1. Development and validation of an analytical strategy to quantify ABC transporters in EVs.
We developed a UPLC-MS/MS method aimed at the simultaneous quantification of P-gp, MRP2 and BCRP. The method was validated according to international standards (EMA, FDA). Surrogate peptides for each transporter were identified and the best chromatographic conditions for these analytes and our UPLC-MS/MS hardware were identified. Linearity, specificity and selectivity were confirmed.
2. Determination of ABC transporter expression in MDCK cells and EVs
Our analytical method was tested on MDCK cells (i.e. renal epithelial cells) with and without overexpression of each individual transporter. EVs and fully reproduced the transporter overexpression of the cells of origin. These results, confirm the suitability of our analytical method for the simultaneous detection of ABC transporters in cells and their derived EVs.
3. Determination of ABC transporter expression and activity in cell lines, immortalized cells and their derived-EVs under situations of up- and down-regulation
We applied our UPLC-MS/MS method to determine whether EVs reflected changes in the transporter expression in the cells of origin. First of all, suitable situations of regulation had to be identified. We used rifampicin, as modulator. Eight different cells were screened. For a cell type to be suitable for further analysis, at least one transporter needed to be expressed and regulated in the cells and detected in EVs. LS180 (up-regulation of P-gp), HepG2 (down-regulation of MRP2) and MCF7 cells (up-regulation of BCRP) fulfilled the conditions.
In HepG2 cells, the decrease in MRP2 expression by rifampicin (and hypericin, a second compound used for confirmation purposes), was fully reflected by the EVs. In MCF7 cells, instead, the BCRP up-regulation by rifampicin was not reproduced by EVs. Although, our results support the potential of EVs as surrogate for ABC transporter regulation, important cell- and transporter factors need to be considered.
4. EVs as surrogates for ABC transporter expression in a multicellular system
We used blood-brain-barrier organoids, consisting of endothelial cells, pericytes and astrocytes, as a model to investigate whether cell-specific EVs can be isolated and, analyzed.
First of all, the expression of P-gp, MRP2 and BCRP was determined in lysates from the constituent cells and their EVs. While endothelial cells exhibited detectable levels of all transporters, none of them was detected in pericytes, astrocytes or EVs. The lack of transporter expression in EVs, specially in the case of astrocytes and pericytes, is likely attributed to the intrinsically low expression in the cells of origin. Thus, blood-brain-barrier organoids do not constitute a good model to study ABC transporters in EVs.
