The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target.黑色素瘤（Melanoma）仍是致死性最高的皮肤癌，其主要成因在于肿瘤对治疗具有高度耐药性。侧群细胞（side population, SP）广泛存在于多种癌症中，通常富集于治疗耐药及致瘤性细胞群体内。本研究在一系列涵盖不同进展阶段的人类黑色素瘤样本中，成功鉴定出可外排Hoechst染料的SP细胞。SP细胞的占比并不随疾病分期发生变化，但与原发性（皮肤性）肿瘤的预后指标‘布雷斯洛厚度（Breslow’s depth）’呈显著相关。将SP细胞注射至免疫缺陷小鼠体内后，连续两代实验均显示，相较于主体细胞群（main population, MP）黑色素瘤细胞，SP细胞可形成体积更大的肿瘤，且在更低的细胞接种量下即可展现致瘤能力。此外，SP细胞可重建人类A375黑色素瘤细胞系的异质性组成，其克隆形成活性较MP细胞高出2.5倍。基因表达分析显示，相较于MP细胞，黑色素瘤SP细胞中与化疗耐药及抗凋亡相关的基因表达显著上调。与上述分子特征相符的是，当A375细胞暴露于黑色素瘤标准化疗药物达卡巴嗪（dacarbazine）以及缺氧这一恶化应激条件时，SP细胞的占比均出现上升。此外，SP细胞中与细胞侵袭、迁移相关的基因，以及与潜在（黑色素瘤）癌症干细胞（cancer stem cells, CSC）相关的基因（包括ABCB1与JARID1B）的表达均出现上调。在人类黑色素瘤的诸多肿瘤细胞中均可检测到ABCB1免疫反应阳性信号，尤其集中于原发性肿瘤侵袭前沿的细胞簇中。综合以上结果，本研究证实人类黑色素瘤SP细胞富集了致瘤性与化疗耐药性这两大癌症干细胞的核心特征。因此，黑色素瘤SP细胞有望成为极具研究价值的治疗靶点。