Verification of ALDH activity as a biomarker in colon cancer stem cells-derived HT-29 cell line

Background: Recent evidence has suggested that epithelial cancers including colorectal cancer [CRC] have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells [CSCs] which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 [ALDH1] activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer

Objectives: The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line

Materials and Methods: In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice

Results: According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers [P < 0.001]. Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells [P < 0.05]. In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells [P < 0.05]. Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor

Conclusions: For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line

[Characterization of stemness properties of colonospheres in colon adenocarcinoma patients]

Proliferation and expansion of cancer stem cells as spheroids were proved in previous studies. But, capability of primary tumor-derived stem cells to keep their unique properties in vitro is still disputed. So, the goal of this study was to isolate, expand and characterize of colon cancer-derived stem cells. In the present work, colon cancer stem cells markers including CD44 and EPCAM in spheroid and parental cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and parental cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated and used implantation of tumor xenografts into nude mice. Our data shows 79% of spheroids were CD44+/EpCAM+, while parental cells only expressed 20% of CD44/EpCAM markers [p< 0.01]. In compared with the parental cells, the expression levels of "stemness" genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells [p< 0.05]. Furthermore, as little as 1000 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x10[6] of parental cells was needed to generate tumor. Sphere formation assay is a useful method to enrich cancer stem cells. Spheroid cells showed increasing expression of stemness genes and tumorigenic activity in nude mice