Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis - PubMed (original) (raw)

Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis

Emina H Huang et al. Cancer Res. 2009.

Abstract

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC), investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly, aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom, where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma, ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells, which are more numerous and broadly distributed in normal crypts, showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not), (b) generated them after implanting as few as 25 cells, and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus, ALDH1 seems to be a specific marker for identifying, isolating, and tracking human colonic SC during CRC development. These findings also support our original hypothesis, derived previously from mathematical modeling of crypt dynamics, that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development.

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Figures

Figure 1

Figure 1

ALDH1, CD44, and CD133 expression in colonic tissues. A, immunohistochemical analysis of normal colonic mucosa, normal-appearing colonic mucosa from FAP patients, adenomas, and colon carcinomas. B, staining indices for ALDH1, CD44, and CD133. Although CD44 and CD133 profiles were similar, they were considerably different than ALDH1. AUCs for CD44, CD133, and ALDH1 profiles were as follows: normal crypts, 40.3%, 41%, and 5.6%; normal-appearing FAP crypts, 55.7%, 56.7%, and 14%; and adenomatous crypts, 76.9%, 75.9%, and 29.3%.

Figure 2

Figure 2

Coexpression of SC markers in normal and malignant colonic tissues. A, immunofluorescence staining of normal colonic epithelium shows nuclear staining (DAPI), membranous CD44 expression (green, FITC), and cytoplasmic ALDH1 expression (red, phycoerythrin). The merged image shows a pink-orange color from the overlap of membranous CD44 and cytoplasmic ALDH1 staining, showing that the ALDH1+ population is a subset of the CD44+ population. B, staining of normal colonic epitheliumshows nuclear staining (DAPI), membranous CD133 expression (green, FITC), and cytoplasmic ALDH1 expression (red, phycoerythrin). The merged image reveals a pink-orange color showing overlap of membranous CD133 and cytoplasmic ALDH1 staining and showing that the ALDH1+ population is a subset of the CD133+ population. C, staining of an invasive colon carcinoma shows nuclear staining (DAPI), ESA expression (green, FITC), and ALDH1 expression (red, phycoerythrin). The merged image reveals an orange-yellow color that shows overlap of ESA and ALDH1 staining. Doubly positive ESA and ALDH1 cells tended to be present at the invasive front of colonic malignancies.

Figure 3

Figure 3

Flow cytometric isolation of cancer-initiating cells based on ALDH enzymatic activity. A, FACS histogram showing Aldefluor assay done on human CRC cells in the presence (A) and absence (B) of ALDH1 enzyme inhibitor. The pattern of background ALDH activity (R7-gated population) was used to select ALDH+ cells. B, ALDH+ cells represent 3.5% (outside R7-gated population of ESA+ cells). ESA+ selection limited the ALDH+ population to carcinoma cells. C, H&E stains of primary human CRC (C) and the resulting xenograft (D) revealing similar histopathology.

Figure 4

Figure 4

Tumorigenicity of cells isolated using individual SC markers. ESA was used to select carcinoma cells from colon cancers before selection using other markers. Further sorting generated the following additional subpopulations: ALDH+ and ALDH− cells, CD44+ and CD44− cells, and CD133+ and CD133− cells. A, representative experiment showing latency of tumor development after implanting three different doses (500, 1,000, or 10,000) of ALDH+ cells or 10,000 ALDH− cells. B, log dose-response curve for generating xenograft tumors. Xenograft tumors were generated by cancer cells obtained from all patient cases (n = 4). C, representative experiment showing latency of tumor development after implantation of 10,000 ALDH+, CD44+, or CD133+ cells. All cells, including ESA+ alone, were sorted from a single colon cancer.

Figure 5

Figure 5

Tumorigenicity of cells isolated using multiple SC markers. ESA was used to select carcinoma cells from colon cancers before selection using other markers. A, isolation of cells using ALDH and CD44. Left image gives proportions of ALDH+ and CD44+ cells in colon carcinomas. Only a minority (3.5%) were ALDH+. A larger proportion (19.5%) were CD44+. The proportion positive for both was 1.3%. Percentages (mean ± SE) are averages of four experiments. The middle plot shows percentage of implants that produced a xenograft tumor after sorting using ALDH and CD44 markers. Percentages represent a summation of data across xenograft tumor assays using cells isolated from four different cancers involving 10 implants per cell subpopulation category. The same range of implant doses (50–1,000 cells) was used for each category. Percentage for ALDH+ alone was deduced from data on percentage tumor formation and proportions of cells for ALDH+/CD44+ and ALDH+/CD44−. Tumors generally did not result from ALDH−/CD44+ or ALDH−/CD44− cells. Right plot depicts a representative experiment showing latency of tumor development after implantation of two different doses (1,000 and 100) of ALDH+/CD44+ or ALDH+/CD44− cells. No tumors were generated from ALDH−/CD44+, ALDH−/CD44−, or ESA+ cells. B, isolation of cells using ALDH and CD133. Left image gives proportions of ALDH+ and CD133+ cells in colon carcinomas. Only a minority (3.5%) were ALDH+. A larger proportion (24.8%) were CD133+. The proportion positive for both was 0.9%. Percentages (mean ± SE) are averages of four experiments. The middle plot shows percentage of implants that produced a xenograft tumor after sorting using ALDH and CD133. Percentages represent a summation of data across xenograft tumor assays using cells isolated from three different cancers involving nine implants per cell subpopulation category. The same range of implant doses (50–500 cells) was used for each category. Percentage for ALDH+ alone was deduced from data on percentage tumor formation and proportions of cells for ALDH+/CD133+ and ALDH+/CD133− . Tumors did not form from ALDH−/CD133+ or ALDH−/CD133− cells. Right plot depicts a representative experiment showing latency of tumor development after implantation of different doses (50, 250, and 500) of ALDH+/CD133+ or ALDH+/CD133− cells. No tumors were generated from ALDH+/CD133−, ALDH−/CD133+, ALDH−/CD133−, or ESA+ cells.

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References

    1. Boman BM, Wicha MS. Cancer stem cells: a step toward the cure. J Clin Oncol. 2008;26:2795–2799. - PubMed
    1. Singh SK, Clarke ID, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63:5821–5828. - PubMed
    1. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature. 2004;432:396–401. - PubMed
    1. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100:3983–3988. - PMC - PubMed
    1. Boman BM, Huang E. Human colon cancer stem cells: a new paradigm in gastrointestinal oncology. J Clin Oncol. 2008;26:2828–2838. - PubMed

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