Jin Wang1, Bertrand Z Yeung2, Minjian Cui2, Cody J Peer3, Ze Lu4, William D Figg3, M Guillaume Wientjes5, Sukyung Woo6, Jessie L-S Au7. 1. Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA; Institute of Quantitative Systems Pharmacology, Carlsbad, CA 92008, USA. 2. Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA; Institute of Quantitative Systems Pharmacology, Carlsbad, CA 92008, USA; Optimum Therapeutics LLC, Carlsbad, CA 92008, USA. 3. Clinical Pharmacology Program, National Cancer Institute, NIH, Bethesda, MD 20892, USA. 4. Optimum Therapeutics LLC, Carlsbad, CA 92008, USA. 5. Institute of Quantitative Systems Pharmacology, Carlsbad, CA 92008, USA; Optimum Therapeutics LLC, Carlsbad, CA 92008, USA. 6. Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA. 7. Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA; Institute of Quantitative Systems Pharmacology, Carlsbad, CA 92008, USA; Optimum Therapeutics LLC, Carlsbad, CA 92008, USA; College of Pharmacy, Taipei Medical University, Taipei, Taiwan. Electronic address: jau@optimumtx.com.
Abstract
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our 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.
PURPOSE: 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. METHODS:Humancancer 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. RESULTS: 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. CONCLUSIONS: Our 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.
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