Self-Renewal Assessment and Isolation of CD133 Positive Cancer Stem Cells from the Ovarian A2780 Cell Line

Background: Ovarian cancer (OC) is the 7th most common type of cancer and the 5th cause of cancer-related death among women worldwide. It is a heterogeneous disease which is quite variable from the genomic and histopathological aspect. In addition to the usual treatments for ovarian cancer, its recurrence is quite common, mainly due to lack of complete eradication of cancer stem cells. These cells have different properties such as self-renewal ability and stemness property, including proliferation.Method: In the present study, we isolated cancer stem cells with the CD133 surface marker from the ovarian A2780 cell line and examined the stemness property and self-renewal ability of these cells. Initially, CD133 surface marker expression in this cell line was assessed by the flow cytometry technique. Then, the isolation of these cells was performed by the Magnetic-activated cell sorting (MACS) method. Flow cytometry (FCM) was also used to confirm the isolation efficiency. The levels of mRNA expression were evaluated in several stem cell markers in CD133+ cells compared with CD133- cells. Moreover, the self- renewal ability of the isolated cells was investigated in serum-free culture medium.Results: Ovarian cancer stem cells (OCSCs) with CD133 surface marker showed high expression of some stemness markers such as Sox2, Nanog, Oct4, ABCG2, ALDH1, LGR5 and Msi. These cells also had the ability to regenerate themselves in a serum-free environment.Conclusion: Cancer stem cells can be isolated through surface markers by the MACS technique; they have stemness and self-renewal properties. So, the CD133 surface marker can be introduced as a key CSC marker in the isolation, characterization and targeted therapy of ovarian cancer patients.

Silencing HSF4 can inhibit the proliferation of HCC cells

Purpose: To explore the regulatory role of HSF4 in hepatocellular carcinoma stemness property maintaining and analysis the clinical significance of HSF4 in hepatocellular carcinoma. Materials and methods: Tumor spheroid formation assay was conducted to assess stemness property and enrich hepatocellular carcinoma stem-like cells. qRT-PCR and Western Blot was used to detect genes mRNA and protein expression level. KM-plotter database wad used to analyze the correlation between HSF4 and overall survival (OS) in HCC patients. Result: mRNA level of HSF4, as well as stemness-associated genes, was significantly higher in hepatocellular carcinoma tissue then in adjacent normal tissue. Positive correlation between expression level of HSF4 and SOX (r=0.1668, p<0.05), NANOG (r=0.7, p<0.05), POUSF1(r=0.7362, p<0.05), CD44(r=0.0128, p<0.05) was observed. The KM plotter showed that there is no significant difference between HSF4 high patients and HF4 low patients in term of overall survival (OS)(p=0.48). However, significant difference in terms of progression-free survival (PFS)(p=0.019) and relapse-free survival (RFS)(p=0.005) between these two groups was observed. In vitro assay results also suggest the positive correlation between HSF4 and stemness-associated genes. Increased HSF4 expression confers HCC enhanced tumor spheroid formation ability. Conclusion: HSF4 is significantly increased in liver cancer stem-like cells, indicating the possible contribution of HSF4 to stemness properties maintenance in liver cancer stem-like cells. Silencing HSF4 inhibits proliferation of hepatoma cells.