**4.2. Analysis of EVs uptake and binding by flow cytometry**

studies, we have combined cell tracking with GFP expression together with analysis of apoptosis by fixable viability dye and detection of intracellular level of Grp78 protein (**Figure 5**). Cells were co-cultured and the subpopulation expressing high level of GFP (GFP+) was gated and further analyzed. Additionally, as cells were treated with anti-leukemia drug, this proce-

**Figure 5.** Combination of cell tracking by expression of GFP protein (GFP+, green), with analysis of apoptosis by using fixable viability dye and estimation of intracellular level of Grp78 protein (red histograms). Subpopulation expressing high level of GFP was gated (GFP+) and further analyzed. Cells were untreated (control) or treated with antileukemic

dure enabled to estimate sensitivity to treatment by detection of dead cells.

28 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

repair, immunotherapy, and drug delivery has also been evaluated.

**by extracellular vesicles**

**4.1. Extracellular vesicles**

drug for 24 h.

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

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

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 Multiple studies underline functional relevance of different extracellular vesicles. Thus, a significant aspect of research is analysis of fluorescently labeled EVs uptake or binding to different cells, both in *in vitro* and *in vivo* experiments. It allows to confirm biological relevance of experiments and exclusion of indirect effects on target cells. Different analytical techniques are used to study extracellular vesicles, as there is no one single method which can be proposed to analyze these particles [50]. Confocal microscopy is widely used to analyze EVs uptake by cells [51]. However, the flow cytometry provides a very good and fast method for quantification of EVs uptake or binding to cells.

Flow cytometry allows to quickly assess if association of EVs with target cells is dose- and timedependent. This may incline biologically active doses of EVs and thus help choose appropriate amounts of EVs for further functional studies. Multicolor flow cytometry allows to monitor association of EVs to different subpopulations of cells, especially *in vivo* or in *ex vivo* cultures of, for example, immune cells. This is doable if a combination of fluorescent EVs labeling is used in parallel with staining of protein markers of subpopulations with fluorochrome-conjugated antibodies. Moreover, flow cytometry analysis enables correlation of EVs uptake with various biological parameters, such as cell viability, apoptosis, proliferation, and expression of signaling molecules. Such approach allows more precise and direct observation of functional effects of EVs. On the other hand, opposed to confocal microscopy, flow cytometry does not distinguish between uptake and binding of extracellular vesicles to target cells, but rather gives information on association of EVs with target cells. Such information may be relevant in terms of mechanism of influence on target cells, as solely binding to surface of target cell suggests regulation through mediators (mostly proteins) on EVs surface rather than by, for example, non-coding RNAs in the lumen.

Various methods and reagents may be used to fluorescently label extracellular vesicles for analysis using flow cytometry (**Table 2**). EVs staining can either lead to labeling of all types of


was used for EVs labeling to track time-dependent uptake of mantle cell lymphoma-derived exosomes by different immune cells and cell lines and to investigate their possible therapeutic potential. Lai et al. took another approach on staining of EV membrane by fusing GFP at N-terminus with palmitoylation signal and thus directing it to plasma and EV membrane. This enabled more specific staining than with the use of PKH67 dye. Additionally, it allows effective *in vivo* tracking of EVs released by tumors [51]. Furthermore, fusion of specific EV-enriched proteins (such as tetraspanins—CD9, CD63, CD81; ESCRT proteins—Tsg101; proteins widely expressed on cells producing EVs—e.g. HLA proteins) with GFP/RFP or other fluorescent proteins can be applied. CD63-GFP and CD81-GFP have already been used

Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry

http://dx.doi.org/10.5772/intechopen.76481

31

Flow cytometry has emerged as one of the tools for analysis of extracellular vesicles and their composition. As detection limit of conventional flow cytometers is around 500 nm, bead capture of EVs has been used to facilitate, for example, analysis of proteins expressed on their surface. However, direct flow cytometry of extracellular vesicles has also been widely performed [55]. As light scatter triggering itself is not sufficient to distinguish nanoscale vesicles, fluorescence triggering has been used to directly identify EVs on flow cytometers. Commercially available flow cytometer (BD Influx) has been adapted for high-throughput analysis of fluorescently labeled vesicles. PKH67 staining of EVs allowed fluorescence-based thresholding of events, and by modifying cytometer elements, the authors were also able to observe size distribution of exosomes in the FSC channel. The described technique allowed to identify molecules expressed on the surface of EVs [56] and then apply the method to perform quantitative and qualitative analyses of dendritic cell-released EVs upon different stimulations and culture conditions [57]. Direct flow cytometry of fluorescently labeled EVs using conventional flow cytometers has been applied to study mesenchymal stromal cell markers (CD90, CD44, CD73) on EVs released by HS-5 and K562 cells as well as to analyze surface molecules expressed on microvesicles isolated from patient sera with hematological tumors (CD20, CD38, CD30) [48]. However, direct flow cytometry of EVs needs to be used cautiously, as different sample parameters and cytometer settings may affect measurements. It was demonstrated that due to high concentration of very small molecules in suspension, the measurement may not be precise, due to analysis of multiple events as one. Flow cytometry is emerging as a tool for direct analysis of EVs and their surface markers, allowing fast and precise measurements. Various aspects of EVs analysis by FACS are demonstrated in a special issue of Cytometry Part A Journal "Measurement of Extracellular Vesicles and Other Submicron Size Particles by Flow Cytometry" [58]. It is noteworthy that due to small size of EVs precise fluorescent labeling needs to be applied and conventional flow cytometer settings need to be cautiously optimized, for example, by using fluorescent beads of different sizes (e.g. 200 and 100 nm beads).

**4.4. Fluorescent tracking and flow cytometry analysis of direct, intercellular transfer** 

Recently, a novel way of intercellular communication has been described. This is mediated by the long intercellular connections called tunneling nanotubes (TNTs), which link two cells

**of vesicular cargo**

by different groups to track exchange of EVs using flow cytometry [54].

**4.3. Flow cytometry analysis of extracellular vesicles**

**Table 2.** Fluorescent dyes used to fluorescently label extracellular vesicles for analysis by flow cytometry; function, excitation, and emission spectra as well as full name are shown.

vesicles or any of the specific populations, like exosomes or microvesicles. To stain EVs lumen, widely used dyes such as CFSE and CalceinAM may be used. Those dyes in non-fluorescent form passively enter EVs and due to esterase activity in the EV lumen are transformed into fluorescent molecules. Gray et al. demonstrated that CalceinAM stains only intact EVs, which may be advantageous for its use in both EVs uptake studies and direct flow cytometry of EVs [52]. CFSE staining was applied to quantitatively show differential association of cancer EVs with myeloid cells depending on their differentiation into various stages of monocyte and dendritic cell development [53]. In our experiments, we have used CFSE labeling to study the dose-dependent uptake of EVs released by murine BCR-ABL-expressing cells by thymocytes (**Figure 6**). It is clearly visible that increasing amounts of fluorescently labeled EVs correlate with increased fluorescence of CFSE signal detected in acceptor thymocytes.

Membrane staining is also widely used to label EVs with the use of dyes such as PKH67, PKH26, DiD, Dil, and DiO. They allow a wider choice of fluorescence spectra but, as they are lipophilic, can form various aggregates and micelles. Thus, in this case, EVs need to be carefully washed to avoid adding unbound dye which may nonspecifically label cells. PKH67

**Figure 6.** CFSE labeling to study uptake of EVs. Leukemia-released EVs were labeled with CFSE and different amounts of EVs were added to thymocytes. Fluorescence of CFSE in thymocytes was detected by flow cytometry in FITC channel.

was used for EVs labeling to track time-dependent uptake of mantle cell lymphoma-derived exosomes by different immune cells and cell lines and to investigate their possible therapeutic potential. Lai et al. took another approach on staining of EV membrane by fusing GFP at N-terminus with palmitoylation signal and thus directing it to plasma and EV membrane. This enabled more specific staining than with the use of PKH67 dye. Additionally, it allows effective *in vivo* tracking of EVs released by tumors [51]. Furthermore, fusion of specific EV-enriched proteins (such as tetraspanins—CD9, CD63, CD81; ESCRT proteins—Tsg101; proteins widely expressed on cells producing EVs—e.g. HLA proteins) with GFP/RFP or other fluorescent proteins can be applied. CD63-GFP and CD81-GFP have already been used by different groups to track exchange of EVs using flow cytometry [54].
