Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b

ABSTRACT

Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts.

KEYWORDS:

• Extracellular vesicles
• microRNAs
• cancer-associated fibroblasts
• breast cancer

Acknowledgments

We sincerely thank Dr. Jiahai Shi, Dr. Leo Chan, Dr. Liang Zhang and Jianbo Yue (City University of Hong Kong), Prof. Randy Schekman (University of California Berkley), Prof. Harvey Lodish (Whitehead Institute), Dr. Ng Shyh Chang, Dr. Wai Leong Tam (Genome Institute of Singapore), Dr. Soh Boon Seng (Institute of Molecular and Cell Biology, Singapore) and Andrew Kung (Columbia University) for providing valuable reagents and advices. We also thank Waqas Muhammad Usman, Chanh Tin Pham, Lawrence Chan, Limin Feng, Dr. Amy Fong (City University of Hong Kong), Zhan Xu, Dr. Natasha Barteneva, Dr. Radiana Trifonova and Kenneth Ketman (Boston Children's Hospital), Hazel Kwok (Queen Elizabeth Hospital, Hong Kong) and Morayma Temoche (University of California Berkley) for technical assistance.

Author contribution

L.T.V. and B.P. performed the experiments, collected and analysed the data and prepare the manuscript with assistance from D.X.Z. (EV purification, EV characterization, imaging and animal experiments), V.M. (histopathology, immunostaining and in situ hybridization), C.A.M. (animal experiments, FACS analysis and immunostaining), C.K.L. (EV purification and characterization, digital drawing), T.K. (EV purification and characterization) and J.J. (sequencing).

M.T.L. conceptualized the project, obtained funding, trained and supervised the students, designed the experiments, analysed the data and wrote the manuscript with help from J.L. (funding, training and supervision), W.C.C. (resources and supervision), L.H. (sequencing data analysis), A.G. (supervision), and L.M. (supervision).

The authors declare no competing interests.

Geolocation

Hong Kong, China; Ithaca, New York, USA; Boston, Massachusetts, USA; and Tübingen, Germany

Additional information

Funding

This project is funded by City University of Hong Kong (grant 9610343, 9667133, and 7200475), the Hong Kong Research Grants Council (21106616), the National Natural Science Foundation of China (81602514 and 81773246) and a private donation by Miss Everly Cheryl Choi, Hong Kong.