Supplementary file 2_Mesenchymal stem cells promote cisplatin resistance in non-small cell lung cancer through IL-6/MEK-ERK/macrophages axis: construction of prognostic signature and experimental investigation.docx

BackgroundCisplatin is a primary treatment for non-small cell lung cancer (NSCLC), however, cisplatin resistance develops readily, leading to tumor progression. Mesenchymal stem cells (MSCs) distributed in the tumor microenvironment (TME), yet the effects and mechanisms of cisplatin on MSCs and their subsequent impact on TME remains unclear.
MethodsThe MSC-related prognostic signature was developed through Cox and LASSO regression analysis. Validation was performed by survival analysis, ROC analysis and nomogram construction. TME evaluation was carried out by GSEA and immune infiltration analysis. The effect of cisplatin on MSCs was investigated by polymerase chain reaction (PCR) array, qRT-PCR, ELISA and Western blot. The migration of RAW 264.7 macrophages was analyzed by transwell assays and the polarization was analyzed by flow cytometry.
ResultsHigh-risk of MSC-related prognostic signature (PDGFB, ANPEP, CD40) was significantly linked to poor prognosis in patients under cisplatin treatment for NSCLC and other cancers. Patients with high IL-6 expression demonstrated poor response to cisplatin therapy. MSCs were linked with an immunosuppressive TME characterized by macrophage infiltration, particularly M2 macrophages. Cisplatin upregulated IL-6 expression in MSCs via the MEK-ERK pathway. The ability of MSCs to promote RAW 264.7 macrophages recruitment and polarization was enhanced by cisplatin.
ConclusionWe established MSC-related prognostic signature for cisplatin therapy in NSCLC. MSCs and IL-6 were associated with cisplatin resistance and macrophages infiltration. Thus, MSCs and IL-6 were potential targets for NSCLC therapeutic intervention.

背景：顺铂（cisplatin）是非小细胞肺癌（non-small cell lung cancer, NSCLC）的一线治疗药物，但极易产生顺铂耐药性，进而导致肿瘤进展。肿瘤微环境（tumor microenvironment, TME）中分布有间充质干细胞（mesenchymal stem cells, MSCs），但顺铂对MSCs的作用与机制，以及其后续对肿瘤微环境的影响仍尚不明确。
方法：本研究通过Cox回归与最小绝对收缩和选择算子（LASSO）回归分析，构建了与MSCs相关的预后特征。验证环节采用生存分析、受试者工作特征（ROC）分析与列线图（nomogram）构建。通过基因集富集分析（GSEA）与免疫浸润分析评估肿瘤微环境。采用聚合酶链反应（PCR）芯片、实时定量聚合酶链反应（qRT-PCR）、酶联免疫吸附测定（ELISA）与蛋白质印迹（Western blot）探究顺铂对MSCs的作用。通过Transwell实验分析RAW 264.7巨噬细胞的迁移能力，并通过流式细胞术分析其极化状态。
结果：高风险MSCs相关预后特征（包含PDGFB、ANPEP、CD40基因）与接受顺铂治疗的非小细胞肺癌及其他癌症患者的不良预后显著相关。IL-6高表达的患者对顺铂治疗的响应较差。MSCs与以巨噬细胞浸润（尤其是M2型巨噬细胞）为特征的免疫抑制性肿瘤微环境密切相关。顺铂可通过MEK-ERK通路上调MSCs中IL-6的表达。顺铂能够增强MSCs促进RAW 264.7巨噬细胞招募与极化的能力。
结论：本研究构建了针对非小细胞肺癌顺铂治疗的MSCs相关预后特征。MSCs与IL-6与顺铂耐药及巨噬细胞浸润相关。因此，MSCs与IL-6可作为非小细胞肺癌治疗干预的潜在靶点。