CD73 expression on cancer-associated fibroblasts of Head and Neck Cancers shapes the immune landscape

摘要头颈部鳞状细胞癌是一种侵袭性极强的多因素疾病。 全球每年有60万名患者。酒精、烟草和HPV暴露是已知的致病因素 HNSCC，但HPV-HNSCC约占发病率的80%，且与临床不良有关 结果，尤其是那些表现为间充质表型的结果。尽管PD-1/PD取得了重大进展- L1和CTLA-4检查点抑制剂在为部分癌症患者提供疗效方面的有效率 在HNSCC患者中，这些检查点抑制剂的使用是有限的。除了高度的异质性和免疫力 细胞可塑性，一种高度免疫抑制的肿瘤微环境(TME)是导致 HNSCC的临床结局不佳，这突显了定义额外的免疫检查点的必要性 对于PD-1/PD-L1通路是冗余的。CD73是一种主要的胞外-5‘-核苷酸酶，也是一种限速酶 将免疫刺激的三磷酸腺苷转化为腺苷(ADO)，腺苷抑制T细胞活化，诱导细胞凋亡。 因此，CD73是维持免疫抑制的一个普遍的免疫检查点。临床上，高CD73 在所有HNSCC组织中的表达与预后不良有关。然而，主要的贡献者 HNSCC肿瘤微环境(TME)中CD73活性的影响尚未确定，应予以解决 通过对每种细胞类型的严格和比较检查，因为观察到CD73在 总的肿瘤组织可能是一种细胞亚型中CD73水平增加或增加的结果 这种细胞类型的频率。最近，我们观察到癌症相关成纤维细胞(CAF)，显著的 非造血性间质中CD73的表达水平最高，而CD73的表达水平较低。 HNSCC TME的其他细胞成分变化很大。此外，HNSCCs的免疫细胞可塑性， 包括TME中T细胞的丢失，与高CAF丰度和高CAF-CD73水平有关。 鉴于HNSCCs细胞成分的高度异质性，我们建议进行全面的比较 CD73在不同细胞类型中表达的相对丰度和生物分布分析 将对CD73总活性的主要贡献者(S)以及CAF丰度的影响提供更多的洞察 免疫细胞的可塑性。使用新鲜的HNSCC标本和最先进的技术方法，我们将 解决关键问题，即CAF-CD73是否以及如何调节免疫细胞的可塑性 目的：(1)通过流式细胞仪检测CD73在CAF上的表达水平，并将其与免疫细胞可塑性联系起来。 (2)证明HNSCC TME中CAF和免疫细胞的可塑性 从典型的HNSCC和间充质样HNSCCs中提纯的肿瘤、CAF和TIL的转录组图谱， 以及证明它们的CD73酶活性和免疫抑制功能的差异。结果是 我们的系统和全面的分析将使我们能够构建HNSCC的整体免疫格局 并提供有关CD73检查站的主要贡献者(S)的宝贵信息。

Head and Neck squamous cell carcinomas (HNSCCs) are highly aggressive multi-factorial diseases affecting 600,000 patients worldwide each year. Alcohol, tobacco, and HPV exposure are known causative factors for HNSCCs, but HPV- HNSCCs represent about 80% of the incidence and are associated with poor clinical outcomes, especially those that manifest a mesenchymal phenotype. Despite the major advances of PD-1/PD- L1 and CTLA-4 checkpoint inhibitors in providing curative benefits for some cancer patients, the response rate in HNSCC patients to these checkpoint inhibitors is limited. Besides the high levels of heterogeneity and immune cell plasticity, a highly immunosuppressive tumor microenvironment (TME) is another contributing factor to the poor clinical outcomes of HNSCCs, which underscores the need for defining additional immune checkpoints non- redundant to the PD-1/PD-L1 pathways. CD73 is a major ecto-5'-nucleotidase and a rate limiting enzyme that converts the immune stimulatory ATP to adenosine (ADO), which inhibits T cell activation and induces apoptosis. Thus, CD73 serves as a prevalent immune checkpoint for maintaining immunosuppression. Clinically, high CD73 expression in total HNSCC tissues has been implicated in poor prognoses. Nevertheless the major contributor of CD73 activity in the HNSCC tumor microenvironment (TME) has not been identified and should be addressed by rigorous and comparative examination of each cell type because the observed elevated CD73 expression in total tumor tissues could be a result of either an increase in CD73 levels in one cellular subtype or an increase in the frequency of this cell type. Recently, we observed that cancer-associated fibroblasts (CAFs), the prominent non-hematopoietic stroma in the TME, expressed the highest levels of CD73, whereas CD73 expression was highly variable in other cellular constituents of the HNSCC TME. Furthermore, immune cell plasticity in HNSCCs, including the loss of T cells in the TME, was associated with high CAF abundancy and high CAF-CD73 levels. Given the highly heterogeneous cellular constituents of HNSCCs, we propose that a comprehensive comparative analysis of CD73 expression in different cell types in the context of their relative abundancy and bio-distribution will provide more insight into the major contributor(s) of the total CD73 activity and the effects of CAF abundancy to immune cell plasticity. Using fresh HNSCC specimens and state-of-the-art technical approaches, we will address the crucial questions of whether and how CAF-CD73 modulates immune cell plasticity with two specific aims: (1) to define and correlate the CD73 expression levels on CAFs with immune cell plasticity via FACs and multiplexed IHC; and (2) to demonstrate the plasticity of CAFs and immune cells in the HNSCC TME via transcriptome profiling of purified tumors, CAFs, and TILs from typical HNSCCs and mesenchymal-like HNSCCs, as well as to demonstrate their differential CD73 enzymatic activity and immunosuppressive function. The results of our systemic and comprehensive analyses will allow us to construct an overall immune landscape of HNSCC TME and to provide invaluable information about the dominant contributing population(s) to the CD73 checkpoint.