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. 2017 Oct 6;8(56):96035-96047.
doi: 10.18632/oncotarget.21635. eCollection 2017 Nov 10.

TGFβ1 in fibroblasts-derived exosomes promotes epithelial-mesenchymal transition of ovarian cancer cells

Wenqian Li  1 , Xiaoxue Zhang  1 , Ji Wang  1 , Mengchen Li  1 , Canhui Cao  1 , Jiahong Tan  1 , Ding Ma  1 , Qinglei Gao  1
Affiliations

TGFβ1 in fibroblasts-derived exosomes promotes epithelial-mesenchymal transition of ovarian cancer cells

Wenqian Li et al. Oncotarget. .

Abstract

Cancer-associated fibroblasts (CAF), a major component of the tumor microenvironment, play an important role in interacting with neoplastic cells to promote ovarian cancer progression. Exosomes are nano-sized vesicles that mediate the cross-talk between different cell types. An increasing number of studies have focused on the fact that tumor cell-derived exosomes influence stromal cells. However, the mechanism by which CAF-derived exosomes modulate cancer cells in ovarian cancer remains obscure. To investigate the role of CAF exosomes in ovarian cancer, we examined the exosomal content of paired primary, metastatic and normal fibroblasts from seven stage IIIC ovarian cancer patients by ELISA. We found that in ovarian CAF-derived exosomes, TGFβ1 was upregulated compared to normal omentum fibroblasts (NOF). Exosomes derived from CAF were taken up by ovarian SKOV-3 and CAOV-3 cell lines during co-culture and induced malignant behaviors in cancer cells, including an enhanced migration and invasion ability and the promotion of epithelial-mesenchymal transition (EMT) by activating the SMAD signaling pathway. Our results indicate that the role of TGFβ1 in CAF exosomes triggers ovarian cancer cells into a more aggressive phenotype, suggesting that targeting CAF exosomes could be a potential treatment in ovarian cancer.

Keywords: CAF; TGFβ1; epithelial-mesenchymal transition; exosomes; ovarian cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors disclose no potential competing interests.

Figures

Figure 1
Figure 1. Characterization of exosomes derived from primary stromal fibroblasts
(A) Transmission electron microscopy images of ovarian CAF-derived exosomes and NOF-derived exosomes with the exosomes diameter distribution represented in histogram. (B) NanoSight measurement of particle size distribution and concentration in exosomes. (C) Representative images of western blot for the indicated CD63 and TSG101 proteins in fibroblast-derived exosomes lysates. (D) CD63 was present in exosomes derived from patient fibroblasts at different exosomal protein concentrations.
Figure 2
Figure 2. Fibroblast-derived exosomes enter and stimulate migration and invasion of ovarian epithelial cell lines
(A) Exosomes uptake experiment. SKOV-3 and CAOV-3 cells were cocultured with Dil labeled CAF-derived exosomes for 6h. (B) The migration and invasion ability of exosome-treated SKOV-3 and CAOV-3 cells were determined using the Transwell assay. Patients CAF-derived exosomes induced significantly more migration ability and invasiveness than NOF-derived exosomes. Representative images were showed on the left (magnification, ×200), data analysis represented on the right. (C) Analysis of tumor cells migration by scratch assay, wound closure rate represent at least three experiments. * p<0.05, ** p<0.01, *** p<0.001.
Figure 3
Figure 3
Exosomal expression of MMP2 (A), MMP9 (B), TGFβ1 (C), TGFβ2 (D) and CA-125 (E) derived from the supernatant of ovarian fibroblasts were quantified by ELISA. The box plots represent the 25th to the 75th quartiles, with the band inside representing the median. The ends of the extended lines indicate the maximum and minimum values. The expression were measured in paired NOF, CAF-p and CAF-m samples from seven high grade serous ovarian cancer patients.
Figure 4
Figure 4. Fibroblast-derived TGFβ1 induces EMT in ovarian cancer cells
(A) Morphological changes of SKOV-3 cells co-cultured with PBS as control, 10 ng/mL pharmacologic TGFβ1, and CAF-derived exosomes for 72h. Reversal of EMT was formed with TGFβ1 inhibitor treatment. (B) Compared with PBS treated control cells, the CAOV-3 co-incubation with fibroblasts exosomes or recombinant TGFβ1 had significantly elongated pseudopodia after 72h. Filopodia formation (arrows) were zoomed in on the upper left panel. Magnification x200. (C, D) Cell migration and invasion ability were measured by Transwell assay. Patients CAF-derived exosomes or recombination TGFβ1 induced significantly more migration ability and invasiveness than control group. Data analysis represented on the bottom. (E) Ovarian cancer cell lines were stimulated with exosomes or TGFβ1, the expression of EMT markers, E-cadherin and vimentin, and total SMAD2/3, phosphorylation-SMAD2/3 were detected by Western blot. (F, G) The expression of EMT-associated transcription factors were detected in the SKOV-3 (F) and CAOV-3 cells (G) by RT-PCR. * p<0.05, ** p<0.01 and *** p<0.001. (H) Representative bioluminescence images of mice (n=5 each group) bearing SKOV-3-Luc cells alone, co-injection with CAF, or with TGFβ1 antibody at 4 weeks after tumor implantation. Bar graph showing the quantification of normalized total photon counts of the subcutaneous xenografts in mice of each group.
Figure 5
Figure 5. EMT transition of cells cultured with PBS, TGFβ1, CAF-derived exosomes and exosomes with neutralizing TGFβ1 antibody in immunofluorescence
(A) For SKOV-3 cells, expression of E-cadherin (upper) and Vimentin (lower) were changed during EMT, with F- actin and nuclear DAPI staining demonstrated in merge figures. (B) The expression of E-cadherin (upper) and Vimentin (lower) on CAOV-3 cells incubated with different treatments were detected by immunofluorescence staining. Similar variation of protein expressions were observed in both cell lines.
Figure 6
Figure 6. A schematic diagram illustrating the cross-talk of CAF and ovarian cancer cells
CAF-derived TGFβ1 transported into cancer epithelial cells and activated the SMAD2/3 signaling to induce EMT, thus promoting tumor peritoneal metastasis.
See this image and copyright information in PMC

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