Cancer-derived exosomes from HER2-positive cancer cells carry trastuzumab-emtansine into cancer cells leading to growth inhibition and caspase activation

Jorma Isola,Gyorgy Vereb,Janos Szollosi,Mark Barok,Heikki Joensuu,Maija Puhka

doi:10.1186/s12885-018-4418-2

Abstract

BackgroundTrastuzumab emtansine (T-DM1) is an antibody-drug conjugate that carries a cytotoxic drug (DM1) to HER2-positive cancer. The target of T-DM1 (HER2) is present also on cancer-derived exosomes. We hypothesized that exosome-bound T-DM1 may contribute to the activity of T-DM1.MethodsExosomes were isolated from the cell culture medium of HER2-positive SKBR-3 and EFM-192A breast cancer cells, HER2-positive SNU-216 gastric cancer cells, and HER2-negative MCF-7 breast cancer cells by serial centrifugations including two ultracentrifugations, and treated with T-DM1. T-DM1 not bound to exosomes was removed using HER2-coated magnetic beads. Exosome samples were analyzed by electron microscopy, flow cytometry and Western blotting. Binding of T-DM1-containing exosomes to cancer cells and T-DM1 internalization were investigated with confocal microscopy. Effects of T-DM1-containg exosomes on cancer cells were investigated with the AlamarBlue cell proliferation assay and the Caspase-Glo 3/7 caspase activation assay.ResultsT-DM1 binds to exosomes derived from HER2-positive cancer cells, but not to exosomes derived from HER2-negative MCF-7 cells. HER2-positive SKBR-3 cells accumulated T-DM1 after being treated with T-DM1-containg exosomes, and treatment of SKBR-3 and EFM-192A cells with T-DM1-containing exosomes resulted in growth inhibition and activation of caspases 3 and/or 7.ConclusionT-DM1 binds to exosomes derived from HER2-positive cancer cells, and T-DM1 may be carried to other cancer cells via exosomes leading to reduced viability of the recipient cells. The results suggest a new mechanism of action for T-DM1, mediated by exosomes derived from HER2-positive cancer.

Highlights

Trastuzumab emtansine (T-Derivative of maytansine (DM1)) is an antibody-drug conjugate that carries a cytotoxic drug (DM1) to HER2-positive cancer
At immuno-electron microscopy, T-DM1 was present on the surface of Type A exosomes derived from the HER2-positive cell lines (SKBR-3, SNU-216) and treated with T-DM1, but not on any of the control Type A exosomes (SKBR-3 or SNU216 exosomes treated with phosphate-buffered saline (PBS), or MCF-7 or fetal bovine serum (FBS) exosomes treated with T-DM1)
In a flow cytometry analysis, where exosome-bound T-DM1 was detected by staining it with Alexa Fluor 488 (A488)-goat antihuman IgG, high amounts of T-DM1 were found in Type A exosomes derived from the culture media of the HER2positive cell lines (SKBR-3, SNU-216) and treated with T-DM1 compared to exosomes from the HER2-negative cell line MCF-7 or FBS treated with T-DM1, or to SKBR-3 or SNU-216 exosomes treated with PBS (Fig. 2a)

Summary

Introduction

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that carries a cytotoxic drug (DM1) to HER2-positive cancer. The target of T-DM1 (HER2) is present on cancer-derived exosomes. Trastuzumab emtansine (T-DM1, Kadcyla®), an antibodydrug conjugate (ADC), was designed to deliver the cytotoxic drug DM1 (a derivative of maytansine) to human epidermal growth factor receptor-2 (HER2)-positive cancer cells. Exosomes are small (30 to 200 nm) extracellular vesicles covered by a lipid bilayer membrane. They are generated by exocytosis of multivesicular bodies [9]. Exosomes secreted by cancer cells are called cancer-derived exosomes [9]. Cancerderived exosomes have been implicated in the modulation of cancer growth and metastasis, tumor angiogenesis, and anti-cancer immunity [13, 14]

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