RESUMO
Extracellular vesicles (EVs) can mediate intercellular communication, including signaling between the placenta and maternal tissues. Human placental explant culture is a versatile in vitro model system to investigate placental function. We performed systematic studies in different tissue culture media types and oxygen tensions to identify a defined serum-free culture condition that supports high trophoblast viability and metabolism, as well as the release of similar populations of EVs, compared to traditional undefined conditions that contain media additives potentially contaminated with exogenous EVs. We also determined the time frame in which trophoblast viability and functionality remain optimal. Multiplex vesicle flow cytometry with classical EV and placenta-specific markers revealed three separate populations of explant-derived EVs: small CD63+ EVs; large PLAP+ EVs; and CD63-/PLAP- EVs. These culture and analytical approaches will enable in vitro modeling of short-term effects of environmental perturbations associated with pregnancy complications on placental function and EV release.
RESUMO
Although proteolysis targeting chimeras (PROTACs) have become promising therapeutic modalities, important concerns exist about the potential toxicity of the approach owing to uncontrolled degradation of proteins and undesirable ligase-mediated off-target effects. Precision manipulation of degradation activity of PROTACs could minimize potential toxicity and side effects. As a result, extensive efforts have been devoted to developing cancer biomarker activating prodrugs of PROTACs. In this investigation, we developed a bioorthogonal on-demand prodrug strategy (termed click-release "crPROTACs") that enables on-target activation of PROTAC prodrugs and release of PROTACs in cancer cells selectively. Inactive PROTAC prodrugs TCO-ARV-771 and TCO-DT2216 are rationally designed by conjugating a bioorthogonal trans-cyclooctenes (TCO) group into the ligand of the VHL E3 ubiquitin ligase. The tetrazine (Tz)-modified RGD peptide, c(RGDyK)-Tz, which targets integrin αvß3 biomarker in cancer cells, serves as the activation component for click-release of the PROTAC prodrugs to achieve targeted degradation of proteins of interest (POIs) in cancer cells versus noncancerous normal cells. The results of studies accessing the viability of this strategy show that the PROTAC prodrugs are selectively activated in an integrin αvß3-dependent manner to produce PROTACs, which degrade POIs in cancer cells. The crPROTAC strategy might be a general, abiotic approach to induce selective cancer cell death through the ubiquitin-proteasome pathway.
