Radiation‐induced extracellular vesicles from cancer‐associated fibroblasts drive oesophageal squamous cell carcinoma metastasis via the miR‐193a‐3p/PTEN/Akt pathway

BACKGROUND: The recurrence and metastasis of oesophageal squamous cell cancer (ESCC) following radiation therapy are major treatment challenges. Cancer-associated fibroblasts (CAFs) are key in the ESCC microenvironment, yet their role in post-radiation recurrence remains unclear. MATERIALS AND METHODS: KYSE150 ESCC cells were co-implanted with non-irradiated (0 Gy) or irradiated (8 Gy) CAFs in nude mice. CAF-derived extracellular vesicles (EVs) were isolated via differential centrifugation and analysed by electron microscopy and immunoblotting. Transwell assays evaluated EVs' effects on ESCC cell migration and invasion in vitro. RNA sequencing identified differentially expressed microRNAs, and functional experiments verified the role of miR-193a-3p. Plasma samples from 32 ESCC patients and tissue samples from 76 ESCC patients were analysed for miR-193a-3p expression. RESULTS: Irradiated CAFs promoted the lung metastasis of ESCC cells in vivo, and their EVs enhanced ESCC cell invasion, migration and metastasis. Elevated miR-193a-3p levels in EVs from irradiated CAFs increased miR-193a-3p expression in ESCC cells. This effect was effectively attenuated by RNase and Triton X-100 (degrading microRNAs encapsulated in EVs), or GW4869 (inhibiting EVs biogenesis and secretion)-indicating that miR-193a-3p functions in an EV-dependent manner. Knockdown of miR-193a-3p diminished the invasion, migration and epithelial-mesenchymal transition (EMT)-promoting activities of CAF-derived EVs. Luciferase assays confirmed PTEN as a target of miR-193a-3p; miR-193a-3p overexpression decreased PTEN and increased p-Akt expression. In vivo, coinjection of miR-193a-3p-knockdown CAFs with KYSE150 ESCC cells resulted in smaller tumours, fewer lung metastases, increased PTEN and E-cadherin, and decreased p-Akt and Snail expression. Clinically, radiation increased plasma exosomal miR-193a-3p levels, and high miR-193a-3p expression was correlated with shorter survival, identifying miR-193a-3p as an independent predictor of poor prognosis in ESCC patients. CONCLUSION: EVs from irradiated CAFs promote ESCC metastasis via the miR-193a-3p-mediated PTEN/Akt signalling pathway. Targeting this EVs-mediated interaction represents a promising strategy for improving ESCC radiotherapy outcomes. KEY POINTS: The poor prognosis of oesophageal squamous cell carcinoma (ESCC) is largely driven by recurrence and metastasis following radiation therapy. Irradiated cancer-associated fibroblasts (CAFs) drive ESCC recurrence and metastasis through extracellular vesicles (EVs), highlighting their critical role in the post-radiation tumor microenvironment. CAF-derived EVs deliver miR-193a-3p to ESCC cells, suppressing PTEN and activating Akt signaling, thereby enhancing invasion, migration, epithelialmesenchymal transition (EMT), and metastatic potential. High plasma exosomal miR-193a-3p levels predict poor prognosis in ESCC patients and may guide therapeutic strategies after radiotherapy.