Fig. 3

Radiotherapy- or chemotherapy-induced DNA repair triggers the biogenesis of cancer stem cells of human solid tumors. A Percentage of tumorsphere formation of gastric cancer stem cell-like cells (GCSCs) and non-stem cells (GCNSCs). Three GCSCs (GCSC-1, GCSC-2 and GCSC-3) and three GCNSCs (GCNSC-1, GCNSC-2 and GCNSC-3) were isolated from the solid tumors of three patients with gastric cancer, respectively. Then, the isolated potential GCSCs and GCNSCs were subjected to tumorsphere formation assays. B Tumorsphere formation assay of a single cell from GCSCs. Days indicated the culture time. C Upregulation of NME2 in gastric cancer stem-like cells (GCSCs). The expression level of NME2 in GCSCs and gastric cancer non-stem cells (GCNSCs) sorted from 3 gastric cancer patients was examined using Western blot. β-tubulin was used as a control. D Identification of the proteins interacted with the promoter of NME2 gene. Nuclear proteins extracted from GCSC-1 were incubated with the biotin-labeled dsDNA of NME2 promoter and streptavidin-agarose beads. An intragenic double-stranded DNA coupled to streptavidin-agarose beads alone was used as a control. The proteins bound to the NME2 promoter were separated by SDS-PAGE with Coomassie staining and then identified by mass spectrometry. Arrows indicated the identified proteins bound to the NME2 promoter. M, protein marker. E Western blot of the proteins bound to the NME2 promoter in GCSC-1. The proteins of the pulldown eluates using the NME2 promoter were analyzed using Western blot. Histone H3 was used as a control. F Expressions of DNA repair genes in radiotherapy- or chemotherapy-treated gastric cancer non-stem cells. GCNSCs were treated with doxorubicin, cisplatin or X-ray. At 48 h after treatment, the gene expression in cells was examined with Western blot. The cells without any treatment were used as controls. β-tubulin was included in Western blots as a loading control. G Percentage of tumorsphere formation of radiotherapy- or chemotherapy-treated GCNSCs. The cells were treated with cisplatin, doxorubicin or X-ray and then cultured for 7 days. The tumorsphere formation efficiency of radiotherapy- or chemotherapy-treated GCNSCs was evaluated. The data were the average values of three independent experiments (*p < 0.01). H Tumorsphere formation of a single tumor cell. A single cell from a tumorsphere of radiotherapy- or chemotherapy-treated GCNSCs was subjected to tumorsphere formation assay. At different time after culture, the tumorsphere was examined. Scale bar, 20 μm. I Serial tumorsphere formation of a single cell from cisplatin-, doxorubicin- or X-ray-induced GCSCs. A single cell from cisplatin-, doxorubicin- or X-ray-induced GCSCs was subjected to primary tumorsphere formation assay. A single cell from a tumorsphere of primary tumorsphere formation assay was assayed in the secondary tumorsphere formation assay. A single cell from a tumorsphere of secondary tumorsphere formation assay was characterized in the tertiary tumorsphere formation assay. At different time after culture, the tumorsphere was examined. Scale bar, 20 μm. J Western blot analysis of stemness genes in the cisplatin-, doxorubicin- or X-ray-treated GCNSCs. GCNSCs were treated with cisplatin (300 μM), doxorubicin (0.5 μM) or X-ray (20 Gy). At different time after treatment, the expression levels of stemness genes in GCNSCs were detected using Western blot. β-tubulin served as a control. K Influence of DNA damage on the stemness of GCSCs. GCSCs were treated with cisplatin, doxorubicin and X-ray for 24 h. Then, the expression levels of stemness markers in GCSCs were determined using Western blot. β-tubulin was used as a control. L Flow cytometry plots showing ALDH activity of GCNSCs with or without cisplatin, doxorubicin or X-ray treatment. GCNSCs were treated with cisplatin, doxorubicin or X-ray for 48 h. The ALDH1-positive cells were GCSCs (P4 region) and the ALDH1-negative cells were GCNSCs (P3 region). M Representative images of immunofluorescence staining of GCNSCs treated with or without cisplatin, doxorubicin and X-ray. GCNSCs were treated with cisplatin, doxorubicin or X-ray for 48 h, and then the cells were analyzed using immunofluorescence analysis with the specific antibody against Lgr5 or Klf4. Nuclei were stained with DAPI. Scale bar, 5 μm. N Expression levels of stemness genes in radiotherapy- or chemotherapy-treated and actinomycin D-treated GCNSCs. GCNSCs were simultaneously treated with cisplatin, doxorubicin or X-ray and DNA repair inhibitor actinomycin D for 24 h. GCNSCs treated with cisplatin, doxorubicin or X-ray alone were used as controls. Then, the expression profiles of stemness genes in cells were examined using Western blot. O Examination of stemness genes’ expressions in radiotherapy- or chemotherapy-induced GCSCs using Western blot. β-tubulin was used as a control. P Upregulation of stemness genes in the sorted GCSCs. Western blot was conducted to examine the expressions of stemness genes in GCSCs and GCNSCs. Q Tumor growth curves in mice inoculated with the cisplatin-, doxorubicin- or X-ray-induced GCSCs, as well as the sorted GCSCs and GCNSCs. The tumor volumes of 5 mice of a treatment were evaluated every week. R Tumor sizes of different treatments. The mice inoculated different cells were killed at 50 day after the inoculation of cells. Scale bar, 1 cm. S Model for the chemotherapy- and radiotherapy-induced transition of gastric cancer non-stem cells to stem cells