Fig 2: Exosomes contribute to the dedifferentiation of differentiated CRC cells and subsequent drug resistance. a Electron micrograph of exosomes isolated from 18Co cells and CAFs (top; scale bar, 100 nm) and immunoblotting analysis of the exosome marker CD81 (bottom). b The sphere-forming capacity of CD133-/lo SW620 or XhCRC2 cells treated with indicated concentrations of exosomes during chemotherapy (5-Fu or OXA), with CD133+/hi cells as the positive control. *P < 0.05, **P < 0.01. c CM derived from GW4869-pretreated fibroblasts was added to CD133-/lo CRC cells in a sphere formation assay, and CD133+/hi CRC cells were used as a positive control. *P < 0.05, ***P < 0.001. d Effects of exosomes on the growth of CD133-/lo CRC cells (1 × 105 SW620 cells or 4 × 105 XhCRC2 cells) inoculated into immunocompromised mice (n = 5) upon administration of 5-Fu or OXA. Tumor growth curves are shown, and CD133+/hi cells were used as a positive control in SW620 cells. *P < 0.05. e The sphere-forming capacity of GFP-/lo SW620 cells with or without 18Co-exosomes during chemotherapy (5-Fu or OXA), with GFP+/hi cells as a positive control. TOP-GFP expression of spheres was analyzed by flow cytometry. **P < 0.01. f Immunoblotting of total ß-catenin and S552-phosphorylated ß-catenin in CD133-/lo SW620 cells after stimulation with 18Co-CM or exosomes, and CD133+/hi CRC cells as positive control. g Immunoblotting of total ß-catenin and S552-phosphorylated ß-catenin in GFP-/lo SW620 cells after stimulation with 18Co-CM or exosome for 48 h and in the absence of CM or exosome for another 48 h. h mRNA levels of several differentiation markers (mucin2, cytokeratin 20, FABP2) and CSC makers (CD133, Lgr5, CD44, Nanog, OCT4, SOX2, ALDH1 and Bmi1) in exosome-treated spheres in CD133-/lo XhCRC cells. *P < 0.05. **P < 0.01, ***P < 0.001. i Tumor-initiating frequency of exosome-treated CD133-/lo XhCRC cells in NOD/SCID mice

Fig 3: Overall survival in the patients with different IRS scores for CD133 (A), CD24 (B), CD44 (C), and ALDH1A1 (D).

Fig 4: 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.

Fig 5: (A) Colocalization analysis of VEGFR2 with p16INK4A (tumor cell marker of HPV-positive tumors) and VEGFR2 with p53 (tumor cell marker of HPV-negative tumors) by immunofluorescence labeling of OPSCC. Antibody stainings were visualized by confocal microscopy. Colocalization of p16INK4A (a) with VEGFR2 (b) was detected in the cytoplasm and cell nuclei of HPV-positive tumor cells (d). Colocalization of p53 (e) with VEGFR2 (f) was identified in the cytoplasm and cell nuclei of HPV-negative tumor cells. Note, that VEGFR2 was detected in numerous tumor cell nuclei of HPV-positive OPSCC (d) compared to HPV-negative OPSCC (h). Tumor cell nuclei were stained with DRAQ5 (c,g). Scale bar: 20 µm. (B) Colocalization analysis of VEGFR2 with ALDH1A1 (CSC marker) by immunofluorescence labeling of OPSCC. Immunohistochemical staining against VEGFR2 of a representative consecutive HPV-positive (a–f) and HPV-negative tumor section (g–l). (a,g) Overview and (b,h) details. A subpopulation of tumor cells with strong VEGFR2 immunoreactivity can be observed. Colocalization of VEGFR2 (d,j) with ALDH1A1 (f,l) was detected only in a subpopulation of tumor cells and at the subcellular level mainly in the cytoplasm (asterisks). The cells were distributed around the blood vessels (f,l). In HPV-positive OPSCC, some migrating cells were detected at the blood vessel wall and one cell is visible intravasally, while others were recognized in the fibrous tissue (f). Cell nuclei were stained with DRAQ5 (e,k). Scale bars: (a,g) 200 µm; (b,h) 50 µm; (c–f), (i–l) 20 µm.