Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma

Abstract

Recently, exosomes secreted by menstrual mesenchymal stem cells have been identified as inhibitory agents of tumor angiogenesis and modulators of the tumor cell secretome in prostate and breast cancer. However, their direct effect on endothelial cells and paracrine mediators have not yet been investigated. Using a carrier-based cell culture system to test the scalability for exosome production, we showed that different types of endothelial cells present specific kinetics for exosomes internalization. Exosome-treatment of endothelial cells increased cytotoxicity and reduced VEGF secretion and angiogenesis in a dose-dependent manner. Using the hamster buccal pouch carcinoma as a preclinical model for human oral squamous cell carcinoma, we demonstrated a significant antitumor effect of intra-tumoral injection of exosomes associated with a loss of tumor vasculature. These results address up-scaling of exosome production, a relevant issue for their clinical application, and also assess menstrual stem cell exosomes as potential anti-angiogenic agents for the treatment of neoplastic conditions.

Figure 1

Carrier-based cell culture increases exosome yield. (A) Schematic overview of exosome production process. MenSC were expanded on 2D cell culture dishes and then seeded on BioNOC II. After 72 h in DMEM with 10% FBS, the medium was changed to serum-free DMEM for 72 h for exosome production. For purification, the supernatant was collected and processed in serial centrifugations. (B) Western Blot with 15 µg of cell lysate (Cells) and exosomes (Exo). To confirm the purity of the exosomes, positive exosomal markers CD63, Syntenin and CD9 and negative exosomal markers Vinculin (Vinc.) and Calreticulin (Calr.) and β-Actin (actin) were analyzed. (C) Scanning electron micrograph of purified exosomes, magnification 60,000x. (D) Size distribution of exosomes determined by nanosight showing that the highest abundance of particles was below 200 nm. (E) Hoechst-stained MenSC on BioNOC II carrier, showing a typical confluence for exosome production. (F) Yield of purified exosomes in PBS as Particles (part)/ml of initial cell culture supernatant (SN) are shown for 2D (N = 32) and 3D culture (N = 10). Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.

Figure 2

MenSC-exosomes rapidly enter endothelial cells in culture. (A,B) Representative histograms and quantification of exosome uptake analyzed via FACS for HUVEC (A) and HMEC-1 (B) at different time points. (C) HUVEC and HMEC-1 were incubated with DiR labeled exosomes and analyzed by confocal microscopy. Scale bar: 50 µm. Bar graphs show average values, error bars: SEM. N = 3.

Figure 3

Exosomes induce cell death in endothelial cells. (A,B) Percentage cytotoxicity determined by LDH activity in HUVEC (A) and HMEC-1 (B) supernatant after 2, 4 and 8 h using 20 µg/ml and 40 µg/ml exosome treatment, respectively. (C) Annexin V/7-AAD viability stain analyzed HUVEC cells after 8 h of exosome treatment (20 µg/ml). (D) Annexin V/7-AAD viability for HMEC-1 cells after 8 h of exosome treatment (40 µg/ml), viable cells correspond to lower bottom quadrant and apoptotic cells to top and bottom right quadrant. Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.

Figure 4

Inhibition of angiogenesis in vitro by exosome treatment. (A) VEGF secretion by HUVEC after 24 h treatment with 20 µg/ml MenSC-exosomes. (B) Representative photographs of HUVEC tube formation on matrigel in vitro, using 24 h HUVEC supernatant without (control) and with 20 µg/ml exosomes, magnification 10x. The negative and positive controls were DMEM and EGM, respectively. (C) Total loops. (D) Percentage covered area. (E) Total branching points. (F) VEGF reduction in HMEC-1 after 24 h treatment with 40 µg/ml MenSC- exosomes. Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.

Figure 5

Tumor growth and angiogenesis is significantly reduced by exosome treatment. (A) Scheme of experimental design. Tumors were induced with four weeks of DMBA treatment and four injections of exosomes were administered every 3–4 days (B,C) Tumor growth in mm₃ tumor volume and relative tumor growth. indicates days of exosome treatment. Control tumors ar e shown as triangles and exosome treated tumors as circles. N = 9 (control), N = 8 (Exo). (D) Histological sections of tumors at day 25 (end-point) with Hematoxylin and eosin stain (H&E). Quantification of vessel density based on H&E sections is shown below. ▲ indicates vessels. (E) Dextran-Fitc (green), VE Cadherin (red) and Hoechst (blue) stained histological sections of tumors at day 25 (end-point). Upper panel: complete tumors, lower panel: zoom of selected tumor area. VE-Cadherin is only shown for zoom (lower panel). Graph shows quantification of vascular area based on dextran stain. Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.