Cell fusion with type 2 macrophage induce heterogeneity of melanoma cells that potentiate immunological escape from cytotoxic T lymphocytes

Evasion from immunity is a major obstacle for achievement of successful cancer immunotherapy. Hybrids derived from cell-cell fusion is a theory associated with tumor heterogeneity and progression by conferring novel properties to tumor cells, such as drug resistance or metastatic capacity; however, its impact on immune evasion remains still unknown. Here, we investigated the potency of hybrids in immune evasion using tumor-macrophage hybrids. Hybrids were established by co-culture of a melanoma cell line, A375 and type 2 macrophages. The hybrids showed higher migration ability and higher tumorigenicity than those of the parental melanoma cells. We found that the hybrids were less sensitive to T cell receptor (TCR) specific for NY-ESO-1 transduced T cells (TCR-T cells) than parental melanoma cells, although hybrids and parental melanoma cells showed equivalent NY-ESO-1 expression. An in vitro tumor heterogeneity model revealed that TCR-T cells preferentially killed parental cells than hybrids and the survival rate of hybrids were higher than that in parental cells indicating hybrids evade from killing by TCR-T cells efficiently. A single cell analysis data set revealed that a few macrophage cells expressed melanoma differentiation antigens including gp100, MART-1 and tyrosinase, indicating hybrids exist in primary melanoma, and number of potential hybrids were corelated with poorer response to immune checkpoint blockade. These results provide evidence that melanoma-macrophage fusion has a role in tumor heterogeneity and immune evasion. Overall design: Comparative gene expression profiling analysis of CAGE-seq data for A375 cell line and its hybrid clone fused with peripheral monocyte derived macrophages.

免疫逃逸是实现成功癌症免疫治疗的主要障碍。细胞融合衍生的杂交细胞相关理论，与肿瘤异质性及肿瘤进展密切相关——该理论提出，杂交细胞可赋予肿瘤细胞全新特性，例如耐药性或转移能力；然而其对免疫逃逸的具体影响仍未明确。本研究利用肿瘤-巨噬细胞杂交细胞，探究了杂交细胞在免疫逃逸中的功能潜力。研究人员通过共培养黑色素瘤细胞系A375与2型巨噬细胞构建了杂交细胞。相较于亲本黑色素瘤细胞，杂交细胞展现出更强的迁移能力与致瘤性。尽管杂交细胞与亲本黑色素瘤细胞的NY-ESO-1表达水平相当，但杂交细胞对转导了靶向NY-ESO-1的T细胞受体（T cell receptor, TCR）的T细胞（TCR-T细胞）的敏感性更低。体外肿瘤异质性模型实验显示，相较于杂交细胞，TCR-T细胞更优先杀伤亲本细胞，且杂交细胞的存活率高于亲本细胞，表明杂交细胞可有效逃逸TCR-T细胞的杀伤。单细胞分析数据集显示，少量巨噬细胞表达包括gp100、MART-1及酪氨酸酶（tyrosinase）在内的黑色素细胞分化抗原，这表明杂交细胞存在于原发性黑色素瘤中，且潜在杂交细胞的数量与免疫检查点阻断治疗的不良应答呈正相关。上述结果证明，黑色素瘤-巨噬细胞融合在肿瘤异质性与免疫逃逸中发挥重要作用。整体实验设计：对A375细胞系及其与外周血单核细胞衍生巨噬细胞融合得到的杂交克隆进行CAGE-seq数据的比较基因表达谱分析。