**4. Application of flow cytometry in the studies of cell-cell cross talk by extracellular vesicles**

## **4.1. Extracellular vesicles**

In recent years, extracellular vesicles (EVs) have emerged as an important mean of intercellular communication, both in physiological conditions and in disease. They seem to play a crucial role in the cell-cell communication between cancer cells and the neighboring cells and they have been shown to be important for the development of cancer [37, 38]. Extracellular vesicles are released by most cell types and contain lipids, proteins, and different nucleic acids—mRNAs, ncRNAs (including miRNAs), and fragments of DNA. EVs are classified depending on their origin: released from multivesicular bodies (exosomes, 30–100 nm), shed from plasma membrane (microvesicles, usually 50–500 nm), or formed during apoptosis (apoptotic bodies, about 1 μm) [39, 40]. EVs can influence targeted cells both locally and in distant tissues. They regulate multiple biological processes, such as cell differentiation, tissue homeostasis, immune response, and cell signaling, and can contribute to disease progression, for example, by affecting tumor microenvironment [41]. Recently, the role of EVs in tissue repair, immunotherapy, and drug delivery has also been evaluated.

Extracellular vesicles play a significant role in pathology of hematological malignancies, including chronic myeloid leukemia (CML) [42]. It has been demonstrated that exosomes produced by CML cells support leukemic cell growth and survival in an autocrine manner, both *in vitro* and *in vivo* [43]. Moreover, factors facilitating leukemic transformation have been identified within vesicles released by leukemia cells [44]. Extracellular vesicles not only directly supported leukemia cell growth but also potently modified components of CML microenvironment to further promote leukemia progression. CML-derived exosomes activated EGFR signaling in stromal cells, which led to IL-8 production by stroma bone marrow fibroblasts and which in turn stimulated leukemia cell adhesion to stromal cells [45, 46]. CML exosomes have been widely studied in terms of their influence on neovascularization process, which has previously been shown to support progression of leukemia [47]. Our own unpublished data suggest that extracellular vesicles released by murine BCR-ABL-expressing progenitor cells may also influence function of cells in distant tissues. Altogether, this supports the notion that leukemic EVs constitute an important mechanism of disease progression both by direct influence on CML cells and by indirect modification of leukemic niche components and immune cells. Moreover, EVs components, such as proteins and microRNAs, have recently become of interest as biomarkers in hematological malignancies [42, 48, 49]. Thus, analysis of EVs number, size, and composition may soon be used to diagnose, stage, and even analyze relapse of hematological malignancies in the clinical setting.
