Tumor-endothelium reciprocal interactions in co-culture: changes of genetics and phenotypic characteristics of endothelial and laryngeal cells

Abstract Objectives This study aims to elucidate the interactions between laryngeal cancer cells (HEp-2) and endothelial cells (HUVEC) within the head and neck squamous cell cancer (HNSCC) microenvironment using direct and indirect in vitro co-cultivation systems. Methods Conditioned mediums (CM) of HEp-2 and HUVEC were generated, followed by the construction of co-culture systems. Confocal microscopy, XTT assay, trypan blue viability assay, real-time cell analysis system (RTCA), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to assess cell morphology, proliferation, migration, invasion, and the expression of cancer-associated genes ( CDH2 , CCL21 , CXCL8 , ITGB , MMP2 , MMP9 , and VIM ). Results We observed that the HEp2 co-culture effect altered HUVEC morphology, also reduced cell proliferation significantly, increased migration (p<0.05), and invasion of the cells. The mRNA levels of CXCL8 and ITGB were downregulated significantly, while the expression level of CCL21 mRNA was markedly upregulated (88.4-fold) compared to mono-culture HUVECs (p<0.05). Meanwhile, the HEp-2s showed a spindle-shaped and scattered morphology after co-culture with HUVEC. In addition, a higher relative migration rate (p<0.05) but significantly reduced proliferation and invasion rates of HEp2s were detected. Conclusions Reciprocal interactions between HEp2 and HUVEC significantly modulate cellular behaviors and gene expression, suggesting that these interactions play a crucial role in laryngeal cancer progression and offering potential insights for HNSCC treatment strategies.