CD133+ colon cancer cells are more interactive with the tumor microenvironment than CD133- cells

Abstract

Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133-negative (CD133-) cells. We hypothesized that CD133-positive (CD133+) cells, compared with CD133-, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma-associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and CD133- cells using fluorescence-activated cell sorter. The CD133+ cells formed large tumors in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133- cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs CD133- cells. RT-PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs - cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2. The CAF highly express the respective ligands: stromal-derived factor-1 (SDF-1), vitronectin and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in intracellular calcium in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension compared with only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew more colonies compared with vehicle, as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared with CD133-, cells is due to their increased ability to interact with their neighboring CAF.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1

Phase contrast image of characteristic features of primary colon cancer cells and CAF isolated from a patient with Stage II disease maintained. (A) Mixed cell population with two phenotypes in cell culture, consisting of (B), a non-adherent colon cancer cell line that form spheres in culture (phase contrast microscopy) that is cytokeratin-positive (pan- cytokeratin AE1/3), and (C) carcinoma-associated fibroblasts (CAF) that stain for vimentin, but not cytokeratin, with a subpopulation that are α-SMA-positive. Original Magnification 400X.

Figure 2

Immunohistochemical characterization of the original patient tumor sample (400× original magnification), compared to the colospheres maintained in vitro (200× original magnification).

Figure 3

(A) Comparison of fluorescence by colon cancer cells labeled with AC133, an antibody to CD133 conjugated to R-phycoerythrin (PE) and istotype-matched IgG controls labeled with PE (Miltenyi Biotec; Auburn, CA). (B) Fractions colon cancer cells with high expression levels of CD133 (positives) and relatively low expression levels (negatives) collected by fluorescence-activated cell sorting (C) CD133+ and CD133− cells were serially diluted, resuspended in Matrigel, and injected subcutaneously in NOD-SCID mice to assay for tumorigenic potential; representative mouse injected with CD133+ cells at sacrifice shown. (D) Summary of tumor volumes measured at 6 weeks comparing CD133− vs. CD133+ tumors. (E) Representative hematoxylin and eosin stain (H&E) of the tumor xenograft (40×). (F, G) Inset from xenograft tumor demonstrates a moderately differentiated tumor and well-differentiated tumor forming glandular structures (400×), respectively. (H) Representative H&E of the tumor from the patient’s tumor block (40×). (I, J) H&E (400X) from patient’s original tumor block showing similar cellular features from the mouse xenograft, moderately and well-differentiated features.

Figure 4

Heat Map Comparing CD133+, CD133− colon cancer cells with the accompanying CAF cells. Unsupervised hierarchical clustering comparing the CD133+, CD133−, and CAF cells.

Figure 5

Colospheres, enriched for CD133+ or relative lack of CD133 (CD133−), sorted by flow cytometry, were maintained in culture for up to 8 weeks. (A) Overlay of histograms (from left to right) comparing fluorescence of CD133+ cells labeled with IgG-APC (negative control), AC133-labelled CD133-negative cells, and AC133-labelled CD133+ (enriched) cells. (B) After 8 weeks, the enriched cells each maintained their respective CD133 phenotype. Co-expression of cell surface CD133 and CXCR4 in CD133+ cells and CD133-negative cells.

Figure 6

Effects of growth conditions (suspension vs. attached) on co-expression of CD133 and CXCR4. (A) Photomicrograph (400×) of colon cancer cells grown in suspension culture (low adhesion flasks). The cells maintain anchorage-independence growth after 9 days. (B) Photomicrograph (400×) of same cells grown in standard tissue culture flasks. After 9–14 days, the cells adhere to the bottom of the flask and exhibit a spread phenotype, consistent with anchorage dependence. (C) FACS analysis demonstrating that when grown in suspension culture, 32% of the cells are double-positive for cell surface CD133 and CXCR4, and that 30% of cells are double-negative for both receptors. (D) When the cells are allowed to adhere and spread, the double-positive cells decrease to 8.9%, while the double-negative cells increase to 46.7%.

Figure 7

Characterization of CD133+/CXCR4+ cells. (A) CD133+/CXCR4+ cells exhibits an increase in intracellular calcium in response to 400 ng/ml of SDF-1 (CXCL12). Subsequent treatment with 10 µM carbachol to insures that the cells were loaded with Fura-2. (B) CD133+/CXCR4− cells do not respond to SDF-1; however, these cells do have muscarinic receptors, as verified by their calcium response to carbachol, indicating that the calcium indicator dye Fura-2 was loaded in the cells. (C) CD133−/CXCR4− cells fail to increase intracellular calcium with SDF-1, but does respond to carbachol. (D, E, F) Effects of cell growth in Matrigel in the presence of SDF-1 (400 ng/ml) or vehicle (0.2% BSA). The average number of cell colonies per well are denoted by the number in the corner of the photo. Photomicrographs (original magnification, 400X) of representative areas of CD133+/CXCR4− (D), CD133+/CXCR4+ (E), CD133−/CXCR4− (F) cell colonies are shown. (G) Bar graph depicting average diameter of the cell colonies, corresponding to figures shown in D, E, F.

Figure 8

Co-staining of CD133 and α-SMA showing the spatial relationship between CD133+ cells and CAF, respectively. (A, B) Immunohistochemical characterization of the original patient tumor sample (600×, original magnification) of a representative area with (A) CD133− colon cancer cells and paucity of CAF and (B) CD133+ colon cancer cells (red stain) surrounded by relative abundance of CAF (brown stain). (C, D) Representative area from another patient in area of (C) normal colonic villi surrounded by α-SMA-positive (brown stain) and (D) malignant tumor with CD133+ (red) and CD133− cells, 200×. (E, F) Colon cancer from third patient in areas with (E) CD133− colon cancer cells and (F) CD133+ colon cancer cells, 100×.

Figure 9

Paired samples of CD133− and CD133+ colon cancer cells from 11 colon cancers. The slides are co-stained for CD133 (red, apical) colon cancer cells and α-SMA (400× and 600×). The arrow demonstrates α-SMA immunopositivity in the walls of small blood vessels (internal positive control).