Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells

Abstract

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

Figure 1. ALDH^hiCD44^hi lung cancer cells showed in vitro TIC characteristics

A, Spheroid formation assay. FACS-isolated lung cancer cell populations with differential ALDH/CD44 expressions and unsorted cell controls were kept in serum-free non-adherent plates for 21 days. B, Matrigel invasion assay. The proportions of invading cells from respective cell subsets were normalized to the unsorted control. C, In vitro differentiation assay. The 4 freshly isolated populations were separately cultured in adhesive plates containing normal medium for 2 weeks. Cells were then freshly harvested and re-analyzed by flow cytometry for ALDH/CD44 expression profile. The central profile represented parental unsorted cells and profiles of the subsets were as labeled. D and E, Normalized mRNA expressions of pluripotency, EMT and other genes by QPCR. F, Pluripotency proteins expression analyzed by flow cytometry. Results were normalized to unsorted control. G, Cell cycle analysis. Freshly isolated ALDH^hiCD44^hi and ALDH^loCD44^lo populations of H1650 were stained with propidium iodide and analyzed by flow cytometry for DNA content. H, Cell proliferation assay. Respective subsets of freshly isolated H1650 cells were analyzed by MTT. I, Expression of CCNB1 and CCNB2 analyzed by QPCR.*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with unsorted; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, compared with ALDH^hiCD44^hi. All data represent the mean ± SD of triplicate experiments.

Figure 2. ALDH^hiCD44^hi population showed TIC properties in vivo

A, Schematic diagram showing transplantation sites of respective ALDH/CD44 populations in SCID mice. B, Representative photographs of H1650 or HCC827 xenografts derived from respective ALDH/CD44 populations after 3 months tumor development. C and D, Tumor growth curves of 1^st generation xenografts derived from 2,500 cells of respective ALDH/CD44 populations. E and F, tumor growth curves of 3 serially transplanted generations of xenografts derived from 2,500 ALDH^hiCD44^hi cells of respective cell lines. Data represent the mean ± SD of tumor volumes at different time points of different groups, there are 6 mice in each group.

Figure 3. ALDH^hiCD44^hi cells showed drug resistance in vitro and in vivo

A, Effects of 30 nM gefitinib treatment for 24 hr on ALDH/CD44 subsets of HCC827 analyzed by flow cytometry. B and C, Proportions of ALDH⁺CD44⁺ and ALDH⁻CD44⁻ cells in drug-resistant HCC827 cells. Resistance was induced by chronic exposure to increasing gefitinib doses (HCC827-GR) or to cisplatin (HCC827-CR). D, In vitro cell viability response. Freshly isolated ALDH^hiCD44^hi and unsorted cells of HCC827 were treated with a range of gefitinib doses for 24 hr and cell viability was assayed by MTT. E, Apoptosis response. Freshly isolated respective populations and unsorted H1650 were treated with 5 μM cisplatin for 24 hr and apoptosis fractions were assayed by Annexin V/PI staining. F, In vivo xenograft response after TKI treatment. Mice bearing HCC827 xenografts from respective ALDH/CD44 subsets as depicted in Figure 2A were given intraperitoneal gefitinib 5 times per week for 5 weeks. G, In vivo tumor response curve. Tumor volumes of respective HCC827 xenografts in TKI-treated (n = 5) or control-treated (n = 3) mice were measured twice weekly. Data represent mean ± SD of tumor volume normalized to pre-treatment size in gefitinib-treated mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control treatment or unsorted cells.

Figure 4. Hedgehog and Notch signaling were involved in ALDH^hiCD44^hi TIC maintenance

A and B, mRNA levels of Hedgehog and Notch signaling genes by QPCR in sorted cells from H1650 and HCC827. C, mRNA levels of HES1 or GLI1 by QPCR in HCC827 after Notch inhibition by RO4929097 or Hedgehog inhibition by cyclopamine. D and E, Proportions of ALDH⁺CD44⁺ by flow cytometry in HCC827 and H1650 after pathway inhibition for 1 or 4 days. F, Spheroid formation assay of HCC827 cells treated with inhibitors. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with unsorted or control; #, p < 0.05; ##, p < 0.01, compared with ALDH^hiCD44^hi. Data represent mean ± SD of triplicate experiments.

Figure 5. CD44 knockdown and ALDH inhibition reduced pluripotency gene expression and sensitized cells to drug treatment

A and B, mRNA and protein expressions of CD44 and pluripotency genes. CD44 was depleted by siRNA and ALDH inhibited by DEAB (100 μM) in H1299, H1650 and PDCL #24, control siRNA and Ethanol were used as control. Histograms represent mean ± SD of mRNA measured by QPCR in triplicates and protein expression was analyzed by western blot. C, Cell viability assay. CD44 and ALDH-inhibited or control cells were treated respectively with cisplatin (H1650, 10 μM; H1299 & PDCL #24, 15 μM) or gefitinib (HCC827 GR, 10 μM; H1650 GR, 20 μM) for 24 hrs. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control. Data represent mean ± SD of triplicate experiments.

Figure 6. ALDH and CD44 expression in clinical lung cancers

A to D, ALDH/CD44 co-expression patterns were variable in different tumors. A and B, Cytoplasmic ALDH expression (A) was observed in only a few tumor cells (→) at the invasion front of this squamous cell carcinoma while membranous CD44 expression (B) was present in all tumor cells in the same cluster. Co-localizing cells showing both ALDH and CD44 expression were few and thus graded as low abundance. C and D, This adenocarcinoma showed co-expression of ALDH (C) and CD44 (D) in almost all tumor cells and was thus graded as high abundance. E to G, Kaplan Meier survival curves comparing recurrence-free survival (RFS) in different tumor groups. For single marker analysis of ALDH (E) or CD44 (F), no significant difference in RFS was observed between tumor groups showing low or high abundance. For ALDH/CD44 dual marker analysis (G), RFS was shorter for patients with high compared to low abundance ALDH/CD44-coexpressing TIC (p = 0.053).