Isolation and characterisation of imatinib-resistant extracellular vesicles

Abstract

Chronic myeloid leukaemia (CML) is a malignant myeloproliferative disorder of the haematopoietic progenitor cells, having an average age of onset of 50-60 years. Imatinib is the first tyrosine kinase inhibitor (TKI) designed as a first-line therapeutic drug for CML, that greatly increases the 5-year relative survival from 20-30% to 50- 90%. However, if given for long periods of time 1/3rd of patients will develop TKI resistance which poses a problem as the patients do not respond optimally and CML will ultimately progress towards the more aggressive pathogenic phenotypes. In previous studies, exosomes from imatinib-resistant (IR) K562 CML cells have been shown to internalize into imatinib-sensitive (IS) K562 CML cells after incubation. This raises the question of whether exosomes from IR cells are able to confer resistance to IS cells. In this study we will compare exosomes from IR cells with exosomes from IS cells via flow cytometry and we will be assessing the viability of cells via MTT assays. In this experiment, both IS and IR cells share two different populations differing in size, likely due to an adaptive phenotypic shift, with IS cells having a larger percentage of the population consisting of smaller cells and IR cells having a higher percentage of the population of larger cells. IR cells show a greater expression of CD63 and CD81 when compared to IS cells, yet both lack expression of CD9. The population consisting mainly of IR cells is CD45 negative, whereas the other population making up IS cells is CD45 positive. Exosomes derived from IR cells have a higher expression of CD63, CD81 and CD9 when compared to exosomes from IS cells. However, CD45 expression was absent on both types of exosomes. From the MTT assays, neither the addition of IR nor IS exosomes mediated IM resistance to IS cells. The observation of phenotypic shift can aid in development of new specifically-targeted treatment options for CML.