OBJECTIVE: To sort CD133(+) subset cells in human gastric cancer (GC) and to identify their tumor initiating cell-like properties. METHODS: The tissues of GC and normal tissues adjacent to GC were obtained from 50 patients. Samples were stained for CD133 by immunohistochemistry. Likewise, assessments of CD133 were undertaken by Western blot. Flow cytometry was used to determine the proportion of CD133(+) cells in four GC cell lines therein the KATO-III was sorted by magnetic activated cell sorting (MACS) method. The growing characteristics and the tumorigenic ability of CD133(+) cells were evaluated in vitro and in vivo. Meanwhile, the growth of single cells in suspension culture was observed and expression of stem cell-specific marker were determined using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of CD133 was demonstrated on the cell membranes in the mucosa and submucosa of primary GC, which were higher than those in the normal gastric tissues adjacent to cancer (P<0.05). Four GC cell lines including KATO-III, SGC-7901, AGS and MKN-45 were found to contain (28 ± 2)%, (17 ± 2)%, (6 ± 2)%, and (4 ± 2)% of CD133(+) cells respectively. In addition, the purity of CD133(+) cells isolated from KATO-III by MACS was (91 ± 3)% and up to(95 ± 2)% after 1-week culture. CCK-8 detection showed that population doubling time of the CD133(+) cells was (21 ± 3)h, significantly shorter than that of the CD133(-) cells[(40 ± 8)h, P<0.05]. Notably, there was a remarkable difference of tumor formation rate between CD133(+) cells (100%), non-sorted cells (80%), and CD133(-) cells(0). The average mass and volume of tumor in group of CD133(+) cells was larger and heavier than those in non-sorted cells (P<0.05, P<0.05). Furthermore, the single cell proliferated well, formed the big sphere and semi-quantitative RT-PCR showed expression of stem cell markers such as Oct-4, Nanog, Sox-2, Musashi-1 and EGFR. CONCLUSIONS: CD133 protein expression in primary lesions is higher than those in the normal gastric tissues. CD133(+) subset cells can be isolated, purified, and amplified in human GC, and possess some properties including the ability of self-renewal, proliferation, and higher tumorigenic ability in vivo and can express some stem cell markers.
OBJECTIVE: To sort CD133(+) subset cells in humangastric cancer (GC) and to identify their tumor initiating cell-like properties. METHODS: The tissues of GC and normal tissues adjacent to GC were obtained from 50 patients. Samples were stained for CD133 by immunohistochemistry. Likewise, assessments of CD133 were undertaken by Western blot. Flow cytometry was used to determine the proportion of CD133(+) cells in four GC cell lines therein the KATO-III was sorted by magnetic activated cell sorting (MACS) method. The growing characteristics and the tumorigenic ability of CD133(+) cells were evaluated in vitro and in vivo. Meanwhile, the growth of single cells in suspension culture was observed and expression of stem cell-specific marker were determined using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of CD133 was demonstrated on the cell membranes in the mucosa and submucosa of primary GC, which were higher than those in the normal gastric tissues adjacent to cancer (P<0.05). Four GC cell lines including KATO-III, SGC-7901, AGS and MKN-45 were found to contain (28 ± 2)%, (17 ± 2)%, (6 ± 2)%, and (4 ± 2)% of CD133(+) cells respectively. In addition, the purity of CD133(+) cells isolated from KATO-III by MACS was (91 ± 3)% and up to(95 ± 2)% after 1-week culture. CCK-8 detection showed that population doubling time of the CD133(+) cells was (21 ± 3)h, significantly shorter than that of the CD133(-) cells[(40 ± 8)h, P<0.05]. Notably, there was a remarkable difference of tumor formation rate between CD133(+) cells (100%), non-sorted cells (80%), and CD133(-) cells(0). The average mass and volume of tumor in group of CD133(+) cells was larger and heavier than those in non-sorted cells (P<0.05, P<0.05). Furthermore, the single cell proliferated well, formed the big sphere and semi-quantitative RT-PCR showed expression of stem cell markers such as Oct-4, Nanog, Sox-2, Musashi-1 and EGFR. CONCLUSIONS:CD133 protein expression in primary lesions is higher than those in the normal gastric tissues. CD133(+) subset cells can be isolated, purified, and amplified in human GC, and possess some properties including the ability of self-renewal, proliferation, and higher tumorigenic ability in vivo and can express some stem cell markers.