Hypoxic regulation of macrophage migration and function via fibroblast reprogramming in pancreatic cancer

项目摘要 胰腺导管腺癌（PDAC）是美国癌症相关死亡的第三大原因。 尽管仅占所有癌症病例的2%。PDAC的特点是广泛的 纤维炎性基质和缺氧微环境，这有助于疾病进展， 治疗抵抗巨噬细胞和癌症相关成纤维细胞（CAF）是主要的细胞类型 在PDAC基质中。巨噬细胞是PDAC中的主要免疫抑制群体。这些细胞 因此，它们的环境在调节它们的功能方面起着重要作用。巨噬 适应缺氧，一种低氧可用性的条件，主要是通过稳定易氧化的 低氧诱导因子（hypoxia inducible factors，HIF）。尽管PDAC极度缺氧， 肿瘤细胞和基质细胞经历缺氧，缺氧和HIF对巨噬细胞及其 在PDAC中与其他基质细胞的通讯在很大程度上仍然未知。我们小组最近发现， 缺氧促进炎症性癌相关成纤维细胞（炎性CAF）的获得，最近的研究表明， 定义的PDAC成纤维细胞亚型，其产生高水平的炎性细胞因子和趋化因子。通过 将缺氧探针注射到携带PDAC的小鼠中，我们观察到炎性CAF和巨噬细胞 与常氧区域相比，T细胞主要存在于低氧肿瘤区域，而T细胞主要存在于低氧肿瘤区域。 排除在缺氧肿瘤区域之外。基于这些初步数据，我假设炎症成纤维细胞 低氧诱导的巨噬细胞浸润促进低氧肿瘤区域， 免疫抑制巨噬细胞表型。将按照以下两个目的对这一假设进行研究： (1)确定缺氧诱导的成纤维细胞分泌因子在调节巨噬细胞迁移中的作用， 在PDAC中的功能和（2）确定巨噬细胞HIF在调节巨噬细胞迁移和功能中的作用 在PDAC。为了完成目标1，我将用来自单核细胞和共核细胞的条件培养基培养巨噬细胞。 胰腺肿瘤细胞和成纤维细胞在缺氧或常氧条件下培养，然后评估 免疫抑制性巨噬细胞标志物的表达以及它们的迁移。为了完成目标2，我将 用新鲜培养基或条件培养基在低氧条件下培养HIF 1 α和HIF 2 α缺陷的巨噬细胞 来自胰腺肿瘤细胞和成纤维细胞在缺氧下的共培养物。然后我会评估 这些巨噬细胞中的免疫抑制标志物，并评估其迁移。最后，我将定义 巨噬细胞HIF介导无髓系小鼠原位胰腺肿瘤发生 HIF 1 α或HIF 2 α的表达。我的建议将提供新的见解缺氧如何促进 免疫抑制PDAC微环境通过调节巨噬细胞成纤维细胞串扰，并最终 为克服对免疫疗法的耐药性提供信息。

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States despite representing only 2% of all cancer cases. PDAC is characterized by an extensive fibroinflammatory stroma and a hypoxic microenvironment, which contributes to disease progression and therapeutic resistance. Macrophages and cancer-associated fibroblasts (CAFs) are the predominant cell types within the PDAC stroma. Macrophages are a major immunosuppressive population in PDAC. These cells are highly plastic and, as a result, their environment plays an important role in regulating their function. Macrophages adapt to hypoxia, a condition of low oxygen availability, primarily through the stabilization of oxygen-liable transcription factors called hypoxia-inducible factors (HIFs). Although PDAC is extremely hypoxic and both the tumor cells and stromal cells experience hypoxia, the effects of hypoxia and HIFs on macrophages and their communication with other stromal cells in PDAC remain largely unknown. Our group has recently found that hypoxia promotes the acquisition of inflammatory cancer-associated fibroblasts (inflammatory CAFs), a recently defined PDAC fibroblast subtype that produces high levels of inflammatory cytokines and chemokines. By injecting a hypoxia probe into mice bearing PDAC, we have observed that inflammatory CAFs and macrophages predominantly reside in hypoxic tumor regions compared with normoxic regions whereas T cells are largely excluded from hypoxic tumor areas. Based on these preliminary data, I hypothesize that inflammatory fibroblasts induced by hypoxia promote macrophage infiltration into hypoxic tumor regions and facilitate an immunosuppressive macrophage phenotype. This hypothesis will be investigated with the following two Aims: (1) to determine the role of hypoxia-induced fibroblast-secreted factors in regulating macrophage migration and function in PDAC and (2) to define the role of macrophage HIFs in regulating macrophage migration and function in PDAC. To complete Aim 1, I will culture macrophages with conditioned media derived from mono- and co- cultures of pancreatic tumor cells and fibroblasts under either hypoxia or normoxia, and then assess the expression of immunosuppressive macrophage markers as well as their migration. To complete Aim 2, I will culture HIF1α-deficient and HIF2α-deficient macrophages under hypoxia with fresh media or conditioned media from co-cultures of pancreatic tumor cells and fibroblasts under hypoxia. Then I will assess the expression of immunosuppressive markers in these macrophages and evaluate their migration. Finally, I will define the role of macrophage HIFs in mediating pancreatic tumorigenesis using orthotopic tumors from mice lacking myeloid expression of HIF1ɑ or HIF2ɑ. My proposal will provide new insights into how hypoxia promotes an immunosuppressive PDAC microenvironment by modulating the macrophage-fibroblast crosstalk, and ultimately inform strategies to overcome resistance to immunotherapy.