DataSheet_1_Neoadjuvant immunochemotherapy improves clinical outcomes of patients with esophageal cancer by mediating anti-tumor immunity of CD8+ T (Tc1) and CD16+ NK cells.zip

BackgroundEsophageal cancer (ESCA) is one of the most common tumors in the world, and treatment using neoadjuvant therapy (NT) based on radiotherapy and/or chemotherapy has still unsatisfactory results. Neoadjuvant immunochemotherapy (NICT) has also become an effective treatment strategy nowadays. However, its impact on the tumor microenvironment (TME) and regulatory mechanisms on T cells and NK cells needs to be further elucidated.
MethodsA total of 279 cases of ESCA who underwent surgery alone [non-neoadjuvant therapy (NONE)], neoadjuvant chemotherapy (NCT), and NICT were collected, and their therapeutic effect and survival period were compared. Further, RNA sequencing combined with biological information was used to analyze the expression of immune-related genes. Immunohistochemistry, immunofluorescence, and quantitative real-time PCR (qRT-PCR) were used to verify the activation and infiltration status of CD8+ T and CD16+ NK cells, as well as the function and regulatory pathway of killing tumor cells.
ResultsPatients with ESCA in the NICT group showed better clinical response, median survival, and 2-year survival rates (p < 0.05) compared with the NCT group. Our RNA sequencing data revealed that NICT could promote the expression of immune-related genes. The infiltration and activation of immune cells centered with CD8+ T cells were significantly enhanced. CD8+ T cells activated by PD-1 inhibitors secreted more IFN-γ and cytotoxic effector factor cells through the transcription factor of EOMES and TBX21. At the same time, activated CD8+ T cells mediated the CD16+ NK cell activation and secreted more IFN-γ to kill ESCA cells. In addition, the immunofluorescence co-staining results showed that more CD276+ tumor cells and CD16+ NK cells were existed in pre-NCT and pre-NICT group. However, CD276+ tumor cells were reduced significantly in the post-NICT group, while they still appeared in the post-NCT group, which means that CD16+ NK cells can recognize and kill CD276+ tumor cells after immune checkpoint blocker (ICB) treatment.
ConclusionNICT can improve the therapeutic effect and survival period of resectable ESCA patients. NICT could promote the expression of immune-related genes and activate CD8+ T and CD16+ NK cells to secrete more IFN-γ to kill ESCA cells. It provides a theoretical basis and clinical evidence for its potential as an NT strategy in ESCA.

背景 食管癌（Esophageal cancer, ESCA）是全球最常见的恶性肿瘤之一，基于放疗和/或化疗的新辅助治疗（neoadjuvant therapy, NT）疗效仍不尽如人意。新辅助免疫化疗（neoadjuvant immunochemotherapy, NICT）如今已成为一种有效的治疗策略，但其对肿瘤微环境（tumor microenvironment, TME）的影响以及对T细胞和NK细胞的调控机制仍有待进一步阐明。
方法 本研究共收集279例接受单纯手术治疗[非新辅助治疗（non-neoadjuvant therapy, NONE）]、新辅助化疗（neoadjuvant chemotherapy, NCT）以及新辅助免疫化疗的食管癌患者，对比各组患者的治疗效果与生存期。进一步采用RNA测序（RNA sequencing）结合生物信息学方法分析免疫相关基因的表达情况；通过免疫组化（immunohistochemistry）、免疫荧光（immunofluorescence）以及实时定量聚合酶链式反应（quantitative real-time PCR, qRT-PCR）验证CD8+ T细胞与CD16+ NK细胞的活化及浸润状态，以及其杀伤肿瘤细胞的功能与调控通路。
结果 与新辅助化疗组相比，新辅助免疫化疗组食管癌患者展现出更优的临床应答率、中位生存期及2年生存率（p < 0.05）。本研究的RNA测序数据显示，新辅助免疫化疗可促进免疫相关基因的表达，以CD8+ T细胞为核心的免疫细胞浸润与活化水平显著提升。经PD-1抑制剂活化的CD8+ T细胞可通过转录因子EOMES与TBX21分泌更多IFN-γ及细胞毒性效应因子。同时，活化的CD8+ T细胞可介导CD16+ NK细胞活化，并分泌更多IFN-γ以杀伤食管癌细胞。此外，免疫荧光共染色结果显示，新辅助化疗前与新辅助免疫化疗前的患者组织中存在更多CD276+肿瘤细胞与CD16+ NK细胞；而新辅助免疫化疗后的患者组织中CD276+肿瘤细胞显著减少，但新辅助化疗后的患者组织中仍可检测到该类细胞，这表明免疫检查点阻断剂（immune checkpoint blocker, ICB）治疗后，CD16+ NK细胞可识别并杀伤CD276+肿瘤细胞。
结论 新辅助免疫化疗可提升可切除食管癌患者的治疗效果与生存期。新辅助免疫化疗可促进免疫相关基因的表达，活化CD8+ T细胞与CD16+ NK细胞并分泌更多IFN-γ以杀伤食管癌细胞，这为其作为食管癌新辅助治疗策略的潜在应用价值提供了理论依据与临床证据。