Mechanical confinement governs phenotypic plasticity in melanoma [HMGB2OE_RNAseq]. Mechanical confinement governs phenotypic plasticity in melanoma [HMGB2OE_RNAseq]

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. Overall design: Bulk RNA-sequencing of A375 human melanoma cells overexpressing HMGB2, relative to empty vector control.

表型切换是细胞可塑性的一种表现形式，指癌细胞可在增殖与侵袭这两种截然相反的极端状态之间发生可逆转换。长期以来学界假设此类切换由外部信号触发，但这类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。

我们借助斑马鱼黑色素瘤模型结合人类临床样本，对肿瘤与周围微环境交界区域的稀有肿瘤细胞开展了表征分析。对这些细胞的形态学观察显示，其细胞核呈扁平椭圆形，提示受到邻近组织的机械约束作用。空间转录组学与单细胞转录组学分析结果表明，交界区肿瘤细胞激活了神经元侵袭相关的基因程序，包括获得一种可在迁移过程中保护细胞核的乙酰化微管蛋白笼结构。

我们鉴定出DNA弯曲蛋白高迁移率族蛋白2（HMGB2）作为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；定量建模分析显示，机械约束会延长HMGB2与染色质的接触时间，进而改变染色质构型，使细胞偏向神经元表型。对HMGB2进行基因敲除实验表明，该蛋白可调控增殖与侵袭状态之间的动态平衡：高表达HMGB2的受限肿瘤细胞增殖能力较弱，但耐药性更强。

本研究结果证实，机械微环境是黑色素瘤表型切换的关键调控机制。

整体实验设计：对过表达HMGB2的A375人黑色素瘤细胞开展批量RNA测序，并以空载转染细胞作为对照。