Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer - PubMed (original) (raw)

Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer

Feng Jiang et al. Mol Cancer Res. 2009 Mar.

Abstract

Tumor contains small population of cancer stem cells (CSC) that are responsible for its maintenance and relapse. Analysis of these CSCs may lead to effective prognostic and therapeutic strategies for the treatment of cancer patients. We report here the identification of CSCs from human lung cancer cells using Aldefluor assay followed by fluorescence-activated cell sorting analysis. Isolated cancer cells with relatively high aldehyde dehydrogenase 1 (ALDH1) activity display in vitro features of CSCs, including capacities for proliferation, self-renewal, and differentiation, resistance to chemotherapy, and expressing CSC surface marker CD133. In vivo experiments show that the ALDH1-positive cells could generate tumors that recapitulate the heterogeneity of the parental cancer cells. Immunohistochemical analysis of 303 clinical specimens from three independent cohorts of lung cancer patients and controls show that expression of ALDH1 is positively correlated with the stage and grade of lung tumors and related to a poor prognosis for the patients with early-stage lung cancer. ALDH1 is therefore a lung tumor stem cell-associated marker. These findings offer an important new tool for the study of lung CSCs and provide a potential prognostic factor and therapeutic target for treatment of the patients with lung cancer.

PubMed Disclaimer

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

FIGURE 1

FIGURE 1

ALDH1-positive lung cancer cells have tumor stem cell properties in vitro. A. FACS analysis of cancer cells using the Aldefluor assay, in which cells were incubated with ALDH substrate, BAAA, which was then converted by intracellular ALDH1 into a negatively charged reaction product BODIPY-aminoacetate. BODIPY-aminoacetate was retained inside cells positively expressing ALDH1, causing the cells to become brightly fluorescent. Brightly fluorescent ALDH1-expressing cells (ALDH1-positive cells) were detected in the green fluorescence channel by using flow cytometry. Cells incubated with BAAA and a specific inhibitor of ALDH, diethylaminobenzaldehyde, were used to establish the baseline fluorescence of these cells (R1) and to define the Aldefluor (ALDH1)-positive region (R2). FACS analysis was done on all cell lines and repeated three times. A. Result from H358 cell line. B. Cell growth curve of parental lung cancer cells and their corresponding ALDH1-positive and ALDH1-negative cancer cells. ALDH1-positive cancer cells grew more rapidly compared with parental and ALDH1-negative cancer cells. Experiments were undertaken on all NSCLC cell lines and repeated three times. B only showed the result from H358 cell line. C. Analysis of cell colony numbers in clonogenicity assays of ALDH1-positive and ALDH1-negative and unsorted cells. ALDH1-positive cell populations formatted larger and more colonies compared with unsorted and ALDH1-negative cells. Experiments were done on all cell lines and repeated three times. C showed the result from H358 cell line. D. Reanalyzing ALDH1-positive and ALDH1-negative cancer cells and measuring their differentiation ability by using the Aldefluor assay and FACS. ALDH1-positive cells (left) gave rise to 52% (52 ± 2.6%) ALDH1-positive population and 48% (48 ± 2.2%) ALDH1-negative cells, whereas ALDH1-negative cells (right) only produced ALDH1-negative cell population, implying that ALDH1-positive cells might undergo asymmetrical division to in vitro self-renew and generate heterogeneous cell populations of both high and low aggressive tumorigenic phenotypes. Experiments were undertaken on all cell lines and repeated three times, whereas D only showed the result from H358 cells. E. Matrigel invasion assay on sorted and unsorted cells. ALDH1-positive and ALDH1-negative lung cancer and unsorted cells (2 × 105) were seeded and incubated for 48 h. Columns, number of cells invaded across the membrane. ALDH1-positive cells were more invasive than ALDH1-negative cells and unsorted parental cancer cells. *, P < 0.05, t test, statistical significance. Each experiment was repeated three times. Points, mean of the independent experiments.

FIGURE 2

FIGURE 2

ALDH1-positive lung cancer cells have tumor stem cell properties in vivo. A. Tumor formation ability of ALDH1-positive cells was greater than that of ALDH1-negative cancer cells. ALDH1-positive and ALDH1-negative H125 cells were implanted into flanks of nude mice. After 4 weeks, the dose of 1 × 105 ALDH1-positive lung cancer cells yielded larger tumors (red arrow) in all five mice with diameters of 30 ± 2.9 mm3, whereas the same dose of ALDH1-negative H125 cell only generated a small tumor mass (5 mm3; green arrow) in only one of five mice. Animal experiments were done by using cell lines H358 and H125 and repeated three times. A only showed the result from H125 cell lines. B. Histopathologic examination of the engrafted tumors formed by ALDH1-positive H125 cancer cells revealed a highly cellular mass with characteristics of adenocarcinoma of lung. C. Dual-color FISH analysis of xenograft tumor cells generated from ALDH1-positive H125 lung cancer cells with a probe (green signal) for FHIT gene and a probe (red signal) for chromosome 3 centromeric region. The majority of cells in tumor cells bore deletion of FHIT indicated by fewer green signals than red ones. D. Reanalyzing cells of engrafted tumors generated from ALDH1-positive and ALDH1-negative cancer cells and measuring their differentiation ability by using the Aldefluor assay and FACS. Cells from the tumor produced by ALDH1-positive H125 cancer cells (left) gave rise to 46% (46 ± 3.1%) ALDH1-positive population and 54% (54 ± 3.3%) ALDH1-negative cells, whereas cells from the tumor formed by ALDH1-negative H125 cells (right) only produced ALDH1-negative cell population, implying that ALDH1-positive cells might undergo asymmetrical division to in vivo self-renew and generate heterogeneous cell populations of both high and low aggressive tumorigenic phenotypes. Aldefluor assay and FACS experiments were undertaken on disaggregated cells of tumor engrafts from H358 and H125 cell lines and repeated three times, respectively, whereas D only illustrated the result from H125 cells.

FIGURE 3

FIGURE 3

ALDH1 overexpression is associated with aggressive biological behavior of NSCLC and a poor prognosis for patients with NSCLC. A. Analysis of TBNA specimen obtained from a lung tumor mass from a stage IV NSCLC patient by using immunohistochemistry showed positive ALDH1 expression. B. immunohistochemical analysis of a paraffin-embedded lung tumor tissue of a patient diagnosed with stage IV lung adenocarcinoma showed that overexpression of ALDH1 occurred predominately in cytoplasm of cancer cells. C. Probability of cancer-specific survival by levels of ALDH1 expression in stage I NSCLC. D. Probability of overall survival by levels of ALDH1expression in stage I NSCLC. Kaplan-Meier method was used to determine the survival probability, and log-rank test was used to compare the survival curves between groups.

References

    1. American Cancer Society. Cancer facts and figures 2007–2008.
    1. Minna JD, Roth JA, Gazdar AF. Focus on lung cancer. Cancer Cell. 2002;1:49– 52. - PubMed
    1. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;1:105– 11. - PubMed
    1. Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med. 2006;12:1253– 61. - PubMed
    1. Berns A. Stem cells for lung cancer? Cell. 2005;121:811– 3. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources