Small Extracellular Vesicles (SEVs) play a role in chemo-resistance in cancer treatment via exocytosis of chemotherapeutics. Ca2+ released from calcium loaded pH-sensitive liposomes (Ca-pSLs) in the endosomes can induce osmotic pressure and might cause endosomal rupture. Previously we have developed a gemcitabine resistant cell line Gr2000 from MIA PaCa-2 pancreatic cancer cell line.
To investigate the potential of Ca-pSLs to suppress SEVs mediated chemo-resistance
MIA PaCa-2 and Gr2000 cells were treated with free gemcitabine for 3 h and then SEVs were isolated by gradient ultracentrifugation of culture medium. The concentration of SEVs were analysed using Nanosight NS300®. After cells were stained with Lysotracker for late endosome and treated with Rh-PE labelled Ca-pSLs or Ca-free-pSLs, the fluorescence was monitored with live cell imaging Olympus FV1000 confocal microscope. Cells were treated with Ca-pSLs or Ca-free-pSLs prepared with thin film hydration method for 2 h, then SEVs were isolated as mentioned above.
MIA PaCa-2 and Gr2000 cells showed a similar production rate of SEVs. Gemcitabine treatment induced significant increase in SEVs production (per 106 cells · day) of Gr2000 (556 ± 19 vs 385 ± 37). In contrast non-resistant cells MIA PaCa-2 showed minor change (368 ± 14 vs 324 ± 24). Ca‑pSLs (drug free) reduced SEVs secretion from both MIA PaCa-2 and Gr2000 cells by 2 and 3 times, respectively. Interestingly, Ca-free-pSLs showed similar inhibition effect. In the Ca-pSLs treated cells, late endosomes fluorescence decreased from 1 to 2h, indicating endosome rupture. In contrast, Ca-free-pSLs did not affect the signal over 2 h.
Ca-pSLs have the potential to overcome gemcitabine resistance by reducing SEVs secretion. Further study is ongoing with gemcitabine loaded Ca-free-pSLs using non pH-sensitive liposomes as references.