ABSTRACT
Cancer stem cells (CSCs) are believed to be responsible for tumor growth, invasion, and metastasis. Submucosal invasion, which greatly enhances metastasis risk, is a critical step in gastric cancer (GC) progression. To identify stem cell-related markers associated with submucosal invasion and lymph node (LN) metastasis in GCs, we investigated the expression of candidate CSC markers (CD133, CD44, and ALDH1A) and intestinal stem cell (ISC) markers (EPHB2, OLFM4, and LGR5) in early GCs that manifested submucosal invasion. We discovered that EPHB2 and LGR5 expression was frequently confined to the basal area of the lamina propria (basal pattern) in mucosal cancer, and the proportion of stem cell marker-positive cells substantially increased during submucosal invasion. CD44 expression showed a focal pattern, ALDH1A was predominantly expressed diffusely, and there was no expansion of CD44 or ALDH1A expression in the submucosal cancer cells. Unexpectedly, no CSC markers showed any associations with LN metastasis, and only loss of EPHB2 expression was associated with increased LN metastasis. Treatment of RSPO2, a niche factor, along with Wnt 3a, to GC cells led to increased EPHB2 and LGR5 mRNA levels. RNA in situ hybridization confirmed specific RSPO2 expression in the smooth muscle cells of the muscularis mucosa, suggesting that RSPO2 is responsible for the increased expression of ISC markers in GC cells at the basal areas. In summary, no stem cell markers were associated with increased LN metastasis in early GCs. Conversely, isolated EPHB2 expression was associated with lower LN metastasis. EPHB2 and LGR5 showed a basal distribution pattern along with enhanced expression in submucosal invading cells in early GCs, which was induced by a niche factor, RSPO2, from the muscularis mucosa.
