Merkel cell carcinoma-derived exosome-shuttle miR-375 induces fibroblast polarization by inhibition of RBPJ and p53
Authors: Jan Gravemeyer
Cathrin Ritter
Kai Horny
Thilo Gambichler
Anja Lange
Ivelina Spassova
David Schrama
Dirk Schadendorf
Selma Ugurel
Kaiji Fan
Niels Ødum
Jürgen C Becker
Journal: Oncogene (2021)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Merkel cell carcinoma; miR-375; extracellular vesicles; fibroblast polarization; cancer-associated fibroblasts (CAFs)
Affiliations: Department of Translational Skin Cancer Research, University Hospital Essen, Essen, Germany., German Cancer Consortium (DKTK), Essen, Germany., German Cancer Research Center (DKFZ), Heidelberg, Germany., Department of Dermatology, Medical University of Graz, Graz, Austria., Research Group Bioinformatics, Faculty of Biology, University of Duisburg-Essen, Essen, Germany., Skin Cancer Center, Department of Dermatology, Ruhr-University Bochum, Bochum, Germany., LEO Foundation Skin Immunology Research Center, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark., Department of Dermatology, University Hospital Würzburg, Würzburg, Germany., Department of Dermatology, University Hospital Essen, Essen, Germany., Department of Translational Skin Cancer Research, University Hospital Essen, Essen, Germany. ., German Cancer Consortium (DKTK), Essen, Germany. ., German Cancer Research Center (DKFZ), Heidelberg, Germany. ., Department of Dermatology, University Hospital Essen, Essen, Germany. .

Abstract:  Merkel cell carcinoma (MCC) is a highly invasive and metastatic skin cancer. While high expression of miR-375 is a characteristic of MCC, it seems not to contribute to the malignant phenotype of MCC cells. miR-375 enrichment in MCC-derived extracellular vesicles suggests its intercellular signaling function. Here, we demonstrate that horizontally transferred miR-375 causes fibroblast polarization toward cancer-associated fibroblasts (CAFs). The polarization is evidenced by phenotypic changes and induction of α-SMA, CXCL2, and IL-1β. Fibroblast polarization is inhibited by specific antagomirs and mimicked by experimental miR-375 expression. Mechanistically, miR-375 downregulates RBPJ and p53, two key players regulating fibroblast polarization. In clinical MCC samples, in situ hybridization located miR-375 in CAFs, which correlated with high α-SMA protein and low RBPJ and TP53 expression; single-cell RNAseq revealed a disparate fibroblast polarization negatively correlating with p53 pathway-related gene expression. Thus, the functional role of miR-375 in MCC is to generate a pro-tumorigenic microenvironment by inducing fibroblast polarization. 

Retracted: No

PubPeer comments:
 I am concerned by the methodology of this paper; Firstly, the authors isolate their EVs from conditioned medium containing 10% FBS. This is never done in EV research, as FBS contains huge quantities of bovine extracellular vesicles, far more than the cells release in culture (in our experience, the nanoparticle concentration of FBS is ~10^14 p mL, whereas cells in 2D culture will release between 10^9-10^11 mL depending on cell type. Either EV-depleted FBS is used or the cells are grown in basal medium. The majority of EVs in their conditioned medium will be from their FBS. Secondly, the isolation technique that they use (polymer precipitation, in this case by the commercial product ExoQuick) is notorious for high levels of co-isolation of contaminating material, including miRNA complexes and bioactive soluble protein. Their EV isolate will contain EVs - and so EV markers will be detectable - but the vast majority of their sample will not be EVs derived from their cultured cells, but material isolated from the FBS. Any effects of the sample cannot be attributed to EVs and are likely artefactual and caused by co-isolating miRNA and growth factors from the FBS. These are fundamental issues that should have been picked up in peer review. , 
 I concur. The methodology employed in this paper does not allow assignment of any effect to exosomes as the purification scheme does not isolate exosomes or extracellular vesicles specifically but rather a mix of vesicles and protein complexes. Additionally, the miRNA from FBS may dwarf that of miRNA contained in exosomes. It is a very widespread problem in the field, probably due to the number of people entering and their inexperience and lack of understanding of some of the basic methodology. , 
 I agree with the previous commenters. The extracellular vesicle isolation procedure, characterization methodologies, and experimental design used in this study do not permit conclusions about EVs and certainly not about exosomes (EVs released from multivesicular bodies). It is unfortunate that certain commercial products continue to be advertised as exosome or EV-specific when they are not. For more information about consensus approaches to EV studies, please see: https: www.tandfonline.com doi full 10.1080 20013078.2018.1535750 ,