CD44+ and CD133+ Non-Small Cell Lung Cancer Cells Exhibit DNA Damage Response Pathways and Dormant Polyploid Giant Cancer Cell Enrichment Relating to Their p53 Status

Abstract

Cancer stem cells (CSCs) play a critical role in the initiation, progression and therapy relapse of many cancers including non-small cell lung cancer (NSCLC). Here, we aimed to address the question of whether the FACS-sorted CSC-like (CD44 + &CD133 +) vs. non-CSC (CD44-/CD133- isogenic subpopulations of p53wt A549 and p53null H1299 cells differ in terms of DNA-damage signaling and the appearance of "dormant" features, including polyploidy, which are early markers (predictors) of their sensitivity to genotoxic stress. X-ray irradiation (IR) at 5 Gy provoked significantly higher levels of the ATR-Chk1/Chk2-pathway activity in CD44-/CD133- and CD133+ subpopulations of H1299 cells compared to the respective subpopulations of A549 cells, which only excited ATR-Chk2 activation as demonstrated by the Multiplex DNA-Damage/Genotoxicity profiling. The CD44+ subpopulations did not demonstrate IR-induced activation of ATR, while significantly augmenting only Chk2 and Chk1/2 in the A549- and H1299-derived cells, respectively. Compared to the A549 cells, all the subpopulations of H1299 cells established an increased IR-induced expression of the γH2AX DNA-repair protein. The CD44-/CD133- and CD133+ subpopulations of the A549 cells revealed IR-induced activation of ATR-p53-p21 cell dormancy signaling-mediated pathway, while none of the CD44+ subpopulations of either cell line possessed any signs of such activity. Our data indicated, for the first time, the transcription factor MITF-FAM3C axis operative in p53-deficient H1299 cells, specifically their CD44+ and CD133+ populations, in response to IR, which warrants further investigation. The p21-mediated quiescence is likely the predominant surviving pathway in CD44-/CD133- and CD133+ populations of A549 cells as indicated by single-cell high-content imaging and analysis of Ki67- and EdU-coupled fluorescence after IR stress. SA-beta-galhistology revealed that cellular-stress-induced premature senescence (SIPS) likely has a significant influence on the temporary dormant state of H1299 cells. For the first time, we demonstrated polyploid giant and/or multinucleated cancer-cell (PGCC/MGCC) fractions mainly featuring the progressively augmenting Ki67^low phenotype in CD44+ and CD133+ A549 cells at 24-48 h after IR. In contrast, the Ki67^high phenotype enrichment in the same fractions of all the sorted H1299 cells suggested an increase in their cycling/heterochromatin reorganization activity after IR stress. Our results proposed that entering the "quiescence" state rather than p21-mediated SIPS may play a significant role in the survival of p53wt CSC-like NSCLC cells after IR. The results obtained are important for the selection of therapeutic schemes for the treatment of patients with NSCLC, depending on the functioning of the p53 system in tumor cells.

Keywords: FAM3C; MITF; cancer stem cells; non-small cell lung cancer; p21; p53; polyploid giant cancer cells; radioresistance; senescence-associated β-galactosidase; γH2AX.

Figure 1

Gating strategy and representative images of CD44−, CD44+, CD133− and CD133+ populations of A549 (a) and H1299 (b) cells using Amnis ImageStreamX Mk II Imaging Flow Cytometer. The first column represents bivariate plot of BF aspect ratio versus BF area, which allows for the selection of single cells and the removal of doublets and small and large debris. Histograms of CD44 and CD133 intensity in the second column allows for the selection of CD44+ and CD133+ cells (third column) from dimly stained (CD44− and CD133−) events (fourth column).

Figure 2

Western-blotting analysis of OCT4 (a) and SOX2 (b) expression in CD-sorted populations of A549 and H1299 cell lines. Data are means ± SD of three independent experiments. Statistical significance is denoted by asterisks, where: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Tumor-spheroid formation in CD-sorted populations of A549 and H1299 cell lines four days after 5 Gy irradiation. Scale bar 200 µm.

Figure 4

Assessment of cell viability using the metabolic MTT assay of the glycolytic NAD(P)H production at different times after IR at 5 Gy. * denotes significant differences at * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

MILLIPLEX^® MAP 7-plex DNA Damage/Genotoxicity Magnetic Bead Panel analytes were detected in CD-sorted populations of A549 and H1299 cell lines 1 h after 5 Gy irradiation. Median Fluorescent Intensities (MFIs) are reported as fold change over the positive control (A549 cells stimulated with 5 μM camptothecin) provided by the manufacturer.

Figure 6

EdU-coupled fluorescence-intensity analysis of distinct cell-cycle characteristics. (a) The proportion of EdU-positive cells in CD-sorted populations of NSCLC cells 24–48 h after irradiation. (b) Using the ImageXpress Micro XL High-Content Screening System, we analyzed EdU fluorescence in PGCC 24–48 h after 0 Gy (blue dots) or 5 Gy (red dots) exposure The nuclear area of 400 nm was used as a threshold for PGCC cells according to the literature. Green circles demonstrate EdU^low area of “quiescent” cells. Data are means ± SD of three independent experiments. Statistical significance is denoted by asterisks, where: **** p < 0.0001.

Figure 6

EdU-coupled fluorescence-intensity analysis of distinct cell-cycle characteristics. (a) The proportion of EdU-positive cells in CD-sorted populations of NSCLC cells 24–48 h after irradiation. (b) Using the ImageXpress Micro XL High-Content Screening System, we analyzed EdU fluorescence in PGCC 24–48 h after 0 Gy (blue dots) or 5 Gy (red dots) exposure The nuclear area of 400 nm was used as a threshold for PGCC cells according to the literature. Green circles demonstrate EdU^low area of “quiescent” cells. Data are means ± SD of three independent experiments. Statistical significance is denoted by asterisks, where: **** p < 0.0001.

Figure 7

Ki67-coupled fluorescence-intensity analysis of distinct cell-cycle characteristics 24–48 h after 0 Gy (blue dots) or 5 Gy (red dots) exposure. The ImageXpress Micro XL High-Content Analysis of Ki67 fluorescence intensities in sorted populations. Green circles demonstrate Ki67^low area of “quiescent” (G0/G1 arrested) cells. Data are means ± SD of three independent experiments.

Figure 7

Ki67-coupled fluorescence-intensity analysis of distinct cell-cycle characteristics 24–48 h after 0 Gy (blue dots) or 5 Gy (red dots) exposure. The ImageXpress Micro XL High-Content Analysis of Ki67 fluorescence intensities in sorted populations. Green circles demonstrate Ki67^low area of “quiescent” (G0/G1 arrested) cells. Data are means ± SD of three independent experiments.

Figure 8

Click-IT EdU- and Ki67-coupled fluorescence measured simultaneously in sorted A549 and H1299 cells. The single-cell EdU and Ki67 quantitative fluorescence analysis is presented to differentiate between slow-cycling/proliferating population (SCP) (Ki67^low/EdU^low) and rapidly cycling/proliferating population (RCP) (Ki67^high/EdU^high) cohorts within each sorted cell population of irradiated (red dots) and non-irradiated (blue dots, control) cells at 24 h and 48h of cultivation after 5 Gy irradiation. Data are representative of three independent experiments.

Figure 9

The proportion of senescence-associated beta-galactosidase (SA-beta-gal)-positive cells in CD-sorted populations of A549 and H1299 cell lines 24 h after 5 Gy irradiation. Data are means ± SEM of more than three independent experiments. Where: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 10

Representative microphotographs of immunofluorescently stained spheroids showing DAPI (blue), FAM3C (GFP, green) and MiTF (Cy5, pink) (a). The expression of MITF (b) and FAM3C (c) in spheroid cultures derived from CD-sorted populations of A549 and H1299 cell lines four days after 5 Gy irradiation. Data are means ± SEM of more than three independent experiments. Where: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.