Exosomes are small membrane vesicles (30-100nm in diameter) secreted by cells into extracellular space. The present study evaluated the effect of chemotherapeutic agents on exosome production and/or release, and quantified the contribution of exosomes to intercellular drug transfer and pharmacodynamics.
Human cancer cells (breast MCF7, breast-to-lung metastatic LM2, ovarian A2780 and OVCAR4) were treated with paclitaxel (PTX, 2-1000nM) or doxorubicin (DOX, 20-1000nM) for 24-48h. Exosomes were isolated from the culture medium of drug-treated donor cells (Donor cells) using ultra-centrifugation, and analyzed for acetylcholinesterase activity, total proteins, drug concentrations, and biological effects (cytotoxicity and anti-migration) on drug-naïve recipient cells (Recipient cells). These results were used to develop computational predictive quantitative pharmacology models.
Cells in exponential growth phase released ~220 exosomes/cell in culture medium. PTX and DOX significantly promoted exosome production and/or release in a dose- and time-dependent manner, with greater effects in ovarian cancer cells than in breast cancer cells. Exosomes isolated from Donor cells contained appreciable drug levels (2-7pmole/106 cells after 24h treatment with 100-1000nM PTX), and caused cytotoxicity and inhibited migration of Recipient cells. Quantitative pharmacology models that integrated cellular PTX pharmacokinetics with PTX pharmacodynamics successfully predicted effects of exosomes on intercellular drug transfer, cytotoxicity of PTX on Donor cells and cytotoxicity of PTX-containing exosomes on Recipient cells. Additional model simulations indicate that within clinically achievable PTX concentrations, the contribution of exosomes to active drug efflux increased with drug concentration and exceeded the p-glycoprotein efflux when the latter was saturated.
Cellular pharmacokinetic/pharmacodynamic models of paclitaxel
Cmedium, free or Ccell, free is free (unbound) drug concentration in medium or cells, respectively. Ccell, tubulin is the tubulin-bound drug concentration. Dfd is the rate constant of passive diffusion of free drug. JmaxPgp is the maximum Pgp-mediated drug efflux rate. KdPgpis the dissociation constant of drug from Pgp. Cmedium, exo or Ccell, exo is the drug concentration in extracellular or intracellular exosomes, respectively. Jmaxinter, exois the maximum rate of receptor-mediated internalization of exosomes. Kdinter, exo is the dissociation constant of exosome from receptor for internalization. ksort, exo is the rate constant of drug sorting into exosomes. krel, exo is the rate constant of exosome release into extracellular space. Btubulin, max is the maximum available drug binding sites in tubulin. ktubulin, on or ktubulin, off are the rate constants of drug association and disassociation with tubulin, respectively. NSB is the proportion constant for the linear nonsaturable drug binding in cells. For PD parameters, IC50 is the tubulin-bound drug concentration needed to generate 50% of maximum drug effect, kkill is the maximal rate constant of cell kill. n is Hill exponent.
These results indicate (a) chemotherapeutic agents stimulate exosome production or release, and (b) exosome is a mechanism of intercellular drug transfer that contributes to pharmacodynamics of neighboring cells.