Targeting extracellular vesicle-mediated tumor-stroma communication
Biochemistry
Evolutionary Cell and Developmental Biology (Zoology)
Final Report Abstract
Interrupting the communication between cancer and stromal cells can prevent tumorigenesis in experimental models. However, our understanding of the mode of tumor-stroma communication is still insufficient. Extracellular vesicles (EVs) provide a potential clue to why tumor-stroma targeting has not been more successful, as EVs transfer a variety of molecules at the same time. EVs are small membrane-encapsulated, DNA-, RNA- and protein-containing structures, which are released from all cell types. Using live-mouse microscopy, it was shown that tumorgrowth-supporting fibroblasts release many-fold more EVs than non-supportive fibroblasts. Flow cytometry further indicates that fibroblast EVs can be taken up by triple negative breast cancer cells in vitro, whereas Cal51 cancer cell-derived EVs are hardly taken up by fibroblasts. In order to visualize uptake of EVs into cells in vitro and in vivo, a method was established to tag the EV-uptaking cells based on Cre-Lox-mediated DNA recombination. It is shown that EV-releasing fibroblasts transfer, in a cell-cell dependent manner, Cre mRNA in EVs to breast cancer cells, thus changing their expression from red to green fluorescent protein. Unfortunately, only a small number of cells showed a successful uptake of EVs, and uptake could not be seen when cells were not in direct cell contact. Because of the small number of cells switching in vitro it was not possible to further analyze the fate and impact of these cells in terms of cellular assays or tumor growth. An EV transfer from fibroblasts to cancer cells in vivo was not apparent in the models used in this project. To decode the language of the EV-mediated communication of tumorsupportive fibroblasts and breast cancer cells, we used a mass spectrometry-based proteomics method to determine key proteins of fibroblast-released EV species. In general, tumorsupportive HFFF2 fibroblasts release way more proteins than non-supportive CCD1112Sk fibroblasts and centrifugation of EVs over an iodixanol gradient is sufficient to gain independent profiles for exosomes and microvesicles. But, most identified proteins are found in another cluster that represents both exosomes and microvesicles. In further studies, this approach can be used to study selective inhibitors of exosome or microvesicle release and its impact on tumor growth.
