Mechanical confinement governs phenotypic plasticity in melanoma [ChIP-seq SKMEL5]

Phenotype switching is a form of cellular plasticity in which cancer cells reversibly move between two opposite extremes - proliferative versus invasive states. While it has long been hypothesised that such switching is triggered by external cues, the identity of these cues has remained elusive. Here, we demonstrate that mechanical confinement mediates phenotype switching through chromatin remodelling. Using a zebrafish model of melanoma coupled with human samples, we profiled tumour cells at the interface between the tumour and surrounding microenvironment. Morphological analysis of these rare cells showed flattened, elliptical nuclei suggestive of mechanical confinement by nearby adjacent tissue. Spatial and single-cell transcriptomics demonstrated that the interface cells adopted a gene program of neuronal invasion, including acquisition of an acetylated tubulin cage that protects the nucleus during migration. We identified the DNA-bending protein HMGB2 as a confinement-induced mediator of the neuronal state. HMGB2 is upregulated in confined cells, and quantitative modelling revealed that confinement prolongs contact time between HMGB2 and chromatin, leading to changes in chromatin configuration that favour the neuronal phenotype. Genetic disruption of HMGB2 showed that it regulates the trade-off between proliferative and invasive states, in which confined HMGB2high tumour cells are less proliferative but more drug resistant. Our results implicate the mechanical microenvironment as a mechanism for phenotype switching in melanoma. ChIP-sequencing targeting HMGB2 in SKMEL5 human melanoma cells expressing V5-tagged HMGB2.

表型切换（Phenotype switching）是细胞可塑性的一种形式，指癌细胞可在增殖与侵袭两种截然相反的极端状态间发生可逆转换。长期以来，学界假设此类切换由外部调控信号触发，但这些信号的具体身份始终未明。本研究证实，机械约束可通过染色质重塑介导黑色素瘤的表型切换。我们结合黑色素瘤斑马鱼模型与人类临床样本，对肿瘤与周围微环境交界区域的肿瘤细胞开展了多维度表征分析。对这类稀有细胞的形态学分析显示，其细胞核呈扁平椭圆形，提示细胞受到邻近组织的机械约束。空间单细胞转录组学分析表明，交界区域的肿瘤细胞激活了神经元侵袭相关基因程序，包括形成可在迁移过程中保护细胞核的乙酰化微管蛋白笼结构。本研究鉴定出DNA弯曲蛋白高迁移率族蛋白B2（HMGB2）作为机械约束诱导的神经元状态介导因子。受限细胞中HMGB2的表达显著上调；定量建模结果显示，机械约束会延长HMGB2与染色质的接触时间，进而改变染色质构象，偏向于促进神经元表型的形成。对HMGB2进行基因功能干扰实验显示，该蛋白可调控增殖状态与侵袭状态之间的平衡：受机械约束的HMGB2高表达肿瘤细胞增殖能力较弱，但耐药性更强。本研究结果表明，机械微环境是黑色素瘤表型切换的重要调控机制之一。本研究在表达V5标签标记HMGB2的SKMEL5人类黑色素瘤细胞中，开展了针对HMGB2的染色质免疫共沉淀测序（ChIP-seq）实验。