尽管肺癌治疗取得了成功，但迫切需要更多新的生物标志物研究来选择患者。本研究旨在利用瘤球作为模型，分析胶质素-3（GAL-3）在肺肿瘤微环境中的作用，并探讨其作为非小细胞肺癌（NSCLC）患者预测和预后生物标志物的潜在作用。对于体外研究，我们培养了早期患者的肺腺癌（LUAD）和肺鳞癌（LUSC）原代培养物以及商业细胞系，使用瘤球形成试验和粘附条件作为对照。我们运用逆转录定量实时PCR（RTqPCR）、免疫印迹、免疫荧光、流式细胞术和免疫分析来分析GAL-3的分泌和表达模式。我们的结果使用三维（3D）肺肿瘤细胞模型揭示了可溶性GAL-3（sGAL-3）的高表达和分泌。为了更准确地模拟肿瘤微环境，我们使用瘤球和成纤维细胞的共培养，揭示GAL-3在调节T细胞调节性功能上在肿瘤微环境中作为免疫调节分子的重要性。在转化阶段，我们确认高表达GAL-3水平的患者具有更多的调节性T细胞，这表明肿瘤可能通过GAL-3招募这一人群。接下来，我们在LUAD和LUSC患者术前评估了sGAL-3水平，假设sGAL-3在早期LUAD患者的总生存期和无复发生存期中可作为独立的预后生物标志物使用。此外，我们还评估了早期LUAD和LUSC患者在接受一线帕博利珠单抗治疗时通过血浆评估sGAL-3的水平来预测临床结果，进一步证明sGAL-3在预测帕博利珠单抗的持久临床反应方面具有高效性，区域曲线下面积（AUC）为0.801（p=0.011）。此外，高水平可能预测抗PD-1治疗的无进展生存期和总生存期下降，从而表明sGAL-3是一个与预后无关的晚期LUAD生物标志物。本文受版权保护。保留所有权利。

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer (NSCLC) patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS ). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS , which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve (AUC) of 0.801 (p=0.011). Moreover, high levels might predict decreased progression-free survival and overall survival to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD.This article is protected by copyright. All rights reserved.