Role of Hypoxia in Shaping the Tumor Stroma in Pancreatic Cancer

摘要 胰腺导管腺癌（PDAC）仍然是最致命和最具侵袭性的癌症类型之一， 因此，迫切需要新的治疗策略。胰腺癌细胞被致密的间质包围 由细胞外基质和非癌细胞组成，包括成纤维细胞和免疫细胞。相互作用 肿瘤细胞和相关基质之间的相互作用对于肿瘤进展和对治疗的抗性是至关重要的。由于 由于PDAC的血管密度异常低，肿瘤细胞和基质细胞都经历缺氧， 氧气供应不足为了科普缺氧应激，肿瘤和间质区室 经历主要由缺氧诱导因子1 β和2 β（HIF 1 β和HIF 2 β）介导的适应性变化。 尽管已知PDAC严重缺氧并且缺氧预示不良的临床结果，但是 缺氧和基质HIF激活对PDAC中肿瘤-基质相互作用的影响还不完全清楚。基于 我们的初步观察表明，炎症成纤维细胞和巨噬细胞优先位于缺氧环境中， 尽管CD 8 + T细胞在很大程度上被排除在PDAC的缺氧区域之外，我们假设， 缺氧通过促进炎性成纤维细胞表型来驱动免疫抑制微环境 进而调节巨噬细胞和CD 8 + T细胞的浸润和功能。具体目标1： 确定缺氧和HIF如何调节PDAC中的炎性成纤维细胞表型， 将共培养物暴露于胰腺肿瘤细胞和成纤维细胞的三维（3D）共培养系统， 缺氧或常氧，以及使用缺乏HIF 1 α或HIF 2 α的成纤维细胞表达的小鼠模型。在特定 目的2，明确缺氧诱导成纤维细胞分泌的因子和巨噬细胞内源性缺氧 通过用条件刺激处理巨噬细胞来调节PDAC中巨噬细胞募集和功能 培养基来源于缺氧条件下的3D肿瘤细胞-成纤维细胞共培养物，并使用缺乏髓样细胞的小鼠模型 HIF 1 α或HIF 2 α的表达。在具体目标3中，我们将确定巨噬细胞和成纤维细胞如何发挥作用， 通过消耗巨噬细胞或阻断从巨噬细胞分泌的因子， 成纤维细胞在同基因原位移植肿瘤，注射缺氧探针到小鼠，并评估CD 8 + 肿瘤乏氧区和常氧区的T细胞浸润和活化。拟议的研究将共同 提供关于肿瘤-间质串扰的缺氧调节的新知识，并为肿瘤-间质串扰的最佳设计提供信息。 PDAC的基质靶向治疗策略。

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest and most aggressive cancer types, and thus new treatment strategies are urgently needed. Pancreatic cancer cells are surrounded by a dense stroma consisting of extracellular matrix and noncancerous cells, including fibroblasts and immune cells. Interactions between tumor cells and the associated stroma are critical for tumor progression and resistance to therapy. Due to the unusually low vascular density of PDAC, both the tumor and stromal cells experience hypoxia, a condition of insufficient oxygen availability. To cope with hypoxic stress, both the tumor and stromal compartments undergo adaptive changes primarily mediated by hypoxia-inducible factor 1ɑ and 2ɑ (HIF1ɑ and HIF2ɑ). Although PDAC is known to be severely hypoxic and hypoxia predicts poor clinical outcome, the effects of hypoxia and stromal HIF activation on tumor-stroma interactions in PDAC are not fully understood. Based on our preliminary observation that inflammatory fibroblasts and macrophages are preferentially located in hypoxic tumor regions whereas CD8+ T cells are largely excluded from hypoxic regions in PDAC, we hypothesize that hypoxia drives an immunosuppressive microenvironment by promoting an inflammatory fibroblast phenotype and in turn modulating infiltration and function of macrophages and CD8+ T cells. In Specific Aim 1, we will determine how hypoxia and HIFs regulate an inflammatory fibroblast phenotype in PDAC using a three- dimensional (3D) coculture system of pancreatic tumor cells and fibroblasts and exposing the coculture to either hypoxia or normoxia, as well as using mouse models lacking fibroblast expression of HIF1ɑ or HIF2ɑ. In Specific Aim 2, we will define how hypoxia-induced fibroblast-secreted factors and macrophage-intrinsic hypoxic responses regulate macrophage recruitment and function in PDAC by treating macrophages with conditioned media derived from 3D tumor cell-fibroblast cocultures under hypoxia, and using mouse models lacking myeloid expression of HIF1ɑ or HIF2ɑ. In Specific Aim 3, we will determine how macrophages and fibroblasts contribute to CD8+ T cell exclusion in hypoxic tumor niches by depleting macrophages or blocking a factor secreted from fibroblasts in syngeneic orthotopic transplanted tumors, injecting a hypoxia probe into mice, and assessing CD8+ T cell infiltration and activation in hypoxic and normoxic tumor regions. Together, the proposed studies will provide new knowledge on hypoxic regulation of the tumor-stroma crosstalk and inform the optimal design of stroma-targeting therapeutic strategies for PDAC.