Isolation and identification of chemotherapy-enriched sphere-forming cells from a patient with gastric cancer

Gastric cancer (GC) is the third and fifth cause of cancer-associated mortality for men and women throughout the world, respectively. Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Cancer stem cells (CSCs) due to their pivotal role in tumor initiation, growth, progression, invasion, distant metastasis, recurrence and resistance to anticancer drugs are very appealing targets for cancer therapies. Here, we isolated and identified CSCs from a chemotherapy-treated patient. Small subpopulation of dissociated cells after tissue digestion formed spheroid colonies in serum-free media under the non-adherent condition. These spheroid colonies differentiated into epithelial like cells in serum-containing medium. Few sphere-forming cells carried CD44 and CD54 markers overexpressed DLL4 that is responsible for tumor growth and angiogenesis. Subcutaneous injections of sphere-forming cells in different passages conferred tumorigenicity in nude mice. Sphere-forming cells upregulated CD44 polymorphisms CD44v3, -v6, and -v8 -10, stemness factors OCT4, SOX2, SALL4 and Cripto-1, self-renewal molecules IHh, Wnt, -catenin and BMI1, and epithelial mesenchymal transition (EMT) markers Twist1 and Snail1 in vitro and in vivo. Moreover, these cells similar to sphere-forming cells isolated from a chemotherapy-free patient expressed Oct-4 and -catenin proteins. However, the Twist1 protein was only expressed by sphere-forming cells derived from the chemotherapy-treated patient. Thus, these cells have all the characteristics of stationary and migratory CSCs, including tumorigenicity, self-renewal, pluripotency, invasion and metastasis. Taken together, targeting chemotherapy-enriched CSCs as chemo-resistance cells observed in GC patients can provide more effective therapeutic strategies compared to untreated patients.