Distinct expression levels and patterns of stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1), in human epithelial cancers

Abstract

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792) by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036). Finally, ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.

Figure 1. Expression and distribution of ALDH1 protein in normal human tissues.

A. An FDA normal human organ tissue microarray was used to characterize the expression and distribution of ALDH1 in 24 tissues. B. Strongly positive ALDH1 cells were found in the areas in which epithelial stem/progenitor cells were putatively located in breast, colon and stomach.

Figure 2. Expression and distribution of ALDH1 protein in human epithelial tumors.

Tissue microarrays were used to characterize the expression and distribution of ALDH1 in cancers (n = 353). A. Expression of ALDH1 in the corresponding normal tissues. B. ALDH1+ tumor-infiltrating cells were detected in the stroma. C. Expression of ALDH1 in epithelial tumors. Percentage of ALDH1+ tumor cells was summarized. D. High percentage of ALDH1+ cells was associated with poor clinical outcomes in serous ovarian cancer.

Figure 3. ALDH1 enzymatic activity and expression in established epithelial tumor cell lines.

A. ALDH1 enzymatic activity was detected using the ALDEFLUOR assay. DEAB was used to inhibit the reaction of ALDH with the ALDEFLUOR reagent, providing a negative control. B. Summary of ALDH1 enzymatic activity in established breast (n = 15), ovarian (n = 18) and colon (n = 10) cancer cell lines. C. ALDH1 protein expression was detected by western blots. There was a positive correlation between ALDH1 protein expression and ALDH1 enzymatic activity. Full-length blots are presented in Supplemental Figure S3.

Figure 4. ALDH1 enzymatic activity in primary epithelial ovarian cancer cells.

A. ALDH1 enzymatic activity in cells isolated from one ovarian cancer specimen. Dead cells were eliminated using magnetic beads and a MidiMACS separator. For immunophenotyping of ALDH^br ascites cells, APC-CD45 or APC-CD326 was used for counterstaining. DEAB was used to inhibit the reaction of ALDH with the ALDEFLUOR reagent, providing a negative control. B. Summary of the percentages of ALDH^br in each cell population from 16 late-stage ovarian cancer patients.

Figure 5. ALDH^br tumor cells exhibit cancer stem cell properties.

A. ALDH^br cell populations (Blue) generated significantly higher numbers of mammospheres compared to the ALDH^low population (Yellow). B. Tumorigenicity of the ALDH^br and ALDH^low cells were examined in vitro using colony formation assays. C. Tumorigenicity of the ALDH^br and ALDH^low cells were examined in vivo. D. Histology of the ALDH^br and ALDH^low BT-474 tumors was examined by HE staining. E. Proliferation of the ALDH^br and ALDH^low BT-474 tumors was examined by Ki-67 immunostaining.

Figure 6. ALDH^br tumor cells are resistant to the chemotherapeutic drug platinum.

A. ALDH^br cells were remarkably expanded in platinum resistant cell lines (A2780/CP70, A2780/C200 and A2780/C30) compared to their parental platinum sensitive line (A2780/WT). B. In vitro platinum treatment significantly increased the ALDH^br cell population in ovarian and breast cancer cell lines after three days. C. ALDH^br cells were resistant to platinum treatment. ALDH^br and ALDH^low cells were isolated by FACS sorting. D. ALDH^br tumor cell population was resistant to chemotherapy in vivo. The q7d×4 i.p. treatment schedule was used for the experimental therapy. Tumors were collected and disassociated by enzyme digestion. Percentage of the ALDH^br population was analyzed by ALDEFLUOR assay.