Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell–cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

中文翻译：

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前列腺肿瘤微环境的综合表征将 CXCR4/CXCL12 串扰确定为前列腺癌的新型抗血管生成治疗靶点

肿瘤细胞和基质细胞之间的串扰促进了前列腺癌 (PCa) 的进展和传播。对细胞间通信网络的洞察为促进 PCa 肿瘤微环境 (TME) 改变的模具过程提供了新的治疗途径。在这里，我们对 PCa 肿瘤内皮细胞 (TEC) 进行了详细表征，以描绘 TEC 和 PCa TME 之间的细胞间串扰。从 67 个新鲜的根治性前列腺切除术 (RP) 标本中分离出的 TEC 进行了多组学离体表征，并通过免疫组织化学 (IHC) 和免疫荧光 (IF) 对 TEC 功能和关键标志物进行了正交验证。为了识别 TEC 中的细胞-细胞相互作用靶点，我们对四名接受 RP 的 PCa 患者进行了单细胞 RNA 测序 (scRNA-seq)，以对细胞 TME 成分进行分类。使用 IHC、公开可用的数据集、细胞培养实验以及 PCa 异种移植小鼠模型对目标进行了交叉验证。与相邻的正常内皮细胞 (NEC) 相比，大量 RNA-seq 分析显示与 TEC 中的肿瘤脉管系统、胶原蛋白修饰和细胞外基质重塑相关的基因上调。PTGIR、PLAC9、CXCL12 和 VDR 被确定为 TEC 标志物，并在独立患者队列中通过 IF 和 IHC 确认。通过 scRNA-seq，我们确定了 27 种细胞（亚）类型，包括具有动脉、静脉和未成熟特征的内皮细胞 (EC)，以及血管生成尖端 EC。一项重点分子分析显示，与所有其他 TME 细胞（亚）群相比，动脉 TEC 表现出最高的 CXCL12 mRNA 表达水平，并显示出负面的预后作用。受体-配体相互作用分析预测动脉 TEC 衍生的 CXCL12 及其同源受体 CXCR4 在血管生成尖端 EC 上的相互作用。通过突出 CXCR4 抑制剂 AMD3100 在小鼠 PCa 中的血管数量和密度降低活性以及体外 TEC 增殖和迁移的抑制作用，CXCL12 在体外和体内被验证为可操作的 TEC 靶标。总体而言，我们的综合分析确定了新的 PCa TEC 靶点，并强调 CXCR4/CXCL12 相互作用是干扰 PCa 中肿瘤血管生成的潜在新靶点。通过突出 CXCR4 抑制剂 AMD3100 在小鼠 PCa 中的血管数量和密度降低活性以及体外 TEC 增殖和迁移的抑制作用，CXCL12 在体外和体内被验证为可操作的 TEC 靶标。总体而言，我们的综合分析确定了新的 PCa TEC 靶点，并强调 CXCR4/CXCL12 相互作用是干扰 PCa 中肿瘤血管生成的潜在新靶点。通过突出 CXCR4 抑制剂 AMD3100 在小鼠 PCa 中的血管数量和密度降低活性以及体外 TEC 增殖和迁移的抑制作用，CXCL12 在体外和体内被验证为可操作的 TEC 靶标。总体而言，我们的综合分析确定了新的 PCa TEC 靶点，并强调 CXCR4/CXCL12 相互作用是干扰 PCa 中肿瘤血管生成的潜在新靶点。