C57BL/6 Mouse Vein Endothelial Cells

Conditioned Media from Gastric Cancer Spheroid Cells Promotes Angiogenesis

The previous work demonstrated that KRAS activation in gastric cancer cells induces epithelial-to-mesenchymal transition (EMT) and generates cancer stem-like cells (CSCs). Here, Yoon et al. investigated how KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric CSCs were found to secrete pro-angiogenic factors such as VEGF-A, and KRAS inhibition markedly reduced their secretion.

They utilized a genetically engineered mouse model of gastric adenocarcinoma (GA) driven by Trp53 loss, Cdh1 loss, and oncogenic KrasG12D in gastric parietal cells (Tcon mice). These mice developed intestinal and diffuse-type GAs with 100% penetrance and metastases to lymph nodes, lung, and liver. Tumor-derived organoids and cell lines (Tcon3077 and Tcon3944) were generated from two Tcon tumors. As spheroid culture enriches for CSCs, they compared conditioned media from Tcon3944 monolayer versus spheroid cells in tube formation assays with mouse vein endothelial cells (MVEC). Spheroid-conditioned media induced 2.2-fold greater tube formation than monolayer-conditioned media (Fig. 1A), with similar results for Tcon3077 cells. Human GA cell lines (AGS, MKN-45, SNU668, KATOIII) grown as spheroids similarly showed 1.9-4.3-fold increased HUVEC tube formation compared to monolayers (Fig. 1B). To determine if KRAS drives angiogenic factor secretion, KRAS was knocked down using shRNA (efficiency confirmed by Western blot; Fig. 1C). KRAS knockdown in Tcon3944 spheroid cells reduced MVEC tube formation by 86.3% (Fig. 1D), with comparable results in Tcon3077 cells and human GA spheroid cells (75-91% reduction; Fig. 1E). These data indicate that KRAS activation in GA spheroid cells promotes pro-angiogenic factor secretion.