ALDH1A1 is a marker for malignant prostate stem cells and predictor of prostate cancer patients' outcome

Abstract

Prostate cancer (PCa) contains a small population of cancer stem cells (CSCs) that contribute to its initiation and progression. The development of specific markers for identification of the CSCs may lead to new diagnostic strategies of PCa. Increased aldehyde dehydrogenase 1A1 (ALDH1A1) activity has been found in the stem cell populations of leukemia and some solid tumors. The aim of the study was to investigate the stem-cell-related function and clinical significance of the ALDH1A1 in human PCa. ALDEFLUOR assay was used to isolate ALDH1A1(+) cells from PCa cell lines. Stem cell characteristics of the ALDH1A1(+) cells were then investigated by in vitro and in vivo approaches. The ALDH1A1 expression was also analyzed by immunohistochemistry in 18 normal prostate and 163 PCa tissues. The ALDH1A1(+) PCa cells showed high clonogenic and tumorigenic capacities, and serially reinitiated transplantable tumors that resembled histopathologic characteristics and heterogeneity of the parental PCa cells in mice. Immunohistochemical analysis of human prostate tissues showed that ALDH1A1(+) cells were sparse and limited to the basal component in normal prostates. However, in tumor specimens, increased ALDH1A1 immunopositivity was found not only in secretory type cancer epithelial cells but also in neuroendocrine tumor populations. Furthermore, the high ALDH1A1 expression in PCa was positively correlated with Gleason score (P=0.01) and pathologic stage (P=0.01), and inversely associated with overall survival and cancer-specific survival of the patients (P=0.00093 and 0.00017, respectively). ALDH1A1 could be a prostate CSC-related marker. Measuring its expression might provide a potential approach to study tumorigenesis of PCa and predict outcome of the disease.

Fig. 1

ALDH1A1⁺ PCa cells had tumor stem cell properties in vitro. A. FACS analysis of cancer cells using the Aldefluor assay. The brightly fluorescent ALDH1A1-expressing cells (ALDH1A1⁺ cells) were detected in the green fluorescence channel. The cells incubated with DEAB were used to establish the baseline fluorescence of these cells (R1) and define the ALDEFLUOR (ALDH1A1)-positive region (R2). The Fig.1A showed the result from PC3 cell line. B. ALDH1A1⁺ PCa cells possessed significantly higher colony-forming efficiency compared with ALDH1A1⁻ PCa populations. ALDH1A1⁺ and ALDH1A1⁻ cells were plated in a six-well dish. Two weeks after plating, clones were counted and results were presented as percent cloning efficiency (Y axis). Column indicates mean from three independent experiments; bars, SE. *, P < 0.0001. C. ALDH1A1⁺ PCa populations formatted larger and more colonies as compared with the ALDH1A1⁻ cells by clonogenicity assays. Spheres were counted three weeks after plating in triplicate at 1,000 per well in a six-well plate coated with soft agar. The experiments were undertaken on all PCa cell lines and repeated three times. The Fig.1 only showed the result from PC3 cell line.

Fig. 2

ALDH1A1+ PCa cells had tumor stem cell properties in vivo. A. Tumor formation ability of ALDH1A1⁺ cells was greater than that of ALDH1A1⁻ cancer cells. 5 × 10² and 5 × 10⁴ ALDH1A1⁺ PC cells, ALDH1A1⁻ PC cells, or unsorted corresponding PC cells were injected into the dorsal prostate of ten NOD/SCID mice, respectively, as indicted in X axis. Tumor incidence refers to the number of tumors developed in the ten mice, as shown in the Y axis. After four weeks, the 5 × 10² ALDH1A1⁺ PC3 cells yielded tumors in all mice, whereas the same amount of ALDH1A1⁻ PC3 cell didn’t produce tumor mass in any mouse.The 5 × 10⁴ ALDH1A1⁺ PC3 cells yielded tumors in all mice, whereas the same amount of ALDH1A1⁻ PC3 cell produced a small tumor mass in only one mouse. B. ALDH1A1⁺ PCa cells generated larger tumors compared with ALDH1A1⁻ cancer cells. 5 × 10⁴ ALDH1A1⁺ PC3 cells yielded tumors with an average of 28±1.5 mm³, whereas the same dose of ALDH1A1⁻ PC3 cell produced a small tumor mass (8 mm³) in only one of the mice. C. Histopathologic examination of the engrafted tumors formed by the ALDH1A1⁺ PC3 cells revealed a highly cellular mass with characteristics of adenocarcinoma of prostate. D. ALDH1A1⁺ PCa cells could generate PCa tumors with heterogeneity in vivo. Reanalyzing cells of the engrafts generated from the ALDH1A1⁺ and ALDH1A1⁻ cells by using the Aldefluor assay showed that the cells from the tumor produced by ALDH1A1⁺ PC3 cancer cells (left panel) gave rise to 50% ALDH1A1⁺ population and 46% ALDH1A1⁻ cells. However, the cells from the tumor formed by ALDH1A1⁻ PC3 cells (right panel) only produced ALDH1A1⁻ PC3 population.

Fig. 3

ALDH1A1⁺ PCa cells produced heterogeneous populations of tumorigenic phenotypes. A. Immunofluorescence analysis of ALDH1A1⁺ cells freshly enriched from cultured LNCaP cells showed that most of the cells were negative for CD44 (weak red fluorescent staining). B. Part of the disassociated cells of engraft developed from the ALDH1A1⁺ LNCaP cells were positively stained for CD44, as illustrated by red fluorescence staining of the cell membrane (arrows). C. ALDH1A1⁺ cells newly isolated from the same cultured LNCaP cells demonstrated that the majority of the cells were positive for androgen receptor (AR) (green fluorescent staining of the nuclei). D. Only fraction of the disaggregated cells of engraft produced from the ALDH1A1⁺ LNCaP cells were positively stained for AR, as demonstrated by green fluorescent staining of the nucleus (arrowheads). 4′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei.

Fig. 4

ALDH1A1 expression was sparse and limited to the CD44⁺ basal component in normal prostates. A. Immunohistochemical study of adjacent sections of a normal prostate tissue specimen for the expression of ALDH1A1 and CD44 showed that only a few ALDH1A1⁺ cells (arrows in left panel), which displayed positive cytoplasmic staining, resided in the CD44 positive basal cells (arrowheads in right panel). B. Comparison of expression of ALDH1A1 and CD44 in 18 normal prostate tissues. Bars reflect the mean percentage of positive staining cells ± SE for each antibody. *, P < 0.0001.

Fig. 5

ALDH1A1 expression existed in PCa tumors with heterogeneous phenotypes and was associated with a poor prognosis for the patients. A. Immunohistochemical study of adjacent sections of a PCa tumor for the expression of ALDH1A1 and AR showed that ALDH1A1⁺ cells (arrows in left panel) exist in portion of secretory type cancer epithelial cells that had strong AR immunopositivity (arrowheads in right panel). B. ALDH1A1⁺ cells (arrows in left panel) were present in part of neuroendocrine tumor cells that had positive chromogranin A (arrowheads in right panel). C. Probability of overall survival (left panel) and cancer-specific survival (right panel) by levels of ALDH1A1 expression in the 99 PCa patients.