Sexual dimorphism and the immuno-modulatory role of estrogen signaling in HNSCC

摘要 头颈癌(HNSCC)主要困扰男性。患上HNSCC的女性患者往往是 绝经后，使用绝经激素治疗可降低罹患该病的风险。我们证明了 绝经前女性HNSCC患者对包括免疫治疗(IO)在内的治疗的反应有所改善。 使用临床试验数据，我们显示对IO和睾酮的反应没有差别。同样，我们的临床前数据 性腺去势没有显示IO反应效果。但0.1毫克雌二醇(E_2)可改善去势后的反应 雄性小鼠对雌性小鼠的影响。我们的总体目标是了解绝经前女性激素是如何 E2有助于病情进展和治疗耐药。我们的数据显示，雌二醇对癌细胞没有影响 活性，表明肿瘤微环境(TME)是作用部位。众所周知，雌激素2在 自身免疫和炎症，在那里它调节树突状细胞(DC)和调节性T细胞(Treg) 分化和功能，淋巴管成熟。这些免疫细胞群调节反应 对于HNSCC的免疫治疗，但它们是如何受性激素影响的，在很大程度上仍不清楚。我们的 临床前数据还显示，与雄性小鼠相比，雌性小鼠DC增加，抑制树突状细胞减少， 对免疫治疗策略的反应更好，具有增强的TJeff功能。这样的响应被移除 卵巢切除(OVX)，但用0.1 mg的E_2挽救。OVX还将TME更改为 免疫抑制类，具有增强的免疫抑制Treg活性，与雄性小鼠相似。最后， 在TME内，OVX减少TME中高内皮微静脉(HEV)的形成和引流淋巴 结节(DLN)，这加强了Tef细胞的启动和随后进入TME的贩运。我们假设 性二型性反应是由E2通过其受体作用于树突状细胞和树突状细胞来驱动的 抗原递呈和增加TJeff激活，从而选择性地确定对 免疫疗法。我们还假设，E2将增加抗原特异性T细胞的归巢和运输 通过促进HEV的形成和成熟来促进细胞。在Aim1中，我们将询问监管机制 用基因工程小鼠模型(GEMM)、体外检测和体外实验研究E2‘S对DC生物学的影响 蛋白质组学分析检测DC分化、成熟和分化的受体亚型和分子介质 抗原呈递。目的2将剖析E_2‘S对Treg的影响如何驱动反应和 选择针对性别的免疫疗法。我们将用GEMM、小鼠嵌合体和药理学来测试这一点 在体内和体外共培养实验中进行操纵，以识别信号和下游效应器。 聚焦于AIM 3中T细胞的转运，我们将检测E2‘S对HEV形成的影响，以及T细胞从AIM 3中流出 使用流动和免疫荧光技术相结合的DLN到TME和体循环 GEMM和体外系统。这些研究将共同阐明促进性行为的基本机制-- HNSCC中定向的新生物标志物和治疗方法。

ABSTRACT Head and neck cancer (HNSCC) afflicts mostly men. Female patients who develop HNSCC tend to be postmenopausal, with menopausal hormone therapy lowering their risk of developing the disease. We show that premenopausal female HNSCC patients have an improved response to therapy including immunotherapy (IO). Using clinical trial data, we show no difference in response to IO from testosterone. Similarly, our preclinical data show no IO response effect from gonadal castration. But 0.1 mg estradiol (E2) improves response in castrated male mice to that of female mice. Our overall objective is to understand how the premenopausal female hormone E2 contributes to progression and treatment resistance. Our data show that E2 had no effect on cancer cell viability, suggesting the tumor microenvironment (TME) as the site of action. E2 is known to play a major role in autoimmunity and inflammation, where it modulates dendritic cell (DC) and regulatory T cells (Treg) differentiation and function, and lymphatic vessel maturation. These immune cell populations regulate response to immunotherapy in HNSCC, but how they are affected by sex hormones remains largely uncharacterized. Our preclinical data also show that compared to male mice, females have increased DCs, less suppressive Tregs, and respond better to immunotherapeutic strategies with enhanced Teff function. Such response is removed with oophorectomy (OVX) but rescued with E2 of 0.1mg. OVX also changes the TME towards an immunosuppressive one with enhanced immunosuppressive Treg activity, similar to that of male mice. Finally, within the TME, OVX reduces the formation of high endothelial venules (HEVs) in the TME and draining lymph nodes (dLN), which enhance Teff cell priming and subsequent trafficking into the TME. We hypothesize that the sexually dimorphic response is driven by E2 acting via its receptors on Tregs and DCs to enhance antigen presentation and increase Teff activation, thus selectively determining response to immunotherapy. We also hypothesize that E2 will increase homing and trafficking of antigen specific T cells by enhancing HEV formation and maturation. In Aim1, we will interrogate the regulatory mechanisms of E2's effects on the DC biology by using genetically engineered mouse models (GEMMS), in vitro assays, and proteomic analysis to examine receptor subtypes and molecular mediators of DC differentiation, maturation, and antigen presentation. Aim 2 will dissect mechanisms of how E2's effect on Tregs can drive response and selection of sex-specific immunotherapies. We will test this with GEMMS, mouse chimeras, and pharmacological manipulation in in vivo and in vitro co-culture experiments, to identify signaling and downstream effectors. Focusing on T cell trafficking in Aim 3, we will examine E2's effect on HEV formation, and T cell egress from the dLN to the TME and systemic circulation using a combination of flow and immunofluorescence techniques in GEMMs and in vitro systems. These studies will collectively elucidate the basic mechanisms for advancing sex- directed novel biomarkers and therapeutics in HNSCC.