PO.TB10.02 · 肿瘤生物学
Hippo/YAP1 通过 IL6/gp130/STAT3 轴介导晚期胃癌中肿瘤-基质-免疫抑制性 TME 的相互作用
Hippo/YAP1 mediated interplay of tumor-stromal-immune suppressive TME through IL6/gp130/STAT3 axis in advanced gastric cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
摘要
背景：腹膜转移（PM）发生于约 45% 的晚期胃癌（GC）患者，伴有使人衰弱的症状，中位生存期不足六个月，然而维持 GC 伴 PM（GCPM）免疫抑制性转移微环境的肿瘤-基质-免疫机制仍知之甚少。对患者来源 PM 的单细胞 RNA 测序显示，除肿瘤上皮细胞簇外，一个亚群的活化癌相关成纤维细胞（CAF）高表达 Hippo 通路共激活因子 YAP1，该表达与 CAF 活化程序及不良生存相关。我们假设 YAP1 高表达的 CAF 编排了一个 IL-6 依赖性的免疫抑制性转移微环境，且可作为治疗靶点。
方法：从 GCPM 种植灶和恶性腹水中建立原代 CAF。使用 CRISPR/Cas9 对 YAP1 进行基因敲除，并通过细胞因子芯片、多重 ELISA 和 qPCR 评估其对 CAF 分泌组的影响。采用 GC 细胞系与 YAP1 高表达或 YAP1 KO 的 CAF 共培养实验，以确定 CAF 中 YAP1 对肿瘤细胞恶性行为（包括肿瘤侵袭、生长和肿瘤球形成）的影响；将 YAP1 高表达或 YAP1 KO 的 CAF 与外周血 PBMC 或恶性腹水来源的 CD45+ 免疫细胞共培养，以评估 CAF 中 YAP1 对 T 细胞功能的影响。体内实验中，在 YAP1 高表达的患者来源异种移植（PDX）和患者来源原位（PDO）模型以及 KP-luc2 同基因 GC 模型中，使用 TEAD 抑制剂 VT00278、STAT3 抑制剂 WP1066 及其与 anti-PD-1 的联合方案，检验对 YAP1 的基因和药理学抑制。
结果：CAF 中 YAP1 敲除在体外减少了共培养肿瘤细胞的侵袭和生长，并在与 PBMC 共培养中增强了 T 细胞活化。细胞因子芯片的分泌组分析确定白细胞介素-6（IL-6）为最强 YAP1 依赖性的细胞因子，IL-6 在 YAP1 高表达的 CAF 中高表达并在恶性腹水中富集，而 IL6R/gp130 在 PM 样本内的耗竭 CD4+/CD8+ T 细胞、成纤维细胞和肿瘤细胞中上调。YAP1 缺失降低了 IL-6 的表达和分泌，而对 IL-6/gp130/STAT3 轴的药理学阻断部分模拟了 YAP1 缺失对肿瘤和 T 细胞功能的作用。正在进行的 PDX、PDO 和 KP-luc2 研究正在评估 YAP1/TEAD 与 STAT3 双重抑制、联合或不联合 PD-1 阻断，作为重塑 PM 微环境的策略。
结论：这些数据支持一个模型，即 YAP1 高表达的 CAF 驱动 GCPM 中一个 IL-6/gp130/STAT3 依赖性的免疫抑制性转移微环境。对 YAP1/TEAD 和 IL-6/STAT3 的双重靶向，可能联合免疫检查点阻断，是一种合理的新型治疗策略，将被优先用于 GC 伴 PM 患者的临床转化。
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Abstract
Background : Peritoneal metastases (PMs) occur in ~45% of patients with advanced gastric cancer (GC) and are associated with debilitating symptoms and median survival of less than six months, yet the tumor-stromal-immune mechanisms sustaining the immune suppressive metastatic niche in GC with PM (GCPM) remain poorly defined. Single-cell RNA sequencing of patient-derived PMs revealed that, in addition to tumor epithelial clusters, a subset of activated cancer-associated fibroblasts (CAFs) expresses high levels of the Hippo pathway coactivator YAP1, which associates with CAF activation programs and poor survival. We hypothesized that YAP1 high CAFs orchestrate an IL-6-dependent immunosuppressive metastatic niche that can be therapeutically targeted.
Methods : Primary CAFs were established from GCPM implants and malignant ascites. YAP1 was genetically ablated using CRISPR/Cas9, and effects on the CAF secretome were assessed by cytokine arrays, multiplex ELISA, and qPCR. Co-culture assays with GC cell lines and CAFs with YAP1 hgih or YAP1 KO to determine the effects of YAP1 in CAFs on tumor cell malignant behaviors including tumor invasion, growth and tumor sphere formation; Co-culture of CAFs with YAP1 hgih or YAP1 KO with PBMC from peripheral blood or malignant ascites-derived CD45 + immune cells were used to evaluate the effects of YAP1 in CAFs on T-cell function. In vivo, genetic and pharmacologic YAP1 inhibition were tested in YAP1 high patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models and in the KP-luc2 syngeneic GC model using the TEAD inhibitor VT00278, the STAT3 inhibitor WP1066, and their combinations with anti-PD-1.
Results : YAP1 knockout in CAFs reduced invasion and growth of co-cultured tumor cells in vitro and enhanced T-cell activation in co-culture with PBMC. Secretome profiling by cytokine array identified interleukin-6 (IL-6) as the most strongly YAP1-dependent cytokine, IL-6 was highly expressed in YAP1 high CAFs and enriched in malignant ascites, whereas IL6R/gp130 were upregulated in exhausted CD4 + /CD8 + T cells, fibroblasts, and tumor cells within PM samples. YAP1 depletion decreased IL-6 expression and secretion, and pharmacologic blockade of the IL-6/gp130/STAT3 axis partially phenocopied the effects of YAP1 loss on tumor and T-cell function. Ongoing PDX, PDO, and KP-luc2 studies are evaluating dual YAP1/TEAD and STAT3 inhibition, with or without PD-1 blockade, as a strategy to remodel the PM microenvironment.
Conclusions : These data support a model in which YAP1 high CAFs drive an IL-6/gp130/STAT3-dependent immunosuppressive metastatic niche in GCPMs. Dual targeting of YAP1/TEAD and IL-6/STAT3, potentially combined with immune checkpoint blockade, represents a rational novel therapeutic strategy that will be prioritized for clinical translation in GC patients with PM.
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利益披露 Disclosure
J. Zhao, None..
X. Yao, None..
Y. Fan, None..
D. Athavale, None..
Y. Zhang, None..
F. Spitz, None..
G. Grana, None..
J. A. Ajani, None..
S. Song, None.