Table_1_Identifying specific TLS-associated genes as potential biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in soft tissue sarcoma.xlsx

BackgroundThe tertiary lymphatic structure (TLS) is an important component of the tumor immune microenvironment and has important significance in patient prognosis and response to immune therapy. However, the underlying mechanism of TLS in soft tissue sarcoma remains unclear.
MethodsA total of 256 RNAseq and 7 single-cell sequencing samples were collected from TCGA-SARC and GSE212527 cohorts. Based on published TLS-related gene sets, four TLS scores were established by GSVA algorithm. The immune cell infiltration was calculated via TIMER2.0 and “MCPcounter” algorithms. In addition, the univariate, LASSO, and multivariate-Cox analyses were used to select TLS-related and prognosis-significant hub genes. Single-cell sequencing dataset, clinical immunohistochemical, and cell experiments were utilized to validate the hub genes.
ResultsIn this study, four TLS-related scores were identified, and the total-gene TLS score more accurately reflected the infiltration level of TLS in STS. We further established two hub genes (DUSP9 and TNFSF14) prognosis markers and risk scores associated with soft tissue sarcoma prognosis and immune therapy response. Flow cytometry analysis showed that the amount of CD3, CD8, CD19, and CD11c positive immune cell infiltration in the tumor tissue dedifferentiated liposarcoma patients was significantly higher than that of liposarcoma patients. Cytological experiments showed that soft tissue sarcoma cell lines overexpressing TNFSF14 could inhibit the proliferation and migration of sarcoma cells.
ConclusionThis study systematically explored the TLS and related genes from the perspectives of bioinformatics, clinical features and cytology experiments. The total-gene TLS score, risk score and TNFSF14 hub gene may be useful biomarkers for predicting the prognosis and immunotherapy efficacy of soft tissue sarcoma.

背景
三级淋巴结构（tertiary lymphatic structure, TLS）是肿瘤免疫微环境的重要组成部分，对患者预后及免疫治疗响应具有重要意义。然而，三级淋巴结构在软组织肉瘤中的潜在作用机制仍未明确。
方法
本研究从TCGA-SARC与GSE212527队列中收集了共计256份RNA测序（RNAseq）样本与7份单细胞测序（single-cell sequencing）样本。基于已发表的TLS相关基因集，通过基因集变异分析（gene set variation analysis, GSVA）算法构建了4种TLS评分。采用TIMER2.0与“MCPcounter”算法计算免疫细胞浸润情况。此外，通过单变量、最小绝对收缩和选择算子（least absolute shrinkage and selection operator, LASSO）以及多变量Cox回归分析，筛选出与TLS相关且具有预后价值的核心基因。利用单细胞测序数据集、临床免疫组化结果及细胞实验对核心基因进行验证。
结果
本研究共鉴定出4种TLS相关评分，其中全基因TLS评分可更准确地反映软组织肉瘤中TLS的浸润水平。本研究进一步构建了2个与软组织肉瘤预后及免疫治疗响应相关的核心基因（DUSP9与TNFSF14）预后标志物及风险评分。流式细胞术分析显示，去分化脂肪肉瘤患者肿瘤组织中CD3、CD8、CD19及CD11c阳性免疫细胞的浸润量显著高于脂肪肉瘤患者。细胞学实验表明，过表达TNFSF14的软组织肉瘤细胞系可抑制肉瘤细胞的增殖与迁移能力。
结论
本研究从生物信息学、临床特征及细胞学实验三个维度系统探究了TLS及其相关基因。全基因TLS评分、风险评分及TNFSF14核心基因有望成为预测软组织肉瘤预后及免疫治疗疗效的潜在生物标志物。