Relationship between CCM3 expression and angiogenesis in esophageal squamous cell carcinoma and its possible mechanism.

Abstract

Background: Angiogenesis is essential for esophageal squamous cell carcinoma (ESCC) progression, yet clinically actionable regulators remain limited. We investigated the expression, functional role, and mechanism of CCM3 in ESCC and its relationship with the HIF-1α/VEGFA angiogenic axis.

Methods: CCM3, HIF-1α, VEGFA and microvessel density (MVD, CD31) were assessed by immunohistochemistry in 53 paired ESCC and adjacent non-cancerous tissues. CCM3 knockdown was optimized using four siRNAs in KYSE-70 and KYSE270 cells. CCM3 siRNA 1064-A was selected as the most effective and optimal knockdown achieved at 8 nM, 48 h. Tumor-conditioned HUVECs (referred to as tumor-conditioned endothelial cells, TECs) were generated using conditioned media from knockdown cells; TEC proliferation (CCK8), migration (scratch), invasion (Transwell) and tube formation were evaluated in vitro. A subcutaneous xenograft model using KYSE-70 cells was employed in vivo, followed by intratumoral silencing of CCM3 to assess tumour development, proliferation (Ki-67), and microvessel density (CD31).

Results: High endogenous CCM3 expression was significantly upregulated in ESCC tissues compared to adjacent non-cancerous tissues and correlated with clinicopathological features like depth of invasion, lymph node metastasis and advanced stage. CCM3 expression positively correlated with HIF-1α and VEGFA. CCM3 knockdown siRNA 1064-A attenuates TEC proliferation, migration, invasion and tube formation in vitro. In vivo, CCM3 knockdown reduced tumor growth, tumor weight, Ki-67, CD31 staining and levels of HIF-1α and VEGFA.

Conclusions: High endogenous CCM3 expression associates with aggressive ESCC clinicopathological features and angiogenesis markers. CCM3 knockdown inhibits tumor-conditioned endothelial function, xenograft growth, intratumoral vascularization, and HIF-1α/VEGFA expression, warranting further mechanistic investigation.