MO_V_L: Molecular Sciences V. Lectures
Kelemen Andrea1, Idan Carmi1, Wiener Zoltán1
1Semmelweis University, Department of Genetics, Cell and Immunobiology, Molecular Cancer Research Group, Budapest, Hungary
Colorectal cancer (CRC) is one of the most prevalent cancer types. The intra-tumoral heterogeneity is a major feature of CRC, however, its functional importance has not been known yet. CD44, CD133, PTK7 cell surface proteins mark aggressive CRC subpopulations. Extracellular vesicles (EVs) are membrane-surrounded structures released by virtually all cell types and they play a critical role in the intercellular communication. The in vivo cellular heterogeneity of tumors is maintained in the organoids. Here we compared the release intensity and cargo of EVs derived from different CRC subpopulations.
CRC cells from patient-derived organoids were sorted and they were cultured in Matrigel. We analyzed the gene expression pattern of CRC cells with qPCR, flow cytometry, immunohistochemistry. We collected EVs from the supernatant with differential centrifugation. EV number was determined by Nanoparticle Tracking Analysis (NTA). The identity of EVs was proved by transmission electron microscopy and their miRNA cargo was characterized with TaqMan arrays.
The CD44+ and CD133+ populations had only a partial overlap and PTK7+ cells formed a distinct cell population. The diameters of CD44high and CD133high cell-derived organoids were higher compared to organoids from CD44low and CD133low cells, respectively. Interestingly, no difference in organoid size was detected when sorting CRC cells for high and low PTK7 levels. In contrast to PTK7high and CD133high cells that produced similar amount of EVs compared to PTK7low and CD133low cells, we found a markedly higher EV release from CD44high cell-derived organoids than from the CD44low population. In addition, we identified miRNAs specific for the cargo of EVs released by specific CRC cell subpopulations.
We found that whereas CD44+ and CD133+ populations are partially overlapping, PTK7+ cells form a distinct population. The cargo of EVs released by these subpopulations contained specific miRNAs. Our results suggest that the intratumoral cellular heterogeneity characterized by different markers critically determines not only EV release, but EV cargo as well. These data are important when considering EVs as diagnostic tools. This study was supported by BO/00131/20/8 (Hungarian Academy of Sciences), ÚNKP-20-5-SE-18 and by the Richter Gedeon Excellence PhD Scholarship of Richter Gedeon Talentum Foundation.
Semmelweis University, Doctoral School of Molecular Medicine