METHODS: The immune-modulatory activity of GLI1 was evaluated in a syngeneic B16F10 melanoma mouse model assessing immune populations by flow cytometry. Murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were differentiated from bone marrow cells and their immunosuppressive ability was assessed by inhibition of T cells. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Cytokine array analysis, qPCR and chromatin immunoprecipitation were performed to explore the regulation of CX3CL1 expression by GLI1. Human monocyte-derived dendritic cells (moDCs) were cultured in CM from GLI1-silenced patient-derived melanoma cells to assess their activation and recruitment. Blocking antibodies anti-CX3CL1, anti-CCL7 and anti-CXCL8 were used for in vitro functional assays.
RESULTS: Melanoma cell-intrinsic activation of GLI1 promotes changes in the infiltration of immune cells, leading to accumulation of immunosuppressive PMN-MDSCs and regulatory T cells, and to decreased infiltration of dendric cells (DCs), CD8 + and CD4 + T cells in the TME. In addition, we show that ectopic expression of GLI1 in melanoma cells enables PMN-MDSC expansion and recruitment, and increases their ability to inhibit T cells. The chemokine CX3CL1, a direct transcriptional target of GLI1, contributes to PMN-MDSC expansion and recruitment. Finally, silencing of GLI1 in patient-derived melanoma cells promotes the activation of human monocyte-derived dendritic cells (moDCs), increasing cytoskeleton remodeling and invasion ability. This phenotype is partially prevented by blocking the chemokine CCL7, but not CXCL8.
CONCLUSIONS: Our findings highlight the relevance of tumor-derived GLI1 in promoting an immune-suppressive TME, which allows melanoma cells to evade the immune system, and pave the way for the design of new combination treatments targeting GLI1.
方法:在通过流式细胞术评估免疫群体的同基因B16F10黑色素瘤小鼠模型中评估GLI1的免疫调节活性。从骨髓细胞分化出小鼠多形核骨髓来源的抑制细胞(PMN-MDSC),并通过抑制T细胞来评估其免疫抑制能力。来自GLI1过表达小鼠黑色素瘤细胞的条件培养基(CM)用于培养PMN-MDSC,并通过Transwell侵袭实验和T细胞抑制来评估CM的作用。细胞因子阵列分析,进行qPCR和染色质免疫沉淀以探索GLI1对CX3CL1表达的调节。在来自GLI1沉默的患者来源的黑素瘤细胞的CM中培养人单核细胞来源的树突细胞(moDC)以评估其活化和募集。阻断抗体抗CX3CL1、抗CCL7和抗CXCL8用于体外功能测定。
结果:黑色素瘤细胞固有激活GLI1促进免疫细胞浸润的变化,导致免疫抑制PMN-MDSCs和调节性T细胞的积累,和减少树枝状细胞(DC)的浸润,TME中的CD8+和CD4+T细胞。此外,我们表明,黑色素瘤细胞中GLI1的异位表达使PMN-MDSC扩增和募集,并增加它们抑制T细胞的能力。趋化因子CX3CL1是GLI1的直接转录靶标,有助于PMN-MDSC扩增和募集。最后,GLI1在患者来源的黑色素瘤细胞中的沉默促进人单核细胞来源的树突状细胞(moDC)的激活,增加细胞骨架重塑和侵袭能力。该表型通过阻断趋化因子CCL7而不是CXCL8而被部分阻止。
结论:我们的发现强调了肿瘤来源的GLI1在促进免疫抑制性TME中的相关性,允许黑色素瘤细胞逃避免疫系统,并为针对GLI1的新组合治疗的设计铺平道路。