CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell line HCT-116

Optimization of flow cytometric detection of CD133 expression in cell lines. (A) The teratocarcinoma cell line NTERA-2 was stained as a control for CD133 cell surface expression analysis by flow cytometry using a PE-conjugated anti-CD133 antibody. Membrane defect propidium iodide (PI) positive cells were excluded. (B) The advantage of the application of Fluorescence Amplification by Sequential Employment of Reagents (FASER) is representatively shown for HCT-116 cells. Fluorescence signal is enhanced and thus allows the clear discrimination of CD133⁺ and CD133⁻ HCT-116 subpopulations. This protocol was applied for all cell lines studied. The analysis was adapted individually for each cell line.

Pattern of CD133 expression in 10 different CRC cell lines indicates three different phenotypes. (A) According to the cell surface expression of CD133 in flow cytometry analyses the cell lines can be categorized into three groups, a first group contains cell lines in which >95% of the cells are CD133⁺, a second group without CD133 cell surface presentation and a third group of lines with two distinct subpopulations, i.e. with and without CD133 cell surface expression. Membrane defect propidium iodide (PI) positive cells were excluded. For every cell line a representative flow cytometric dot plot diagram of CD133 fluorescence versus propidium iodide signal (left panel) and a histogram overlay of the CD133 expression relative to a corresponding isotype control are shown (right panel). (B) The pattern of cell surface presentation was confirmed by Western blot analysis using whole cell protein extracts (50μg protein per lane) for most of the cell lines with the exception of DLD1 and HCC2998. The predicted size of CD133 is 97kDa, a higher band of ∼120kDa indicates a glycosylated CD133 protein. β-Actin is detected as control protein.

Protocol to separate CD133 subpopulations by fluorescence activated cell sorting (FACS) with purities >98%. According to flow cytometric setup that includes staining with fluorescence conjugated anti-CD133 antibody, signal enhancement by FASER and the exclusion of membrane defect, PI positive cells, a sort layout for the cell line HCT-116 was defined for FACS isolation of CD133⁺ and CD133⁻ HCT-116 cell populations. Separated subpopulations were routinely analyzed for purity as shown for a representative experiment (right panel). In addition fluorescence signal intensities of reanalyzed subpopulations and original HCT-116 are visualized in a histogram overlay. Whole cell protein was extracted from the separated HCT-116 subpopulations and the original cell line. The corresponding Western blot analysis reveals that no CD133 protein is expressed in the CD133⁻ HCT-116 subpopulation.

CD133⁺ and CD133⁻ HCT-116 cells do not differ in 2-D and 3-D culture. In vitro culture characteristics were monitored for FACS separated CD133⁺ and CD133⁻ HCT-116 subpopulations in comparison to the original mixed HCT-116 cells that underwent sort procedure (run through sorter) or not (original). Representative experiments are shown. Experiments were performed at least in triplicate. (A) Monolayer growth kinetics recorded after seeding of separated HCT-116 subpopulations and HCT-116 reveals no differences in 2-D growth. Average plating efficiencies (+SD) from n=4 independent experiments demonstrate comparable colony forming capacity for HCT-116 cells differing in their CD133 expression in (inlay). (B) Also, spheroid formation capacity of CD133⁺, CD133⁻ and original HCT-116 cells in liquid overlay technique is comparable. (C) Distribution of CD133 subpopulations was monitored during 14days of 2-D and 3-D in vitro culturing according to (A) and (B). Fractions of CD133⁺ and CD133⁻ HCT-116 subpopulations did not change dramatically compared to the original sorted populations shown as FACS purity in the left columns.

No difference in radioresponse of CD133⁺ and CD133⁻ HCT-116 subpopulations. Colony formation assays (CFAs) were performed for CD133⁺, CD133⁻ and originally distributed HCT-116 that underwent sort procedure (run through sorter). CFA set-up: 300 cells/well; culturing in respective media for 10days. (A) Representative dose response curves of CD133⁺, CD133⁻ and original HCT-116 cells after irradiation with a single dose regime (0.5–12Gy) 4h after plating. Clonogenic survival at 0Gy (control) for each condition was set to 100% for normalization. (B) Mean clonogenic survival at 2Gy (SF_2Gy+SD) as determined from n=4 independent experiments according to (A). Radioresponse of CD133⁺, CD133⁻ and non-separated HCT-116 cells do not differ.

No difference in subcutaneous tumor formation of CD133⁺ and CD133⁻ HCT-116 subpopulations. FACS separated HCT-116 cell subpopulations were injected s.c. into NMRI (nu/nu) mice using different cell numbers according to table (A) in experiments 1 and 2. (A) Xenograft tumors were induced in all mice independent of the HCT-116 subpopulation and the number of cells injected. (B) No difference in xenograft tumor growth was detected as shown for the representative set of tumors from experiment 2 after injection of 2.5×10³ cells. Tumors were dissected at an average diameter of 0.6–0.8cm. (C) Tumor material was dissociated and analyzed via flow cytometry after multicolor staining for CD133 (PE), CD326 (FITC; to exclude cells that are not of human epithelial origin) and PI (membrane defect cells). Representative CD133/CD326 dot blot diagrams are shown for tumor samples #133 (CD133⁺ HCT-116 subpopulation injected), #136 (CD133⁻ HCT-116 subpopulation injected) and #141 (originally distributed HCT-116 injected); membrane defect cells were excluded. The average percentage of CD326 negative cells was 3%. (D) The CD133⁺:CD133⁻ distributions following sort (FACS purity) and in xenografts derived from injection of the various subpopulations (experiment 2) are documented. Xenograft tumors originated from CD133⁻ HCT-116 cell populations imply a potential increase in CD133⁺ fraction in vivo, whereas tumor cells derived from CD133⁺ HCT-116 do not change accordingly with respect to their CD133 expression profile.