The study of colon cancer SC would be greatly enhanced by availability of specific markers to identify and isolate these cells. One key finding of this study was that ALDH identifies colonic cells that exhibit known SC properties: (a) immunohistochemistry identifies a small subpopulation of ALDH1+ cells (≤5%) localized to the bottom of normal crypts (where SC reside) and (b) the Aldefluor assay can be used to isolate a subpopulation of malignant colonic cells that generates xenograft tumors (functionally showing the ability for self-renewal). Aldefluor− cells from CRCs did not form xenograft tumors. Thus, our findings substantiate that ALDH1 and Aldefluor are specific markers for SC in normal and malignant colon.
In comparison, CD133 and CD44 positivity on immunohistochemistry and flow cytometry identified a considerably larger subpopulation within normal colonic tissues, subpopulations that were 7-fold the size of the ALDH+
population. These results are consistent with previously reported data (10
). Nevertheless, CD44+
cells from colon cancers were still capable of generating tumor xenografts and could do so with potency similar to that of ALDH1+
cells. These data suggest that CD44 and CD133 label the proliferative cell population that contains both rapidly proliferating cells as well as the much smaller population of colonic SC. That Aldefluor−
cells, even ones that are CD44+
, do not form xenograft tumors suggests that many of the SCs within the CD44+
populations are eliminated by removal of Aldefluor+
cells during sorting. Thus, we conclude that ALDH1 is more specific for SC in the colon than are CD44 or CD133.
Cells that were isolated using multiple SC markers (particularly Aldefluor+/CD133+) had slightly better efficiency in generating tumors than did Aldefluor+ cells. In addition, cells that were positive not only for ALDH but also for CD44 or CD133 tended to generate tumor xenograftsmore rapidly than did ESA+ cells. This suggests that, in cell selection, the addition of CD133 positivity or CD44 positivity to Aldefluor positivity causes somewhat enhanced SC enrichment.
An apparent discrepancy is that no tumors were generated using cells that were isolated as CD44− when CD44 was used as the sole marker for sorting cells, yet there were tumors generated when cells were implanted from populations that were CD44− if they were isolated as ALDH+/CD44− (using these two SC markers in sequence). The same apparent discrepancy applies to CD133− cells. One explanation is that ALDH+/CD44− cells and ALDH+/CD133− cells have tumor-initiating ability because they are ALDH+, but these cells only constitute a small fraction of the total CD44− population and CD133− population. Consequently, implantation of CD44− or CD133− cells (isolated using CD44 or CD133 markers individually) might not generate xenograft tumors because the ALDH+/CD44− and ALDH+/CD133− cells are in such low concentration in the implanted sample. In contrast, when Aldefluor is used to select ALDH+ cells from the ESA+ cell population, and then CD44 or CD133 is used serially for cell selection, the enrichment of ALDH+/CD44− or ALDH+/CD133− cells then becomes high enough to generate tumors.
It is uncertain how the overlap of the CD44+
populations with the ALDH+
population relates to the ability of these populations to initiate tumors. Based on our data, this issue is complex, and our results (, Venn diagrams) suggest that CD44 positivity and Aldefluor positivity are independent variables, as are CD133 and Aldefluor positivity. Although it seems, based on tumorigenic potential, that ALDH is more specific for stemness than CD44 (CD44+
cells are nontumorigenic), the overall high tumorigenic potential of the CD44+
population () cannot be completely explained by a relatively small subset of cells that are also ALDH+
(<10% contained within the CD44+
population). One possible explanation is that CD44+
cells are not by themselves tumorigenic but somehow support the tumorigenic growth of CD44+
cells. This might occur by the implantation site becoming humanized due to CD44+
cells contained in the implanted sample. This humanization effect has been observed when immortalized human fibroblasts are transplanted along with breast cancer cells (22
A second key finding of this study is that ALDH-based markers can be used to track colonic SC during colon tumorigenesis. ALDH1 positivity on immunohistochemistry indicated an increasing number of colonic SC over the course of colon cancer progression, from normal to FAP to adenoma. If 29% of adenoma cells are ALDH1+, and given that an adenoma contains 106 to 108 cells, one can calculate that there is an absolute increase in the number of SC in an adenoma relative to a normal crypt. This trend toward SC overpopulation continued for carcinomas as well. This was deduced from the fact that colon carcinomas typically have 108 to 1013 cells and that our flow cytometric data on Aldefluor indicate that 3.5% of these carcinoma cells are SC.
A third key finding is that this progressive increase in SC number strongly supports our SC overpopulation hypothesis (23
), developed from our mathematical modeling of colon tumorigenesis. In this hypothesis, SC overpopulation is the key event at the cellular level that links the initiating event at the molecular level (typically APC
mutation) to the earliest event at the tissue level (a crypt proliferative abnormality). Our earlier study (28
) using markers for crypt base cells provided biological evidence in support of our SC overpopulation hypothesis. Two other lines of biological evidence support this hypothesis: (a
) a study using methylation patterns as epigenetic markers for colonic cell fate showed that there is enhanced SC survival with CRC initiation in FAP (31
) and (b
) the murine intestinal SC marker, Lgr5, has been reported to be overexpressed in advanced human CRCs (34
). Taken together, these studies suggested that an increased number of colonic SC occur during the initiation and progression of CRC. The current study provides even more compelling biological evidence for our SC overpopulation hypothesis.
Our mathematical modeling also predicted that the overall proliferative cell population (both SC and rapidly proliferating cells) increases during colon tumorigenesis (23
). The increase, with tumor progression, in the proportion of cells along the crypt axis that stain positively for CD44 and CD133 supports that prediction and is our fourth key finding. Comparing results on ALDH1 (that labels SC) with results on CD44 and CD133 (which seem to label SC and proliferating cells) provides an explanation for how colonic SC contribute to tumor initiation and progression (i.e., that expansion of the SC population leads to expansion of the proliferating cell population), and together, they constitute an overall increase in the proliferative cell population during the stepwise progression to colon carcinoma.