研究背景和目的：
食管癌是全球最常见的恶性肿瘤之一，在东亚地区发病率和死亡率较高。在我国，食管鳞状细胞癌（Esophageal squamous cell carcinoma，ESCC）是主要组织亚型，占食管癌病例的90%以上。ESCC具有较高的侵袭性和转移倾向，通常在初次诊断时就处于进展期。ESCC患者预后较差，5年生存率仅为15%-25%。因此，推进ESCC的研究，特别是寻找更有效的治疗策略至关重要。
ESCC的特征在于其在基因组和表观基因组水平上表现出的复杂异质性，对肿瘤进展、耐药和转移起着重要作用。ESCC的肿瘤微环境以多种免疫细胞为特征，包括耗竭性T细胞（TEX）、调节性T细胞（Tregs）和激活的巨噬细胞，它们在肿瘤进展和免疫逃逸中发挥着关键作用。本研究借助前沿技术，系统剖析ESCC患者血液、癌旁组织和癌组织的免疫微环境，通过对比不同样本间免疫微环境的差异，描绘关键免疫细胞亚群特征，深入挖掘这些特征与患者临床预后的关联。研究成果将为开发新型免疫治疗策略提供扎实的理论基础，为食管鳞状细胞癌临床治疗开辟新思路。
研究方法：
收集并总结前期两个临床研究队列，分别探索放化疗、化疗联合免疫治疗效果及生存分析。进一步深入进行免疫治疗机制探索，研究共纳入25例ESCC患者，收集了癌组织、癌旁组织和外周血样本。利用质谱流式细胞术（CyTOF）技术，对超过1000万个细胞进行全面分析，使用了包含42种免疫标志物进行广泛组合检测。通过单细胞分析，详细描绘不同组织内免疫细胞的组成和功能状态。此外，结合单细胞RNA测序（scRNA-seq）和多重免疫荧光技术（mIHC），进一步分析ESCC组织中免疫细胞的空间分布。为了验证中央记忆T细胞（TCM）在ESCC治疗中的潜在作用，在4例ESCC患者来源的样本中分选获得TCM细胞，然后体外培养扩大数量，并与食管鳞癌细胞接触共培养，评估其杀伤肿瘤细胞的能力。通过单细胞转录组数据和单细胞蛋白数据的对比分析，筛选血液、癌旁组织和癌组织三部分免疫抑制的主要标志物。结合多种肿瘤公共数据库验证标志物对免疫治疗和患者生存的预测结果。利用4NQO小鼠模型，构建原发诱导的食管鳞癌小鼠，进行对照组和PD-1组免疫抑制标志物的表达差异。
研究结果：
术前放化疗或化疗联合免疫治疗带来的临床获益情况分析发现，术前治疗均可给患者带来生存获益，但目前免疫治疗在食管癌的治疗中取得了优异的成绩，所以在术前治疗方案中免疫治疗具有更优的潜在应用价值。明确食管癌微环境的特征是确定免疫治疗方案的关键。因此，本研究通过质谱流式细胞术分析，发现ESCC癌组织中免疫细胞的组成与癌旁组织和外周血存在显著差异，癌组织中CD4+ T细胞的存在显著高于癌旁组织。与无淋巴结转移的ESCC患者相比，有淋巴结转移患者的血液中γδT细胞显著增加，表明γδT细胞具有作为转移性疾病的生物标志物的潜力，并可能参与对肿瘤扩散的系统性免疫反应。
TCM细胞具有显著的杀伤肿瘤效果，表明TCM细胞在调节免疫反应和抑制肿瘤生长中具有关键作用。肿瘤微环境中的巨噬细胞高表达PD-L1与免疫治疗反应相关，表明巨噬细胞在介导免疫反应中可能发挥关键作用。肿瘤浸润T细胞中CD39表达的升高与多种癌症类型的有利预后生存和增强免疫治疗反应相关，表明CD39可能作为评估患者生存和治疗反应的潜在生物标志物。
结论：
本研究绘制出ESCC高分辨率免疫图谱，清晰呈现出不同组织中免疫细胞构成的差异。研究表明，补充TCM 细胞数量，能够显著提升机体对肿瘤细胞的杀伤效能。此外，巨噬细胞上PD-L1的高表达，以及肿瘤浸润T细胞中 CD39、CD103的高表达，有望成为预测免疫治疗响应的生物标志物。这些成果系统揭示了癌症免疫治疗的关键免疫要素，为深入理解癌症免疫治疗机制，以及优化免疫治疗方案筑牢了基础，有望革新食管鳞状细胞癌的临床治疗模式。后续研究将着重在临床场景中验证这些发现，探索基于免疫细胞特征指导临床精准治疗的可行性，推动食管鳞状细胞癌治疗向更高效、更个性化方向迈进。

Background and Objectives:
Esophageal cancer is a major global malignancy, with particularly high incidence and mortality rates in East Asia. In China, esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype, accounting for over 90% of cases. Due to its aggressive behavior and strong metastatic potential, ESCC is frequently diagnosed at an advanced stage, leading to a five-year survival rate of only 15–25%. Although cancer treatments have progressed significantly in recent years, therapeutic advancements for esophageal cancer remain relatively lagging behind other malignancies. Therefore, exploring more effective treatment strategies for ESCC is of critical importance.
ESCC is characterized by profound genomic and epigenetic heterogeneity, which plays a pivotal role in tumor progression, drug resistance, and metastasis. The immune landscape of ESCC featuring exhausted T cells, regulatory T cells (Tregs), and activated macrophages contributes significantly to tumor progression and immune evasion. This study aims to comprehensively profile the immune microenvironment of ESCC by analyzing peripheral blood, adjacent non-tumor tissue, and tumor tissue using state-of-the-art single-cell technologies. By identifying key immune cell subsets and correlating them with clinical outcomes, the findings aim to support the development of novel immunotherapeutic approaches.
Methods:
Two prospective clinical cohorts were collected and analyzed to evaluate the clinical efficacy and survival outcomes of neoadjuvant chemoradiotherapy and chemotherapy combined with immunotherapy. Further mechanistic studies were conducted to explore the underlying immune-related mechanisms. A total of 25 patients with ESCC were enrolled in this study. Tumor tissues, adjacent normal tissues, and peripheral blood samples were collected. Mass cytometry (CyTOF) was employed to perform an in-depth analysis of over 10 million cells using a panel of 42 immune markers. Single-cell analysis was used to characterize the immune cell composition and functional states across different tissue types. In addition, single-cell RNA sequencing (scRNA-seq) and multiplex immunohistochemistry (mIHC) were used to investigate the spatial distribution of immune cells within ESCC tissues. To explore the therapeutic potential of central memory T cells (TCM), TCM cells were isolated from tumor samples of four patients, expanded ex vivo, and co-cultured with ESCC cells to assess their cytotoxic function. Integrated analysis of single-cell transcriptomic and proteomic data helped identify key immunosuppressive markers in peripheral blood, adjacent tissues, and tumor tissues. Public cancer databases were used to evaluate the predictive and prognostic value of these markers in the context of immunotherapy. Additionally, a 4NQO-induced ESCC mouse model was established to examine differences in immunosuppressive marker expression between control and anti-PD-1 treatment groups.
Results:
Analysis of clinical benefits from neoadjuvant chemoradiotherapy or chemoimmunotherapy revealed that both strategies provided survival advantages. However, given the remarkable success of immunotherapy in esophageal cancer, its incorporation into neoadjuvant regimens may offer superior clinical value. Characterizing the tumor immune microenvironment is essential for optimizing immunotherapeutic strategies in esophageal cancer. CyTOF analysis revealed distinct differences in immune cell composition between tumor tissues, adjacent normal tissues, and peripheral blood in ESCC patients. Tumor tissues showed a significantly higher abundance of CD4+ T cells compared to adjacent tissues. Moreover, patients with lymph node metastases exhibited a notable increase in γδT cells in peripheral blood, suggesting that γδT cells could serve as biomarkers for metastatic disease and may play a role in systemic immune responses against tumor spread.
TCM cells exhibited potent tumor-killing effects, indicating their critical role in modulating anti-tumor immunity. Tumor-associated macrophages demonstrated high PD-L1 expression, which was associated with responsiveness to immunotherapy, highlighting their importance in shaping the immune response. Additionally, elevated CD39 expression in tumor-infiltrating T cells was linked to favorable prognosis and enhanced response to immunotherapy across multiple cancer types, suggesting CD39 as a promising biomarker for predicting patient outcomes and treatment efficacy.
Conclusion:
This study offers a comprehensive high-resolution immune profile of ESCC, revealing distinct immune cell compositions in blood, adjacent tissues, and tumor environments. The data suggest that enhancing TCM cell levels may boost the body’s anti-tumor capabilities. Furthermore, high PD-L1 expression in macrophages and increased CD39 and CD103 expression in tumor-infiltrating T cells may serve as reliable biomarkers for predicting responses to immunotherapy. These findings shed light on the immunological mechanisms underlying ESCC and lay a robust foundation for refining immunotherapeutic strategies. Future studies will focus on validating these results in clinical settings and exploring the potential for leveraging immune cell profiles to guide personalized ESCC treatment.