Whole-Transcriptome Sequencing Reveals Hypoxic Esophageal Squamous Cell Carcinoma-Derived Migrasomes Driving Cancer-Associated Fibroblast Activation

Cancer-associated fibroblasts (CAFs) activated by intercellular communication contribute to the progression of esophageal squamous cell carcinoma (ESCC). Migrasomes represent a novel mode of intercellular communication. However, the characteristics of ESCC-derived migrasomes in tumor hypoxic microenvironments and their effects on CAFs remain unclear. Migrasomes were isolated from ESCC cells under normoxia/hypoxia, with TSPAN4-GFP labeling, TEM, and western blot for validation. Hypoxic migrasome whole-transcriptome was analyzed by RNA sequencing. Their effect on CAFs was assessed using fluorescence tracing, ELISA, and western blot. We found that ESCC cells could generate migrasomes. Hypoxic conditions did not significantly affect the number, ultrastructure, or expression of CPQ and PIGK of migrasomes. Whole-transcriptome sequencing revealed that hypoxic migrasomes exhibited significant RNA composition changes in mRNA, lncRNA, and circRNA vs normoxic migrasomes. Differentially Expressed (DE) mRNAs were enriched in 'Response to hypoxia', 'Metabolic pathway', 'Cell polarity establishment', and 'HIF-1 signaling pathway'. DElncRNAs were enriched in Golgi to plasma membrane protein transport' and 'Cell adhesion molecules pathway'. DEcircRNAs were enriched in 'ubiquitin binding' and 'chromatin remodeling'. Predicted ceRNA networks screened by RNAhybrid and Miranda contained 659 miRNAs, 24 lncRNAs, and 132 mRNAs. Moreover, fibroblasts were found to take up migrasomes and transition into a CAF phenotype, exhibiting increased secretion and expression of pro-inflammatory cytokines (IL-1 beta, TGF-beta), particularly under the influence of hypoxic migrasomes. This study characterized hypoxic migrasome whole transcriptome landscapes and revealed their role in CAF activation, uncovering a novel ESCC-tumor microenvironment interaction mechanism and offering new perspectives for ESCC research.