The aging microenvironment promotes melanoma cell invasion and therapy resistance

Cancer is a disease of aging, and incidence and mortality from all cancers increase logarithmically after the age of 45. Older patients have a much poorer prognosis for melanomas of equal stage, compared to younger individuals. The role of the tumor microenvironment in modulating cancer behavior is widely recognized. We hypothesized that age-related changes in the tumor microenvironment could affect the progression of melanoma. We demonstrate that the aging microenvironment increases the invasion of melanoma cells. Using skin fibroblasts isolated from healthy donors, we have built artificial skin and demonstrate that melanoma cells invade more rapidly when exposed to aged fibroblasts. In a mouse model of melanoma (YUMM1.7, BrafV600E/Cdkn2a-/-/Pten-/-), congeneic to C57/BL6 mice, melanomas invade faster in aged mice. Gene expression analyses suggest that melanoma cells are undergoing DNA damage when exposed to an aged microenvironment, and we confirm this in cellular assays. Proteomics analysis of aging fibroblasts suggest that fibroblasts secrete molecules (laminin b2, Wnt inhibitors) associated with increased resistance to targeted therapy. Indeed, melanoma cells injected into aged mice respond less well to PLX4720 than those injected into young mice. This suggests that exciting new drugs targeting the BRAF pathway may be less effective in aging patients. To assess the effects of the aging microenvironment on melanoma cell gene expression, we co-cultured dermal fibroblasts from young (<35 years), medium or aged ( >55 years) individuals with three different melanoma cell lines (UACC1273, M93-047 and UACC903) in triplicate using transwell chambers. After 96 h of co-culture, fibroblasts in the upper well were removed, and melanoma cells (lower chamber) were subjected to microarray analysis.

癌症是一种与衰老相关的疾病，所有癌症的发病率与死亡率均在45岁后呈对数级增长。相较于年轻个体，处于相同临床分期的黑色素瘤老年患者预后显著更差。肿瘤微环境（tumor microenvironment, TME）对癌症行为的调控作用已得到广泛认可。我们提出假说：肿瘤微环境的年龄相关性改变可影响黑色素瘤的进展。本研究证实，衰老微环境能够增强黑色素瘤细胞的侵袭能力。我们通过从健康供体中分离的皮肤成纤维细胞构建人工皮肤模型，实验发现，当黑色素瘤细胞暴露于衰老成纤维细胞时，其侵袭速度显著加快。在与C57/BL6小鼠同基因的黑色素瘤小鼠模型（YUMM1.7, BrafV600E/Cdkn2a-/-/Pten-/-）中，老年小鼠体内的黑色素瘤侵袭速度更快。基因表达分析结果显示，黑色素瘤细胞在暴露于衰老微环境时会发生DNA损伤，该结论在细胞实验中得到了验证。对衰老成纤维细胞的蛋白质组学分析表明，成纤维细胞会分泌与靶向治疗耐药性升高相关的分子（层粘连蛋白b2、Wnt抑制剂）。事实上，注射至老年小鼠体内的黑色素瘤细胞对PLX4720的响应性弱于注射至年轻小鼠的黑色素瘤细胞。这提示，针对BRAF通路的新型潜力药物在老年患者中的疗效可能欠佳。为评估衰老微环境对黑色素瘤细胞基因表达的影响，我们采用Transwell小室（transwell chambers），将来自年轻（<35岁）、中年及老年（>55岁）个体的皮肤成纤维细胞与三种不同的黑色素瘤细胞系（UACC1273、M93-047及UACC903）进行共培养，每组设置3个复孔。共培养96小时后，移除上层小室中的成纤维细胞，对下层小室中的黑色素瘤细胞进行微阵列分析。