High aldehyde dehydrogenase activity identifies cancer stem cells in human cervical cancer

Abstract

High aldehyde dehydrogenase (ALDH) activity characterizes a subpopulation of cells with cancer stem cell (CSC) properties in several malignancies. To clarify whether ALDH can be used as a marker of cervical cancer stem cells (CCSCs), ALDH high and ALDH low cells were sorted from 4 cervical cancer cell lines and 5 primary tumor xenografts and examined for CSC characteristics. Here, we demonstrate that cervical cancer cells with high ALDH activity fulfill the functional criteria for CSCs: (1) ALDH high cells, unlike ALDH low cells, are highly tumorigenic in vivo; (2) ALDH high cells can give rise to both ALDH high and ALDH low cells in vitro and in vivo, thereby establishing a cellular hierarchy; and (3) ALDH high cells have enhanced self-renewal and differentiation potentials. Additionally, ALDH high cervical cancer cells are more resistant to cisplatin treatment than ALDH low cells. Finally, expression of the stem cell self-renewal-associated transcription factors OCT4, NANOG, KLF4 and BMI1 is elevated in ALDH high cervical cancer cells. Taken together, our data indicated that high ALDH activity may represent both a functional marker for CCSCs and a target for novel cervical cancer therapies.

Figure 1. ALDH expression in human cervical tissue specimens and cervical cancer cell lines

A, Representative photos of immunostained specimens showing ALDH1 expression in normal cervical (a-d) and cervical cancer (e-h) tissues. a, no ALDH1-positive cells; b, dot-scattered ALDH1 expression; c, focal distribution of ALDH1-positive cells; d, all basal cells of the normal cervix are ALDH1-positive; e, dot-scattered ALDH1 expression; f, focal distribution of ALDH1-positive cells; g, diffuse ALDH1 expression; h, ALDH1-positive cells in xenograft tissue from mice. Red arrows indicate ALDH1-positive cells. S, Stroma; E, Epithelium; Magnifications, 1000×. B, Cells were labeled using the ALDEFLUOR kit, and ALDH^high cells (bright green fluorescence) were detected by fluorescent microscopy. C, ALDH enzyme activity in 4 cervical cancer cell lines was analyzed by flow cytometry. As a negative control, cells were treated with the specific ALDH inhibitor DEAB. The gated cells represent the ALDH^high cells.

Figure 2. ALDH^high cervical cancer cells exhibit enhanced self-renewal capacity

A, Representative photos of tumorspheres formed by ALDH^high and ALDH^low cells are shown. Bar, 200 µm. B, The number of tumorspheres/200 cells was counted from 3 consecutive passages. C, The number of wells containing tumorspheres was counted. ^***, p<0.001. Data represent mean ± SD of triplicate experiments.

Figure 3. Tumorigencity of ALDH^high and ALDH^low cells from 4 cervical cancer cell lines in NOD/SCID mice

A, The volume of xenograft tumors formed by different numbers of ALDH^high and ALDH^low cervical cancer cells was monitored over time. B, Kaplan-Meier plots showing the tumor-free survival after injection. ns, not significant; ^*, p<0.05; ^**, p<0.01; ^***, p<0.001. Data represent mean ± SD of tumor volumes at different time points of 10 mice in each group.

Figure 4. ALDH^high cervical cancer cells are capable of differentiating ^{in vitro} and ^{in vivo}

A-D, ALDH^high and ALDH^low cells were isolated from the SiHa (A), C33A (B), CaSki (C) or HT-3 (D) cell lines and cultured in DMEM medium supplemented with 10% FBS for 2 weeks. The ALDH enzyme activity was then analyzed by flow cytometry. Cells treated with DEAB served as a negative control. The gated cells represent the ALDH^high cells. E, Expression of ALDH1 was detected by IHC in xenograft tumors from ALDH^high and ALDH^low cells. Red arrows indicate ALDH1-positive cells

Figure 5. ALDH^high cells are more resistant to cisplatin than ALDH^low cells

A, The ALDH activity of the cervical cancer cell lines was analyzed after exposure to cisplatin (3 µg/mL for SiHa and C33A cells or 1 µg/mL for CaSki and HT-3 cells) for 3 days. The percentage of ALDH^high cells in the 4 cervical cancer cell lines was analyzed by flow cytometry following exposure to cisplatin for 3 days. B, Cell viability of the ALDH^high and ALDH^low cervical cancer cells was measured using an MTT assay after treatment with different concentrations of cisplatin for 24 h. C, Cell viability of the ALDH^high and the ALDH^low cervical cancer cells was measured using an MTT assay after treatment with a constant dose of cisplatin for 0, 24, 48 or 72 h. ^*, p<0.05; ^**, p<0.01; ^***, p<0.001. Data represent mean ± SD of triplicate experiments.

Figure 6. Expression of stem cell-associated markers in ALDH^high and ALDH^low cells

A, Western blot analysis of the protein levels of the stem cell-associated transcription factors OCT4, NANOG, KLF4 and BMI1 in the ALDHhigh and ALDHlow subpopulations from C33A and HT-3 cells. GAPDH was used as a loading control. B, The expression of stem cell-associated transcription factors in tumorspheres formed by ALDHhigh and ALDHlow SiHa cells was measured by IHC analysis.

Figure 7. Characteristics of ALDH^high cells from primary cervical cancers

A, The ALDH enzyme activity of primary cervical cancer cells was analyzed by flow cytometry. B, Representative photos of tumorspheres formed by ALDH^high and ALDH^low cells sorted from primary cervical cancer are shown. C, The number of tumorspheres formed by ALDH^high and ALDH^low cells isolated from the primary cervical cancer was counted from 3 consecutive passages. ^***, p<0.001. Data represent mean ± SD of triplicate experiments.