Purpose: Despite advances in the treatment, cancer continues to lead to significantly high mortality mainly due to acquired multiple drug resistance (MDR). As natural nanovesicles secreted by cancer cells, exosomes contain a variety of proteins species and surface compositions from their cells of origin. Emerging results have indicated that endogenous exosomes offer significant advantages for the delivery of therapeutic agents with tumor-homing selectivity. In the study, we characterized exosomes released from drug-resistant cancer cells and tested their ability to restore drug uptake to the parental cells.
Methods: Drug-resistant human adenocarcinomic A549 resistant cells (A549/D160) were developed by five sequential treatments with 10, 20, 40, 80, and 160 nM of doxorubicin. Exosomes were isolated from both A549/D160 and A549/WT cell media using ultracentrifugation and their particle size was measured by Deta Nano sizing system. Proteins in exosomes were extracted using a cell lysis buffer and concentrations of total protein were measured by a Pierce BCA assay. Surface proteins on exosomes were evaluated by an Exo-Check array including nine antibodies for known exosome markers including Cis-Golgi matrix protein GM 130, integral membrane protein FLOT1, intercellular adhesion molecule ICMA-1, member of the transmembrane tetraspanin family CD63, member of the transmembrane tetraspanin family CD81, adaptor protein and sort cargo ALIX, FLOT1, ICAM1, epithelial cell adhesion molecule EpCam, marker of apoptosis ANXA5, and tumor susceptibility gene TSG10. The proteomes of two individual exosomes were analyzed by Tandem mass spectra. Exosomes secreted from both A549/D160 and A549/WT cells were labeled by using an Exo-Red Fluorescent Label kit according to the manufacturer’s protocol. Fluorescence-labeled and doxorubicin-loaded exosomes were incubated with resistant and original cancer cells at 37 and 4ºC, respectively.
Results: A549/D160 cells exhibited approximately 34-fold more resistance to doxorubicin than original wild-type cells (A549/WT) with an LD50 value of 234.5±9.2 nM, comparing to 6.2±7.2 nM in A549/WT cells. There was no significant difference in the size and size distribution between these two exosomes. Exosomes derived from resistant A549/D160 cells expressed the highest level of transmembrane protein CD63 (Figure 1). 79 of totally identified 500 proteins were unique for exosomes derived from resistant A549/D160 cells. A high intensity of fluorescence was observed at 37ºC in both cells in an exosome-independent manner. At 4ºC, there was more cell internalization of fluorescent exosomes from A549 resistant cells (Figure 2). Drug-resistant cells were treated with doxorubicin loaded into exosomes and cell viability was measured by MTT assay. Doxorubicin (10 µM) alone did not cause cell death as compared to medium control. Similarly, cells-released exosome alone control (100 µg/ml quantified by the amount of total protein) had no effect on cell viability compared to medium. Doxorubicin delivered by exosomes derived from both resistant and wild-type cells significantly decreased cell viability of 67.9±4.1% and 47.5±4.6%, respectively at 37ºC. Significantly cytotoxic effects (73.4±8.8%) were found with A549/D160 cell-derived exosomes, but not with A549/WT exosomes (94.5±7.0%) at 4ºC (Figure 3). The differences in cellular uptake and delivery efficacy of exosomes between 37°C and at 4°C may be explained by the mechanism that cell-secreted exosomes can use active receptor-mediated endocytosis process and energy-dependent internalization pathway to deliver drugs into cells. As nanovesicles, both types of exosomes used energy-driven transcytosis at 37ºC, but only resistant cells secreted exosomes are concurrently involved in receptor-mediated endocytosis (energy-independent internalization) at 4ºC.
Conclusion: The data in this study provided valuable insights into membrane components of nanosized exosomes that possibly reflect their cellular sources. The outcome will assist in developing useful cells-derived exosomes as carriers to deliver therapeutic agents for overcoming drug resistance, one of the toughest and most challenging tasks in cancer therapy.