Extracellular vesicles (EV) have been proposed to have significant roles in breast cancer growth and metastasis. Therefore they have been evaluated as potential avenues of new therapeutic intervention as well as biomarkers for disease detection.
To resolve the bottlenecks in EV production using in vitro cell line model, we grew a breast cancer cell line BT474 in a two-chamber adhere bioreactor flask (CELLine AD1000). The use of CELLine AD1000 culture flask increased the number of cells and also EVs in a 3D-culture. Advanced culture media (Advanced DMEM/F-12) with the serum replacement (CDM-HD) eliminated the presence of contaminating bovine EVs from fetal calf serum. EVs were isolated by ultracentrifugation and were characterized by their size distribution, EV protein marker expression, and the EV yields were quantified by nanoparticle tracking analysis. We isolated a total of 1.2 e11 micro EV of mean size 145+57nm, and 8.1 e11 small EV of mean size 127+50nm on average from 15 ml of culture supernatants.
We have validated exosome biomarker TSG101 as well as BT474 proteins including HER2 and EpCAM associated with the BT474 derived EV by immunoblotting. GAPDH and HPRT are constitutively expressed in all mammalian cells, these RNA transcripts of GAPDH and HPRT genes1 as well as YB-12 and lncRNA ZFAS13,4 genes were readily detectable by quantitative reverse transcription PCR (qRT-PCR) from BT474 derived EVs while EpCAM and TFF15 genes are selectively retained by cells, suggesting a biological role for these EV-associated RNAs in tumour malignant progression.