Stromal HIF2 Regulated Immune Suppression in the Pancreatic Cancer Microenvironment

We used a dual recombinase mouse model to delete Hif2a in a-smooth muscle actin (aSMA)-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF-Hif2a expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, a HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade. Results. CAF-specific deletion of HIF2, but not HIF1, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = 0.0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models. Conclusions. Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease. Overall design: Examination of Hif2a deletion in aSMA-expressing cancer-associated fibroblasts to understand the role of hypoxia-inducible factors within the PDAC tumor microenvironment. Single-cell RNA sequencing allowed for the identification and deep characterization of multiple cell types in the PDAC tumor microenvironment, such that aSMA-expressing CAFs could indeed be distinguished from other cell types in the scRNA assay.本研究采用双重组酶小鼠模型，在自发性胰腺肿瘤中自发形成的、表达α-平滑肌肌动蛋白（α-smooth muscle actin, αSMA）的癌症相关成纤维细胞（cancer-associated fibroblasts, CAFs）中敲除缺氧诱导因子2α（hypoxia-inducible factor 2α, HIF2A）。通过卡普兰-迈耶分析、实时定量聚合酶链反应、组织学检测、免疫染色，以及批量RNA测序与单细胞RNA测序，评估CAFs中HIF2A表达对肿瘤进展及肿瘤微环境组成的影响。本研究采用缺氧处理及临床在研HIF2抑制剂PT2399处理后的CAFs条件培养基，体外模拟CAF与巨噬细胞的串扰信号。此外，使用同源皮下移植与原位胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）模型，评估HIF2抑制是否可增强免疫检查点阻断治疗的应答效果。 结果：仅敲除CAFs中的HIF2（而非HIF1）即可抑制胰腺导管腺癌的肿瘤进展与生长，使小鼠生存率提升50%（每组n=21~23只小鼠，对数秩检验P=0.0009）。敲除CAFs的HIF2可轻度降低肿瘤纤维化程度，并显著减少瘤内免疫抑制性M2巨噬细胞与调节性T细胞的募集。使用临床在研HIF2抑制剂PT2399处理，可显著降低体外巨噬细胞趋化与M2极化水平，并在两种同源移植胰腺导管腺癌小鼠模型中增强肿瘤对免疫治疗的应答。 结论：综上，本研究数据表明基质HIF2是胰腺导管腺癌病理发生过程中的关键组分，同时也是可靶向的治疗靶点，有望缓解胰腺导管腺癌肿瘤微环境的免疫抑制状态并增强该疾病的免疫应答。 整体实验设计：本研究通过在表达αSMA的癌症相关成纤维细胞中敲除HIF2A，探究缺氧诱导因子在胰腺导管腺癌肿瘤微环境中的作用。单细胞RNA测序可对胰腺导管腺癌肿瘤微环境中的多种细胞类型进行鉴定与深度表征，从而能够在单细胞RNA测序实验中准确区分表达αSMA的癌症相关成纤维细胞与其他细胞类群。