DataSheet_1_Functional status and spatial interaction of T cell subsets driven by specific tumor microenvironment correlate with recurrence of non-small cell lung cancer.docx

BackgroundThe anti-tumoral or pro-tumoral roles of CD4+ and CD8+ T cells typify the complexity of T cell subsets function in cancer. In the non-small cell lung cancer (NSCLC), the density and topology of distinct T cell phenotypes at the tumor center (TC) versus the invasive margin (IM) are largely unknown. Here, we investigated T cell subsets density and distribution within TC and IM regions in NSCLC and its impact on the prognosis.MethodsWe performed multiplex immunofluorescence using a tissue microarray of samples from 99 patients with locally advanced NSCLC to elucidate the distributions of tumor cell, T cell subpopulations (CD4/conventional CD4/regulatory CD4/CD8/cytotoxic CD8/pre-dysfunctional CD8/dysfunctional CD8), microvessel density (MVD), cancer-associated fibroblasts (CAFs) and hypoxia-inducible factor-1α (HIF-1α) in TC and IM tissues. Cell-to-cell nearest neighbor distances and interactions were analyzed using the phenoptrreports R package. Cox regression was used to evaluate the associations between T cell subsets density and proximity to tumor cells and recurrence-free survival (RFS). Correlations between different cell subsets were examined by Spearman’s or Kruskal-Wallis tests.ResultsIn the locally advanced NSCLC, the proportion of tumor cells and CAFs in IM is lower than in the TC, while MVD, CD4+, and CD8+ T lymphocytes were increased, and tumor cells were closer to T lymphocytes and their subsets. The density and proximity of CD4+ and CD8+ T cells in the TC and IM regions were not associated with RFS, but in the IM area, increased density of dysfunctional CD8 and closer regulatory CD4 to tumor cells were independent risk factors for recurrence (HR were 3.536 and 2.884, respectively), and were positively correlated with HIF-1α+CD8 (r = 0.41, P = 0.000) and CAFs (P = 0.017), respectively.sConclusionsIn locally advanced NSCLC, the functional status of T cells in the IM region is closely related to recurrence. The density of dysfunctional CD8 and the proximity of regulatory CD4 to tumor cells were independent risk factors for recurrence, and are positively correlated with the hypoxia response of CD8+ T cells and CAFs. Targeting hypoxia or CAFs is expected to further sensitize therapy.

背景：CD4+和CD8+ T细胞在抗肿瘤或促肿瘤作用方面的角色体现了T细胞亚群在癌症中功能的复杂性。在非小细胞肺癌（NSCLC）中，肿瘤中心（TC）与浸润边缘（IM）处不同T细胞表型的密度和拓扑结构在很大程度上尚不明确。本研究旨在探讨NSCLC中TC和IM区域T细胞亚群的密度与分布，及其对预后的影响。方法：通过使用99例局部晚期NSCLC患者的组织微阵列，我们采用多重免疫荧光技术，揭示了肿瘤细胞、T细胞亚群（CD4/传统CD4/调节性CD4/CD8/细胞毒性CD8/前功能失调CD8/功能失调CD8）、微血管密度（MVD）、癌相关成纤维细胞（CAFs）和低氧诱导因子-1α（HIF-1α）在TC和IM组织中的分布。利用phenoptrreports R包分析细胞间最近邻距离和相互作用。通过Cox回归评估T细胞亚群密度与肿瘤细胞邻近性及无病生存期（RFS）之间的关联。通过Spearman或Kruskal-Wallis检验考察不同细胞亚群之间的相关性。结果：在局部晚期NSCLC中，IM区域的肿瘤细胞和CAFs的比例低于TC区域，而MVD、CD4+和CD8+ T淋巴细胞增加，肿瘤细胞与T淋巴细胞及其亚群的距离更近。TC和IM区域的CD4+和CD8+ T细胞的密度与RFS无关联，但在IM区域，功能失调CD8的增加和调节性CD4与肿瘤细胞的接近是复发的独立风险因素（HR分别为3.536和2.884），且与HIF-1α+CD8（r = 0.41，P = 0.000）和CAFs（P = 0.017）呈正相关。结论：在局部晚期NSCLC中，浸润边缘区域T细胞的功能状态与复发密切相关。功能失调CD8的密度和调节性CD4与肿瘤细胞的接近性是复发的独立风险因素，且与CD8+ T细胞对低氧的响应和CAFs呈正相关。针对低氧或CAFs的治疗有望进一步提高治疗效果。