Single-cell profiling reveals an XBP1s-associated ER proteostasis state linked to metastatic stress adaptation in melanoma.

Metastatic melanoma cells must adapt to matrix detachment and proteotoxic stress during dissemination and colonization, but the stress-adaptive tumor-cell states that support this process remain unclear. Using single-cell RNA sequencing in a B16 melanoma model, we identified a metastasis-enriched ER-stress_tumor state characterized by increased Xbp1 regulon activity. Pseudotime, RNA-velocity and CellRank analyses placed this state in a late terminal-like region of the inferred cell-state transition landscape. Integration of metastasis-associated genes with the Xbp1 regulon defined a Core32 signature enriched for ER proteostasis, ER-Golgi transport and COPII-mediated vesicular trafficking, which was also elevated in human metastatic melanoma datasets. Computational perturbation, parallel genetic perturbation and functional analyses supported preferential but partially overlapping roles for Xbp1 and Atf4 in melanoma stress adaptation, with Xbp1 more closely associated with proteostasis and trafficking programs. CD47/CD230-based enrichment further linked the single-cell-defined ER-stress_tumor state to survival under detachment stress. In these enriched cells, Xbp1 depletion increased caspase-dependent anoikis, whereas disruption of the COPII components SEC23A or SAR1A produced a similar anoikis-sensitizing phenotype. XBP1 loss also impaired clonogenic recovery and experimental lung colonization, whereas XBP1s overexpression increased metastatic burden. Together, these findings identify XBP1s-dependent ER proteostasis and COPII-associated trafficking as components of a stress-adaptive program that limits caspase-dependent anoikis and supports metastatic colonization in melanoma.