Journal: Oncotarget

Article Title: Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells

doi: 10.18632/oncotarget.10574

Figure Lengend Snippet: ( A ) Transmission electron microscopy image of SW480 primary CRC derived exosomes (pCRCexo). Arrows indicate different size nanovesicles. Scale bar, 0.2 μM. ( B ) Western blot analysis of sucrose gradient fractions of pCRCexo blotted for the detection of carcinoembryonic antigen (CEA), tsg101 and CD81 (ubiquitous exosome markers) molecules. The density in which exosomes float corresponds to the tsg101- and CD81-positive fractions, and it is comprised between 0.90 and 1.22 g/ml. Total protein extracts of pCRC cells and their purified exosomes (pCRCexo) were loaded as control. M is the weight molecular protein marker; 1–12 correspond to the twelve fractions from sucrose density gradient. ( C ) Phase contrast microscopy (left panels) and scanning electron microscopy (SEM, right panels) images of colonic MSCs (cMSCs) treated for 6 days with pCRCexo. Arrows, asterisks and dotted circle indicate pseudopods, microvilli and vesicles respectively. 20X magnification in contrast microscopy; in SEM scale bar, 20 μM. Inserts represent a 2X magnification. Representative images of two independent experiments are reported. ( D ) Cell proliferation of cMSCs exposed to pCRCexo or cMSCs derived exosomes (cMSCexo) for 6 and 12 days; arrow indicates the exosomes re-feeding at day 9; proliferation was measured at day 6 and 12. ( E ) Cell proliferation of cMSCs incubated with pCRCexo or cMSCexo for 9 days and then replated in fresh medium without exosomes for other 7 days; proliferation was measured at day 9 and 16. ( F ) Cell proliferation of cMSCs or SW480 primary CRC (pCRC) cells incubated with pCRCexo or cMSCexo for 6 days at 1% FCS and pH 6.5 culture conditions. Results in D, E and F are expressed as optical density (mean ± SD, n = at least three independent sets of experiments (** p ≤ 0.005; (*** p ≤ 0.001;), compared to untreated cMSCs (CTR).

Article Snippet: Membranes were incubated with the following primary antibodies: anti-CEA (EPCEAR7, ab133633, Abcam, Cambridge, UK), anti-V-ATPase H (G-2, Santa Cruz Biotechnology), anti-V-ATPase A1 (Santa Cruz Biotechnology), anti-tsg101 (C-2; Santa Cruz Biotechnology) and anti-actin (Sigma-Aldrich, St. Louis, MO).

Techniques: Transmission Assay, Electron Microscopy, Derivative Assay, Western Blot, Purification, Control, Marker, Microscopy, Incubation

Journal: Oncotarget

Article Title: Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells

doi: 10.18632/oncotarget.10574

Figure Lengend Snippet: ( A ) Transmission electron microscopy image of SW620 metastatic CRC derived exosomes (mCRCexo). Arrows indicate different size nanovesicles. Scale bar, 0.2 μM. ( B ) Western blot analysis of sucrose gradient fractions of mCRCexo blotted with CEA, tsg101 and CD81 (ubiquitous exosome markers). The density in which CEA + exosomes float, correspond to the tsg101 + and to the CD81 + fractions, and it is comprised between 0.95 and 1.25 g/ml. Total protein extracts of mCRC and mCRCexo were loaded as control. 1–12 correspond to the twelve fractions from sucrose density gradient. ( C ) Measurements of the volume of colonic MSC spheroids (CTR), formed after the pCRCexo or mCRCexo treatments at 48 and 72 h. ( D ) pH measurements of colonic MSCs spheroids supernatants, derived from pCRCexo- or mCRCexo-treated spheroids (at 72 hours) compared to supernatants of untreated ones (CTR). Statistical analysis were performed by unpaired Student t -test (* p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.001). ( E ) Confocal laser scanning microscopy of cMSCs spheroids incubated or not (CTR) with pCRCexo and mCRCexo and stained for V-ATPase proton pump molecule, followed by Alexa Fluor ® -488-conjugated secondary Ab (shown in white). Nuclei are reported in blue (DAPI). Scale bar, 40 μM.

Article Snippet: Membranes were incubated with the following primary antibodies: anti-CEA (EPCEAR7, ab133633, Abcam, Cambridge, UK), anti-V-ATPase H (G-2, Santa Cruz Biotechnology), anti-V-ATPase A1 (Santa Cruz Biotechnology), anti-tsg101 (C-2; Santa Cruz Biotechnology) and anti-actin (Sigma-Aldrich, St. Louis, MO).

Techniques: Transmission Assay, Electron Microscopy, Derivative Assay, Western Blot, Control, Confocal Laser Scanning Microscopy, Incubation, Staining