Fig 2: Analyses of MDA-MB-231-TmxR cells for CD44, CD24 and ALDH1A1 stem-like markers following 72-hour exposure to Tmx, ASA, Met, OP and Tmx in a combination of ASA, Met and OP cocktail using immunocytochemistry and flow cytometry. (A) MDA-MB-231-TmxR cells were plated at 150,000 cells/mL and left untreated, treated with Tmx (20 µM), ASA (8 mM), Met (4 mM), OP (300 µg/mL) and Tmx (20 µM) in combination with ASA, Met and OP for 72 hours. Cells were immunostained for markers of CD44, CD24 and ALDH1A1. The results are depicted as a scatter plot of data visualization using dots to represent the indicated marker values obtained from 2 independent experiments of multiple images (n = 4–12). The mean density staining corrected for background (Bkg) ± S.E.M. for indicated marker values is indicated for each group. The density staining values of each group were compared to the untreated control as well as Tmx in combination with drug cocktail with Tmx alone by ANOVA using the uncorrected Fisher’s LSD multiple comparisons test with 95% confidence with indicated asterisks for statistical significance. (B) Flow cytometry of untreated and Tmx (20 µM) treated MDA-MB-231-TmxR cells for 72 hrs. One million cells were immunostained for CD44, CD24, and ALDH1A1. Data are presented as the percentage of cells expressing the markers corrected for background autofluorescence. ns, not significant, *p = 0.0001, P1, all viable cells; P2, antibody of interest.Abbreviations: Tmx, ?tamoxifen; ASA, acetylsalicylic acid; Met, metformin; OP, oseltamivir phosphate; DAPI, 4',6-diamidino-2-phenylindole, IgG, immunoglobulin G; Bkg, background; ALDH1A1, aldehyde dehydrogenase 1 family, member A1; CD44, cancer stem cell marker participates in cell-cell interaction, cell adhesion and migration; CD24, a cell adhesion molecule and a glycophosphatidylinositol-linked sialoprotein that serves as a ligand for P-selectin.

Fig 3: Analyses of MDA-MB-231 cells for CD44, CD24 and ALDH1A1 stem-like markers following 72-hour exposure to Tmx, ASA, Met, OP and Tmx in a combination of ASA, Met and OP cocktail using immunocytochemistry and flow cytometry. (A) MDA-MB-231 cells were plated at 150,000 cells/mL and left untreated, treated with Tmx (20 µM), ASA (8 mM), Met (4 mM), OP (300 µg/mL) and Tmx (20 µM) in combination with ASA, Met and OP for 72 hours. Cells were immunostained for markers of CD44, CD24 and ALDH1A1. The results are depicted as a scatter plot of data visualization using dots to represent the indicated marker values obtained from 2 independent experiments of multiple images (n = 4–12). The mean density staining corrected for background (Bkg) ± S.E.M. for indicated marker values is indicated for each group. The density staining values of each group were compared to the untreated control as well as Tmx in combination with drug cocktail with Tmx alone by ANOVA using the uncorrected Fisher’s LSD multiple comparisons test with 95% confidence with indicated asterisks for statistical significance. (B) Flow cytometry of untreated and Tmx (20µM) treated MDA-MB-231 cells for 72 hrs. One million cells were immunostained for CD44, CD24, and ALDH1A1. Data are presented as the percentage of cells expressing the markers corrected for background autofluorescence. ns, not significant, *p = 0.01, **p = 0.001, ***p = 0.0001. P1, all viable cells; P2, antibody of interest.Abbreviations: Tmx, ?tamoxifen; ASA, acetylsalicylic acid; Met, metformin; OP, oseltamivir phosphate; DAPI, 4',6-diamidino-2-phenylindole, IgG, immunoglobulin G; Bkg, background;ALDH1A1, aldehyde dehydrogenase 1 family, member A1; CD44, cancer stem cell marker participates in cell-cell interaction, cell adhesion and migration; CD24, a cell adhesion molecule and a glycophosphatidylinositol-linked sialoprotein that serves as a ligand for P-selectin.

Fig 4: WIP1 promotes and the WIP1 inhibitor GSK2830371 reduces the tumor-initiating ability of NSCLC cells and the growth rate of xenograft tumors formed by NSCLC cells in p38-dependent manners. a Numbers of tumors formed after subcutaneous injection of six BALB/c nude mice with the indicated numbers of H460 cells transduced with MKK3E or a vector control (BP), and WIP1 or a vector control (pLV). b Tumor weights measured at the end of the study. The tumors were formed following injection of 1 × 106 H460 cells transduced with MKK3E or the corresponding vector control (BP), and WIP1 or the corresponding vector control (pLV). The data are presented as the mean ± SD, and the lines represent the quartiles and mean values of six xenograft tumors from each group. * indicates P < 0.05, and ** indicates P < 0.01 vs. the control determined by Student’s t test. c, d The growth curves of xenograft tumors formed by injection of 1 × 106 H460 cells transduced with MKK3E or the corresponding vector control (BP), and WIP1 or the corresponding vector control (pLV). The data are presented as the mean ± SD of six xenograft tumors from each group. ** indicates P < 0.01 vs. the control determined by Student’s t test. e Numbers of tumors formed after subcutaneous injection of six BALB/c nude mice with the indicated numbers of H460 cells transduced with MKK6A or a vector control (BH), and then treated with either DMSO or GSK2830371 (25 mg/kg via intraperitoneal injection once daily) from day 1 to 30. f Tumor weights at the end of the study. The tumors were formed by injection of 1 × 106 H460 cells transduced with MKK6A or the vector control (BH), and treated with either DMSO or GSK GSK2830371 for 30 days. The data are presented as the mean ± SD, and the lines represent the quartiles and mean values of six xenograft tumors from each group. ** indicates P < 0.01, and *** indicates P < 0.001 vs. the control determined by Student’s t test. g, h The growth curves of xenograft tumors formed by injection of 1 × 106 H460 cells transduced with MKK6A or the vector control (BH) after 30 days of treatment with either DMSO or GSK GSK2830371. The data are presented as the mean ± SD of six xenograft tumors from each group. ** indicates P < 0.01 vs. the control determined by Student’s t test. i, j Quantification of IHC staining scores for SOX2 and ALDH1A1 in the xenograft tumors described in (b) and (f), respectively. The data are presented as the mean ± SD of three xenograft tumors from each group. * indicates P < 0.05, and ** indicates P < 0.01 vs. a control determined by Student’s t test

Fig 5: Schematic flow diagram of the study design and method. After surgery and tissue processing of OPSCC samples, the HPV-status was determined by GP5+/GP6+ polymerase chain reaction (PCR) and p16INK4A-immunohistochemistry (IHC). To test the specificity of the VEGFR2-antibodies, liver, melanoma, papillary thyroid carcinoma, and cervix squamous cell carcinoma tissues were selected as positive controls. First, hematoxylin and eosin (H&E) staining was performed to determine the regions relevant for further analyses. This was followed by staining of consecutive sections with VEGFR2-AB using the avidin-biotin-peroxidase complex (ABC). Subsequently, densitometric analyses of the VEGFR2-staining intensity of blood vessels in tumor regions and tumor-free regions, as well as tumor cells, were performed. The number of VEGFR2-expressing capillaries in a defined microscopic field was counted and statistically analyzed. Consecutive sections of OPSCC were selected for double immunofluorescence analysis to show colocalization of VEGFR2 and CD31 in endothelial cells (EC), to show the localization of VEGFR2 in tumor cells (TC) by double staining for p16INK4A- (in HPV-positive tumors) or for p53 (in HPV-negative tumors). Confocal double immunofluorescence analysis was performed for VEGFR2 and the stem cell marker ALDH1A1.