YBX1 takes actions on triggering M2-like polarization of macrophages and stabilizing CXCL8 mRNA to exhibit its metastatic potential in bladder cancer.

BACKGROUND

Patients diagnosed with bladder cancer (BCa) with lymph node (LN) metastasis face a grim prognosis with limited treatment options. We examined the relationship between Y-box binding protein 1 (YBX1) and tumor-associated macrophages (TAMs) concerning LN metastasis in BCa.

METHODS

Popliteal lymphatic metastasis model was constructed in Balb/c mice. Histological examinations were conducted using hematoxylin and eosin (HE) and Immunohistochemical staining. Flow cytometry detected M1/M2 polarization markers. Immunofluorescence staining tested distribution of interleukin enhancer binding factor 3 (ILF3) and YBX1 and macrophage markers. Transwell and tube formation assays assessed migration and angiogenesis. Enzyme-linked immunosorbent assay (ELISA) measured C-X-C motif chemokine ligand 8 (CXCL8), transforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGFA) and macrophage markers. Levels of mRNA and protein were measured by RT-qPCR and Western blot. Subcellular localization of ILF3 and CXCL8 was detected utilizing fluorescence in situ hybridization (FISH) assay. Exosomes derived from BCa cells were isolated and identified. RNA immunoprecipitation (RIP) and Co-immunoprecipitation (Co-IP) validated molecular interactions.

RESULTS

Knockdown of YBX1 in BCa cells suppressed lymphangiogenesis in vitro and in vivo and reduced M2 macrophage polarization. CXCL8 levels, elevated in BCa patients, were positively correlated with YBX1 and M2 macrophage infiltration, and this elevation was reduced upon YBX1 knockdown. BCa cell-derived exosomes containing YBX1 were internalized by macrophages, where they were crucial for inducing M2-like polarization and promoting CXCL8 expression, ultimately stimulating angiogenesis and lymphangiogenesis. Mechanistically, YBX1 interacted with ILF3 to stabilize CXCL8 mRNA.

CONCLUSION

By inducing macrophage M2-like polarization, YBX1 promoted lymphangiogenesis and lymphatic metastasis in BCa, which may provide novel clinical markers for LN metastatic BCa.