Rapid re-expression of CD133 protein in colorectal cancer cell lines in vitro and in vivo

Abstract

Studies related to the cancer stem cell hypothesis are challenging because of the imperfect tools to identify cell populations of interest and controversy on the usefulness of established cancer cell lines. We previously found CD133 to not be selective for a tumor-propagating or radioresistant population in a near-diploid, microsatellite-instable colorectal carcinoma (CRC) cell line. Because of discrepant literature data, we herein systematically analyzed the behavior of microsatellite-stable cell line subpopulations reflecting the more frequent carcinogenesis pathway in spontaneous CRC. CD133 þ and CD133 À /low populations were isolated by fluorescence-activated cell sorting and further processed. HT29 and SW620 cells were studied in detail in monolayer and/or spheroid culture assays and upon subcutaneous injection in NMRI (nu/nu) mice using a limiting dilution approach. CD133À /low HT29 cells showed a significantly lower clonogenic survival and reduced spheroid formation capacity than their CD133 þ counterparts. However, the cell populations neither differed in growth kinetics and response to treatment in vitro nor in tumor formation capacity when injecting as low as 10 cells. CD133 À /low HT29 cells rapidly re-expressed CD133 protein in vitro and in vivo as shown by flow cytometry and/or western blot analyses, and they also showed a particular survival benefit under tissue normoxic conditions. In contrast, CD133 protein in the CD133 þ population was quite stable throughout culturing. The observation of CD133 re-expression and lack of difference in tumor take rate of subpopulations was confirmed in SW620 cells. Here, we found cell density to affect CD133 re-expression in the CD133À -sorted population. And even SW480 cells, classified as a CD133 À cell line, presented some CD133 protein on their surface upon in vivo engraftment. We conclude that (i) CD133 protein expression shows high plasticity in CRC cell lines, and (ii) in vitro CD133 status on the cell surface neither determines tumorigenic potential nor CD133 profile in vivo.

Alkylphosphocholine Analogs for Broad-Spectrum Cancer Imaging and Therapy

“…5, F and G). Notably, a small percentage of CD133 + cells may be retained in the serum-cultured U251 glioma line (25), and serum-cultured tumor lines may reexpress CD133 after in vivo engraftment (26). Therefore, the presence of CD133 + U251 cells within mouse brain tumor xenografts is not unexpected.…”

CD133 Is Essential for Glioblastoma Stem Cell Maintenance

“…Notably, the impairment in self‐renewal capacity and tumorigenic potential of CD133‐negative cells was lost when these cells re‐expressed CD133 on the plasmamembrane. The rapid re‐expression of CD133 on the surface of CD133‐negative cells has been described in different cancer cell types [27] suggesting a conserved mechanism of CD133 regulation in tumors of different tissues. These results suggest that the localization of CD133 on the plasmamembrane is functionally related to the CSC biology and characterizes cell populations with enhanced self‐renewal and tumorigenic capacity.…”

Potential of a Cetuximab‐based radioimmunotherapy combined with external irradiation manifests in a 3‐D cell assay

“…MDA‐MB435S (melanoma) cells from the ATCC (USA) were used as EGFR‐negative controls. All cell lines were free of mycoplasm (PCR Mycoplasma Kit, Applichem, Germany) and were checked for correct genetic profile at the Institute of Legal Medicine (TU Dresden, Germany) by microsatellite analysis as described earlier . For cell transfer and spheroid initiation, exponential monolayer cultures were dissociated (0.05% trypsin/0.02% EDTA in PBS).…”

“…All cell lines were free of mycoplasm (PCR Mycoplasma Kit, Applichem, Germany) and were checked for correct genetic profile at the Institute of Legal Medicine (TU Dresden, Germany) by microsatellite analysis as described earlier. 29 For cell transfer and spheroid initiation, exponential monolayer cultures were dissociated (0.05% trypsin/0.02% EDTA in PBS). A Casy1 cell analyzer (Roche Innovatis, Germany) was applied for assessment of cell culture quality, cell numbers and volumes in single cell suspensions for further processing.…”