume. Pancreatic tumors are extremely hypoxic due to this dense stromal infiltration. We previously showed that CAFs in PDAC regulate macrophage activity and polarization in a HIF2-dependent manner, revealing the importance of hypoxia signaling in stromal cells. PDAC-CAFs also participate in cross talk with cancer cells inducing cancer malignancy, angiogenesis, metastasis, and drug resistance. In this study, we utilized a novel mouse model to produce genetically defined CAFs to study how HIFs (including HIF1a, HIF2a, and their binding partner ARNT) contribute to the function of CAFs in the pancreatic cancer TME. Methods: LSL-tdTomato mice were obtained from Jackson laboratories. TdTomato mice were bred with aSMACreERT2/+ mice, and their progeny were bred with Hif1afl/fl, Hif2afl/fl and ARNTfl/fl mice to produce LSL-tdTomato; aSMACreERT2/+; HIF1afl/fl (HIF1-KO)/HIF2afl/fl (HIF2-KO)/ARNTfl/fl (ARNT-KO) mice, respectively. KPC tumor cells (KrasG12D;Trp53R172H) were then injected orthotopically into the pancreas of these mice. After one month, tumors were harvested and digested to allow for CAF isolation. Digested single cells were then sorted for tdTomato expression to specifically isolate aSMA+ CAFs. All mice are on a C57BL/6J genetic background. Results: We isolated genetically defined CAFs lacking either HIF1, HIF2 and ARNT in a syngeneic orthotopic tumor model. After dissociating solid tumors and isolating tdTomato+ cells, we isolated early passage CAFs as verified by expression of fibroblast markers (ACTA1, COL1A1, FN1, etc.) without epithelial markers (EPCAM, KRT19). Genetic loss of HIF1, HIF2 or ARNT was verified via western blot and qPCR analysis. Conclusion: We created a Cre-driven model to specifically label CAFs and genetically manipulate hypoxia signaling that can be used with any desired promoter. This model allows the isolation and characterization of spatially and temporally genetically defined stromal populations, which may enable downstream syngeneic transplantation. We believe that this powerful system could be used to interrogate each stromal component in a definitive fashion without the need for more complex multiallelic mouse models. Citation Format: Sahar B. Fattani, Matthew T. Cribb, Abagail M. Delahoussaye, Nefetiti P. Mims, Natividad R. Fuentes, Cullen M. Taniguchi. Producing genetically defined CAFs for the study of hypoxia signaling in the pancreatic cancer TME [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 2347.
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