[摘 要] 目的：探究半乳糖凝集素-9（Gal-9）对胰腺癌细胞的侵袭与迁移功能的促进作用及其机制。方法：基于公开单细胞转录组数据分析胰腺癌中免疫检查点分子的表达谱，结合Western blotting和ELISA验证Gal-9在胰腺癌细胞及临床相关模型中的表达与分泌特征。建立C57BL/6小鼠胰腺原位移植瘤模型，通过免疫组化、免疫荧光染色和流式细胞术分析Gal-9对肿瘤细胞增殖及其对瘤内CD8 + T细胞浸润与功能状态的影响。Gal-9重组蛋白及肿瘤细胞条件培养基（CM）处理肿瘤相关成纤维细胞 （CAF），Western blotting、RT-qPCR和免疫荧光检测CAF中Ⅰ型胶原表达变化。免疫共沉淀、免疫荧光染色和Western blotting分析Gal-9与CD44的相互作用及JAK2/STAT3通路相关蛋白的表达。构建过表达Gal-9胰腺癌细胞与CAF共移植的裸鼠皮下荷瘤模型，结合天狼星红和马松染色评估小鼠肿瘤组织中胶原沉积情况。脱细胞外基质法提取CAF中沉积的细胞外基质（ECM）蛋白；通过细胞划痕、Transwell迁移及侵袭实验，检测CAF来源Ⅰ型胶原对胰腺癌细胞侵袭、迁移功能的影响，并通过siRNA干扰Gal-9表达进行功能反向验证。结果：与经典免疫检查点分子PD-L1和CTLA-4相比，Gal-9在胰腺癌中显著高表达（P < 0.000 1）。 体内实验表明，Gal-9促进肿瘤生长（P < 0.001），上调Ki-67表达，同时减少肿瘤内CD8? T细胞浸润并诱导肿瘤组织中TIM-3?细胞比例升高，Granzyme B、IFN-γ 及TNF-α 表达下降（P < 0.05）。体外实验显示，外源性Gal-9及肿瘤细胞来源的CM均可上调CAF中Ⅰ型胶原表达（P < 0.001）。机制研究表明，在CAF中抗Gal-9抗体免疫沉淀复合物中检测到CD44，且JAK2/STAT3蛋白表达水平增加。CAF来源的Ⅰ型胶原处理胰腺癌细胞后，肿瘤细胞的迁移和侵袭数量增加。相反，肿瘤细胞中敲低Gal-9表达后，CAF来源Ⅰ型胶原水平下降，同时肿瘤细胞侵袭和迁移数量下降。结论：胰腺癌来源的Gal-9具有双重作用：一方面，通过促进肿瘤细胞增殖和诱导CD8 + T细胞耗竭促进肿瘤进展；另一方面，通过结合CAF上的CD44受体，激活JAK2/STAT3信号通路，刺激CAF合成并分泌Ⅰ型胶原，进而增强胰腺癌细胞的侵袭迁移能力。

[Abstract] Objective: To investigate the role of galectin-9 (Gal-9) in promoting invasion and migration of pancreatic cancer cells and to elucidate the underlying mechanisms. Methods: Publicly available single-cell RNA sequencing datasets were analyzed to characterize the expression landscape of immune checkpoint molecules in pancreatic cancer. Western blotting and ELISA were performed to validate the expression and secretion profiles of galectin-9 (Gal-9) in pancreatic cancer cell lines and clinically relevant models. An orthotopic pancreatic tumor model was established in C57BL/6 mice. Immunohistochemistry, immunofluorescence staining, and flow cytometry were employed to evaluate the effects of Gal-9 on tumor cell proliferation, as well as on the infiltration and functional status of intratumoral CD8? T cells. Cancer-associated fibroblast (CAF) were treated with recombinant Gal-9 protein or tumor cell-derived conditioned medium (CM). Changes in type Ⅰ collagen expression in CAF were assessed by Western blotting, RT- qPCR, and immunofluorescence staining. Co-immunoprecipitation, immunofluorescence staining, and Western blotting were conducted to investigate the interaction between Gal-9 and CD44, as well as the activation of the JAK2/STAT3 signaling pathway. A subcutaneous · · 630 [[PAGE_INDEX=13 FILE=08b612ef-59ff-47e1-960e-f599d97bfff7.pdf]] 赵闯闯, 等. 半乳糖凝集素-9介导肿瘤相关成纤维细胞Ⅰ型胶原生成促进胰腺癌细胞侵袭与迁移xenograft model was established in nude mice by co-implanting Gal-9-overexpressing pancreatic cancer cells with CAF. Sirius Red staining and Masson's trichrome staining were performed to evaluate collagen deposition in tumor tissues. Extracellular matrix (ECM) proteins deposited by CAF were isolated using a decellularization approach. Wound healing assays, Transwell migration assays, and invasion assays were conducted to assess the effects of CAF-derived type Ⅰ collagen on the migratory and invasive capacities of pancreatic cancer cells. Functional rescue experiments were performed using siRNA-mediated knockdown of Gal-9. Results: Compared with classical immune checkpoint molecules PD-L1 and CTLA-4, Galectin-9 (Gal-9) was significantly upregulated in pancreatic cancer (P < 0.05). In vivo experiments demonstrated that Gal-9 promoted tumor growth (P < 0.01), accompanied by increased Ki-67 expression. Meanwhile, Gal-9 markedly reduced intratumoral CD8? T-cell infiltration and increased the proportion of TIM-3? cells, while the expression levels of Granzyme B, IFN-γ, and TNF-α were significantly decreased (P < 0.05). In vitro studies revealed that both exogenous Gal-9 and tumor cell-derived conditioned medium (CM) significantly upregulated type Ⅰ collagen expression in CAF (P < 0.001). Mechanistically, CD44 was detected in the immunoprecipitated complexes using anti-Gal-9 antibodies in CAF, and the protein expression levels of JAK2/STAT3 were correspondingly increased, indicating activation of the JAK2/STAT3 signaling pathway. Functional assays showed that treatment of pancreatic cancer cells with CAF-derived type Ⅰ collagen significantly enhanced their migratory and invasive capacities. In contrast, knockdown of Gal-9 in tumor cells resulted in reduced levels of CAF- derived type Ⅰ collagen, accompanied by a marked decrease in tumor cell migration and invasion. Conclusion: Tumor-derived Gal-9 may exert dual pro-tumorigenic functions in pancreatic cancer. It promotes tumor progression by enhancing cancer cell proliferation and inducing CD8? T cell exhaustion. In parallel, Gal-9 binds to CD44 on CAF to activate the JAK2/STAT3 pathway, thereby stimulating type Ⅰ collagen production and secretion, which in turn facilitates pancreatic cancer cell invasion and migration.

[基金项目] 江苏省自然科学基金-优秀青年项目（BK20240128）；江苏省社会发展面上项目（BE2023759）；镇江市社会发展项目（SH2022035）；江苏省研究生科研与实践创新计划（SJCX25_2499）