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. 2020 Jul 10;41(5):625-633.
doi: 10.1093/carcin/bgz124.

Tumor-derived exosomes promote carcinogenesis of murine oral squamous cell carcinoma

Beatrice M Razzo  1   2 , Nils Ludwig  2   3 , Chang-Sook Hong  2   3 , Priyanka Sharma  2   3 , Kellsye P Fabian  4 , Ronald J Fecek  5 , Walter J Storkus  3   5   6 , Theresa L Whiteside  2   3   6   7
Affiliations

Tumor-derived exosomes promote carcinogenesis of murine oral squamous cell carcinoma

Beatrice M Razzo et al. Carcinogenesis. .

Abstract

Circulating tumor-derived exosomes (TEX) interact with a variety of cells in cancer-bearing hosts, leading to cellular reprogramming which promotes disease progression. To study TEX effects on the development of solid tumors, immunosuppressive exosomes carrying PD-L1 and FasL were isolated from supernatants of murine or human HNSCC cell lines. TEX were delivered (IV) to immunocompetent C57BL/6 mice bearing premalignant oral/esophageal lesions induced by the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Progression of the premalignant oropharyngeal lesions to malignant tumors was monitored. A single TEX injection increased the number of developing tumors (6.2 versus 3.2 in control mice injected with phosphate-buffered saline; P < 0.0002) and overall tumor burden per mouse (P < 0.037). The numbers of CD4+ and CD8+ T lymphocytes infiltrating the developing tumors were coordinately reduced (P < 0.01) in mice injected with SCCVII-derived TEX relative to controls. Notably, TEX isolated from mouse or human tumors had similar effects on tumor development and immune cells. A single IV injection of TEX was sufficient to condition mice harboring premalignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor.

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Figures

Figure 1.
Figure 1.
Treatment schema. (A) A schema is provided for 4NQO oral administration in water for tumor initiation and for IV delivery of TEX beginning in week 18. Green, blue and red arrows indicate IV treatments with TEX. A detailed description of the individual treatments can be found in Table I. (B) Representative images of sublingual, lingual and esophageal tumors on gross observation, harvested at weeks 24–26. Black arrows indicate tumors. (C) Representative tumor and EMT images based on H&E histology.
Figure 2.
Figure 2.
Characterization of exosomes derived from mouse SCCVII cells or human SCC90 and PCI-13 HNSCC cell lines. (A) Representative TEM images of SCC90-derived exosomes. (B) Representative TRPS (qNano) size and a concentration distribution plot of SCC90-derived exosomes. (C) Representative TEM images of PCI-13-derived exosomes. (D) Representative TRPS (qNano) size and a concentration distribution plot of PCI-13-derived exosomes. (E) Representative transmission electron microscope images of SCCVII-derived exosomes. (F) Representative TRPS (qNano) size and a concentration distribution plot of SCCVII-derived exosomes. (G) Western blot (WB) profiles of immunoregulatory proteins carried by SCCVII-derived exosomes. Each lane was loaded with 10 μg protein of exosomes lysate. TSG101 is an ESCRT-1 complex protein confirming the endosomal origin of exosomes. (H) Particle concentrations in fraction 4 samples of exosomes derived from SCCVII, PCI-13 or SCC90 cells and normalized to 106 of exosome-producer cells.
Figure 3.
Figure 3.
SCCVII-derived TEX-induced apoptosis (A) and proliferation inhibition (B) of CD4+ or CD8+ splenocytes in vitro. (A) Apoptosis of normal C57BL/B mouse splenocytes 24 h after incubation with increasing concentrations (in μg) of SCCVII-derived TEX. Cells were stained with propidium iodide (PI) and Annexin V and were assessed by flow cytometry. (B) Proliferation as measured by CFSE uptake by Con A-activated versus resting CD4+ and CD8+ splenocytes coincubated with/without SCCVII-derived TEX on day 3 of culture. Data are from a representative experiment of three performed.
Figure 4.
Figure 4.
Mean survival (A) and rates of weight loss (B) for the PBS control group compared with the group receiving 90 μg of exosomes from SCCVII, PCI-13 or SCC90 cells. In C–F, composite in vivo tumor data collected by the gross examination under ×3 magnification of oropharyngeal and esophageal tissue per mouse and reported by group. (C) Average number of tumors per mouse per treatment cohort (receiving different total levels of SCCVII exosomes; in μg protein). (D) Average numbers of tumors per mouse in mice receiving 90 μg or 2× 70 μg of SCCVII-derived TEX, 90 μg SCC90-derived TEX, 90 μg PCI-13-derived TEX or 100 μl PBS as a control. (E) Aggregate volumes of tumors per mouse as measured in groups of mice receiving different total levels of SCCVII-derived exosomes (in μg protein). (F) Aggregate tumor volumes per mouse as measured in groups of mice receiving 90 μg or 2× 70 μg of SCCVII-derived TEX, 90 μg of SCC90-derived TEX, 90 μg PCI-13-derived TEX or 100 μl PBS as control. The tumor volume data in (C and D) are calculated as an ellipsoid (4/3π abc).
Figure 5.
Figure 5.
IFM analysis of the impact of TEX delivery on 4NQO-induced oral carcinoma in mice. (A) Representative IFM staining of tumor sections at week 27/28 from the various indicated treatment cohorts for Ki-67, CD4 and CD8a (green fluorescence) with DAPI counterstaining of nuclei (blue fluorescence) at ×10 magnification (scale bar: 100 μm). Quantitative analysis of IFM staining Ki-67 (B), CD4 (C) and CD8a (D). All data are expressed as the percentage of the area positively stained from the region of interest (% ROI).
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