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. 2008 Jun;118(6):2111-20.
doi: 10.1172/JCI34401.

CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors

Sergey V Shmelkov  1 , Jason M Butler, Andrea T Hooper, Adilia Hormigo, Jared Kushner, Till Milde, Ryan St Clair, Muhamed Baljevic, Ian White, David K Jin, Amy Chadburn, Andrew J Murphy, David M Valenzuela, Nicholas W Gale, Gavin Thurston, George D Yancopoulos, Michael D'Angelica, Nancy Kemeny, David Lyden, Shahin Rafii
Affiliations

CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors

Sergey V Shmelkov et al. J Clin Invest. 2008 Jun.

Abstract

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10-/-CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133- population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133- metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133- cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24-), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133(- )subset, which is also capable of tumor initiation in NOD/SCID mice.

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Figures

Figure 1
Figure 1. Design and validation of CD133lacZ/+ mouse model for tracking CD133+ cells.
(A) Structure of murine CD133 promoter region, genomic structure of murine CD133 gene, and the lacZ knockin strategy. AUG, start codon; Ex1A, exon 1A. (B) X-gal staining of the adult mouse kidney shows high β-galactosidase activity in the proximal tubule (PT), but not distal tubule (DT). Staining with anti-CD133 antibody localizes CD133 protein specifically to the brush border of PT. Coimmunofluorescence of anti-CD133 with anti-CD31 antibodies shows CD133 expression on the parietal layer of Bowman’s capsule, but not the glomeruli (GL). Scale bar: 50 μm.
Figure 2
Figure 2. CD133 expression identifies differentiated luminal epithelium in organs with hollow cavities.
(A–D) CD133 expression in bile ducts of murine liver. X-gal staining (which reflects β-galactosidase expression) of CD133lacZ/+ mouse liver (A) and immunohistochemistry of wild-type mouse liver with anti-CD133 antibody (B) localize CD133 expression to a columnar cells within the hepatic triad morphologically defined as a bile duct (BD). Costaining with X-gal and anti-CD31 antibody (C) and coimmunofluorescence with anti-CD133 (red) and anti-CD31 antibodies (green) (D) demonstrate that CD133 is not expressed on the vessels of hepatic triads. Original magnification, ×200 (A, inset); ×400 (B and C, insets). (E–H) CD133 expression in pancreatic ducts. X-gal staining of CD133lacZ/+ mouse pancreas (E) and immunohistochemistry of wild-type mouse pancreas with anti-CD133 antibody (F) localize CD133 expression to a ductal epithelium. Costaining with X-gal and anti-CD31 antibody (G) and coimmunofluorescence with anti-CD133 (red) and anti-CD31 antibodies (green) (H) demonstrate that CD133 is not expressed on pancreatic vessels. (I–L) CD133 expression in bronchus. X-gal staining of CD133lacZ/+ mouse lung (I) and immunohistochemistry of wild-type mouse lung with anti-CD133 antibody (J) localize CD133 expression ubiquitously to all of the ciliated bronchial epithelium. Costaining with X-gal and anti-CD31 antibody (K) and coimmunofluorescence with anti-CD133 (red) and anti-CD31 antibodies (green) (L) demonstrate that CD133 is not expressed on the mature vessels in lungs. BRN, bronchus; BV, blood vessel; CV, central vein; PD, pancreatic duct; PV, portal vein. Scale bar: 50 μm.
Figure 3
Figure 3. CD133 is widely expressed in murine colonic epithelial lining.
(A–C) X-gal staining shows the distribution of β-galactosidase activity in the colon of CD133lacZ/+ mice (A), and in sagittal (B) and transverse (C) sections of the Lieberkuhn’s crypt. (D–F) Corresponding immunostaining with anti-CD133 antibody confirms the X-gal staining and demonstrates that CD133 protein is localized to the apical surface of mouse colon epithelium (indicated with the arrows). (G) X-Gal staining of a wild-type mouse colon. (H) Magnification of rectangle in E showing the brush border localization of CD133 protein. The inset shows a negative control staining, where arrows point to brush borders on both sides of the lumen. Original magnification, ×200 (inset). (I) Coimmunofluorescence with anti-CD31 antibody (green) and anti-CD133 antibody (red) shows the apical localization of CD133 protein in mouse colon (indicated with arrows). Scale bar: 50 μm.
Figure 4
Figure 4. CD133 is expressed in differentiated epithelium of normal human colon.
(A and B) Immunostaining with CD133 (AC133) antibody (red) demonstrates the luminal localization (indicated with the arrows) of CD133 in normal human colon. (C and D) Coimmunofluorescence with CD133 (AC133) antibody (red) and EpCAM (a marker of epithelial cells) antibody (green) shows wide distribution of CD133 expression on differentiated epithelium in normal human colon, demonstrating consistency with the murine model. C shows a transverse section of the crypt. D shows a saggital section of the crypt. Scale bar: 50 μm.
Figure 5
Figure 5. CD133 is expressed ubiquitously in all malignant cells of both human and mouse primary colon tumors.
(A–D) CD133 expression on human primary colon cancer epithelium is shown by anti-CD133 antibody (A and B) and anti-EpCAM antibody (D); the negative IgG control is shown in C. Note the broad expression of CD133 in all malignant epithelial cells. CD133 protein expression is localized to the apical region of the columnar colonic cancer cells in primary human tumors. (E–H) CD133 expression in primary colon tumor of Il10–/–CD133lacZ mice. H&E staining (E and F) shows the site of the primary tumor in the murine colon; X-gal (G) and anti-EpCAM (H) antibody immunostaining corroborate the human primary colon cancer data. Note the broad expression of CD133 in all transformed epithelial cells in the Il10–/–CD133lacZ mice as shown by the lacZ reporter. Scale bar: 50 μm.
Figure 6
Figure 6. Metastatic colon cancer comprises both EpCAM+CD133+ and EpCAM+CD133– tumor cells.
Costaining of colon cancer metastases to the liver with anti-EpCAM (green) and anti-CD133 (red) antibodies. (A and C) Typical examples of metastatic colon cancer harboring 2 subpopulations of tumor cells, EpCAM+CD133+ and EpCAM+CD133–. (B and D) Metastatic colon cancer completely lacking expression of CD133. The arrows indicate CD133 expression. Scale bar: 50 μm.
Figure 7
Figure 7. Both CD133+ and CD133– subsets of human metastatic colon cancer exhibit long-term tumorigenic potential.
(A) Serial xenograft strategy. (B) FACS analysis of human primary metastatic colon cancer cell subsets demonstrates their purity after separation with CD133 (AC133) antibody. Numbers indicate the percentage of negative and positive cells within the analyzed populations. (C) Subcutaneous tumors derived from the tertiary injection of the CD133– subset of human colon cancer grow faster in NOD/SCID mice than tumors which were derived from the CD133+ subset. Original magnification, ×0.25 (left panel). The units for the right panel of C are centimeters. (D) Growth curve of xenograft tumors in NOD/SCID mice shows that even after 3 serial transplantations CD133– cancer cells are capable of tumorigenesis; they initiate tumor formation earlier and exhibit faster growth rate than the CD133+ fraction. Data are shown as mean ± SEM; n = 3.
Figure 8
Figure 8. Both CD133+ and CD133– xenografts form colonospheres, while CD133– cells display the molecular signature of cancer stem cells.
(A) CD133+ and CD133– human colon cancer xenografts dissociated to a suspension of single cells are capable of forming colonospheres. Original magnification, ×200. (B) Colonospheres were subject to FACS analysis with CD133 antibody to confirm that the expression pattern was maintained in both CD133+ and CD133– colonies after in vitro culture. Both fractions were analyzed for the expression of CD44, CD24, and carcinoembryonic antigen (CEA). The expression pattern of these stem cell markers indicates the lesser level of maturity (CD44+CD24–) and higher level of aggressiveness (CEA+) of the CD133– fraction of human colon cancer. Numbers indicate the percentage of positive cells within each population. Arrows refer to the stained population within the selected gates with the indicated markers.
Figure 9
Figure 9. CD133+ cells do not contribute to long-term tumorigenesis in CD133– human metastatic colon cancer xenografts.
(A) H&E staining shows similar tertiary xenograft morphology in both CD133+- and CD133–-derived tumors. Immunofluorescence demonstrates that CD133 (red) expression is maintained in tertiary CD133+-derived and absent in CD133–-derived tumors. (B) CD133 mRNA expression in CD133+ and CD133– xenografts by quantitative PCR (qPCR) and RT-PCR. (C) FACS analysis of tertiary xenografts of human metastatic colon cancer cell subsets in NOD/SCID mice demonstrates the absence of CD133+ cells in CD133–-derived tumors. Numbers indicate the percentage of negative and positive cells for indicated markers within the analyzed populations. Scale bar: 50 μm.
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