Aldehyde dehydrogenase 1 A1-positive cell population is enriched in tumor-initiating cells and associated with progression of bladder cancer

Abstract

Aldehyde dehydrogenase 1 A1 (ALDH1A1) has recently been suggested as a marker for cancer stem or stem-like cancer cells of some human malignancies. The purpose of this study was to investigate the stem cell-related function and clinical significance of the ALDH1A1 in bladder urothelial cell carcinoma. Aldefluor assay was used to isolate ALDH1A1+ cells from bladder cancer cells. Stem cell characteristics of the ALDH1A1+ cells were then investigated by in vitro and in vivo approaches. Immunohistochemistry was done for evaluating ALDH1A1 expression on 22 normal bladder tissues and 216 bladder tumor specimens of different stage and grade. The ALDH1A1+ cancer cells displayed higher in vitro tumorigenicity compared with isogenic ALDH1A1- cells. The ALDH1A1+ cancer cells could generate xenograft tumors that resembled the histopathologic characteristics and heterogeneity of the parental cells. High ALDH1A1 expression was found in 26% (56 of 216) of human bladder tumor specimens and significantly related to advanced pathologic stage, high histologic grade, recurrence and progression, and metastasis of bladder urothelial cell carcinomas (all P < 0.05). Furthermore, ALDH1A1 expression was inversely associated with cancer-specific and overall survivals of the patients (P = 0.027 and 0.030, respectively). Therefore, ALDH1A1+ cell population could be enriched in tumor-initiating cells. ALDH1A1 may serve as a useful marker for monitoring the progression of bladder tumor and identifying bladder cancer patients with poor prognosis who might benefit from adjuvant and effective treatments.

Fig. 1

Identification of a small ALDH1A1⁺ population in bladder cancer cell lines. 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). B. The ALDH1A1⁺ cell proportion in the bladder cancer cells was significantly lower than that of CD44⁺ cells. The percentage of ALDH1A1⁺ population in the celll lines was from 6.4% to 8.2%. However, the proportion of CD44⁺ cells was from 57% to 72%. C. Merged immunofluorescence imaging showed a high percentage of ALDH1A1⁺ cancer cells positive for CD44 staining. ALDH1A1⁺ cells isolated from HTB-2 cells were positive for ALDH1A1 antibody (green fluorescent staining of the cytoplasm). Fraction of the same cells was positively stained for CD44 as demonstrated by red fluorescent staining of the cell membrane (white arrows). 4',6-diamidino-2-phenylindole (DAPI) was used to stain nuclei. The experiments were undertaken on all bladder cancer cell lines and repeated three times. The Fig.1A and C only showed the result from HTB-2 cell line.

Fig. 2

ALDH1A1⁺ bladder cancer cells had high in vitro and in vivo tumorigenic potential. A. ALDH1A1⁺ cancer cells (left) possessed significantly higher colony-forming efficiency compared with ALDH1A1⁻ populations (right). ALDH1A1⁺ and ALDH1A1⁻ cells were plated in six-well dishes coated with a thin layer of agar. Three weeks after plating, ALDH1A1⁺ cells formatted larger and more colonies as compared with the ALDH1A1⁻ cells. B. Tumor formation ability of ALDH1A1⁺ cancer cells was greater than that of isogenic ALDH1A1⁻ cells. ALDH1A1⁺ and ALDH1A1⁻ HTB-2 cells were implanted into flanks of nude mice. After four weeks, the dose of 1 × 10⁵ ALDH1A1⁺ cells yielded larger tumors (red arrow) in all 10 mice with diameters of 32±2.6 mm³, whereas the same dose of ALDH1A1⁻ cells only generated a small tumor mass (5.5 mm³) (green arrow) in only one mouse. The animal experiments were done by using all cell lines. The Fig. 2B only showed the result from HTB-2 cell line. C. Histopathologic examination of the engrafted tumors formed by the ALDH1A1⁺ HTB-9 cancer cells revealed a highly cellular mass with characteristics of bladder transitional cell carcinoma. D. ALDH1A1⁺ cancer cells created bladder tumors with heterogeneity in vivo. Reanalyzing cells of the engrafted tumors generated from the ALDH1A1⁺ HTB-9 by using the Aldefluor assay showed that the xenograft tumors produced 56.9% ALDH1A1⁺ cells and 29.7% ALDH1A1⁻ cells. The experiments were undertaken on all bladder cancer cell lines and repeated three times. The Fig. 2D only showed the result from HTB-9 cell line.

Fig. 3

High ALDH1A1 expression in human bladder cancer specimens was associated with a poor prognosis for the patients. A and B. ALDH1A1⁺ cancer cells comprised a fraction of the CD44⁺ tumor cell population. Immunohistochemical study of adjacent sections of a bladder tumor for the expression of ALDH1A1 and CD44 showed that a small number of ALDH1A1⁺ cells (arrows in A) were present within the CD44 immunopositive tumor areas (B). C. Survival analysis of 216 patients with bladder urothelial carcinomas based on high ALDH1A1 expression status. C. Cancer-specific survival time; D. Overall survival time. P values were calculated using the log-rank test.