Supporting data for The impact of malignant ascites on macrophage polarization in ovarian cancer peritoneal carcinomatosis

Peritoneal carcinomatosis of epithelial ovarian cancer (EOC), driven by both the endogenous malignancy of the ovarian cancer cells and the exogenous sharping of the tumor microenvironment, is highly associated with an extremely poor prognosis among EOC patients. Clinical evidence disclosed that EOC possesses precedential metastatic tropism towards the adipose-rich omentum and accumulates lipid-enriched ascitic fluid with this progress, suggesting the malignant ascites microenvironment played a pivotal role in facilitating tumor progression and metastasis. Previous studies have suggested that lymphocyte adaptation in the ascites microenvironment not only impairs cytotoxic abilities of T cells and natural killer (NK) cells, but also exhibits tumor-supporting actions. Macrophages, the most abundant immune cells within the tumor microenvironment (TME), are often polarized into tumor-associated macrophages (TAMs). Recently, it has been revealed that TAMs fall on a dynamic spectrum of phenotypes, skewing to M2-like with trophic functions instead of M1-like with anti-tumoral properties, in the promotion of tumor growth, invasiveness, angiogenesis, metastasis and immunosuppression to facilitate tumor progression. TAMs with a high ratio of M2/M1 are therefore correlated to the poor prognosis of EOC and play a critical role in metastatic progression in numerous human cancers. However, the fundamental mechanisms inherent to TAMs polarization in the ascites microenvironment and TAMs-mediated metastatic progression remain poorly understood. In this study, functional and mechanistic characterizations of how the malignant ascites microenvironment contributes to macrophage-mediated pro-metastatic niche formation are being assessed. Results showed that RHOA-GTPase-Hippo/YAP1 signalling regulates M2-like TAMs accumulation and that targeting the Hippo/YAP1 pathway reverses M2-like TAMs polarization to an M1-like phenotype, combating peritoneal carcinomatosis of EOC. I found that the abundant levels of polyunsaturated fatty acids (PUFAs) in malignant ascites increase oxidative phosphorylation (OXPHOS) metabolism and inactivate RHOA-GTPase, leading to MST1 activation and YAP1 cytoplasmic retention in macrophages. Abolish Yap1 in macrophages <i>in vivo</i>, causing high M2/M1 TAMs polarization, suppressing CD8+ T cell infiltration and aggravating peritoneal metastatic progression. Consistently, upregulated MST1 expression and the loss of nuclear YAP1 were observed in metastatic lesions and ascites spheroids in EOC. Pharmaceutical inhibition of MST1/2 restored nuclear YAP1 expression, facilitating M2-like TAMs polarization toward the tumoricidal M1 phenotype and promoting antitumor immunity. Hence, targeting Hippo/YAP1 signalling can prevent peritoneal carcinomatosis in EOC. This study elucidates the mechanism underlying the lipid-enriched ascites microenvironment that governs M2-like TAMs polarization in peritoneal carcinomatosis. Abundant PUFAs in malignant ascites suppress RHOA-GTPase but activate Hippo/YAP1 signalling and OXPHOS metabolism, facilitating M2-like TAMs polarization. Pharmacological targeting of MST1/2 combats peritoneal carcinomatosis by reprogramming M2-like TAMs to M1-like TAMs.

上皮性卵巢癌（epithelial ovarian cancer, EOC）的腹膜播散癌，由卵巢癌细胞的内在恶性特性与肿瘤微环境的外源性塑造共同驱动，与EOC患者极差的预后密切相关。临床证据显示，EOC具有优先向富含脂肪的大网膜转移的亲嗜性，并随疾病进展积累富含脂质的腹水，提示恶性腹水微环境在促进肿瘤进展与转移中发挥关键作用。既往研究表明，腹水微环境中的淋巴细胞适应不仅会削弱T细胞与自然杀伤（natural killer, NK）细胞的细胞毒性功能，还会产生促肿瘤效应。巨噬细胞作为肿瘤微环境（tumor microenvironment, TME）中丰度最高的免疫细胞，常极化为肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）。近期研究揭示，TAMs的表型处于动态谱系之中，在促进肿瘤生长、侵袭、血管生成、转移及免疫抑制以推动肿瘤进展的过程中，偏向于具备营养支持功能的M2样表型，而非具有抗肿瘤活性的M1样表型。因此，M2/M1比值较高的TAMs与EOC不良预后相关，并在多种人类癌症的转移进展中扮演关键角色。然而，腹水微环境中TAMs极化的核心内在机制，以及TAMs介导的转移进展过程，仍有待阐明。本研究对恶性腹水微环境促进巨噬细胞介导的促转移微生态形成的功能与机制特征进行了评估。结果表明，RHOA小GTP酶（RHOA-GTPase）-Hippo/YAP1信号通路可调控M2样TAMs的聚集，而靶向抑制Hippo/YAP1通路可将M2样TAMs重极化为M1样表型，从而对抗EOC的腹膜播散癌。本研究发现，恶性腹水中高水平的多不饱和脂肪酸（polyunsaturated fatty acids, PUFAs）可增强氧化磷酸化（oxidative phosphorylation, OXPHOS）代谢并失活RHOA-GTPase，进而诱导巨噬细胞中MST1激活与YAP1胞质滞留。在体内敲除巨噬细胞的Yap1基因，会导致M2/M1比例升高的TAMs极化，抑制CD8+ T细胞浸润并加重腹膜转移进展。一致性实验结果显示，在EOC的转移病灶与腹水肿瘤球体中，均观察到MST1表达上调以及核内YAP1水平降低。通过药物抑制MST1/2可恢复核内YAP1的表达，促进M2样TAMs向具有抗肿瘤活性的M1表型极化，并增强抗肿瘤免疫。因此，靶向Hippo/YAP1信号通路可预防EOC的腹膜播散癌。本研究阐明了富含脂质的腹水微环境调控腹膜播散癌中M2样TAMs极化的潜在机制：恶性腹水中丰富的PUFAs可抑制RHOA-GTPase活性，但激活Hippo/YAP1信号通路与OXPHOS代谢，从而促进M2样TAMs的极化。通过药理学靶向MST1/2，可将M2样TAMs重编程为M1样TAMs，进而对抗EOC的腹膜播散癌。