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

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

incubation, the fluorescence signal appears in the acceptor cells (GFP+, DiD+), indicating uptake of the vesicular cargo transferred from donor cells (**Figure 7C**, time 24 h). To confirm that vesicles were transferred by direct cell-cell contact, not secreted into the medium, the trans-well control has been performed. In this case, the vesicle uptake has not been observed (**Figure 7C**, transwell). Such methodology has been already successfully used in different

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

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

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Immune responses against hematological cancers are less characterized than those against solid tumors. Among them, chronic myeloid leukemia (CML) is not an exception [72]. Little is known about mechanisms of innate and acquired immunity exerted against CML.Although, the involvement of CD4+ and CD8+ T cell as well as NK cells and humoral response in the immunity against CML is widely accepted. Recently, CML-specific antigens exerting cellular and humoral response have been identified [73, 74]. The growing body of evidences is pointing the role of cytotoxic CD8+ T cell as a central player in the anti-CML response. [3, 8] Moreover, CML is considered as one of the diseases which are most sensitive to immunological manipulation [75].

Analysis of blood of untreated patients diagnosed with CML reveals decreased number of NK-cells, which are antitumor effector cells [76]. Moreover, the increased number of inhibitory cells such as T regulatory (Treg) cells and myeloid-derived suppressor cells were present [77]. Also, it has been shown that CML cells (including CD34+ leukemia stem cells) express programmed death receptor ligand 1 (PD-L1). Binding of PD-L1 to the PD-1 receptor expressed

Immune evasion is a major obstacle for effective anticancer therapy. T regulatory (Treg) cells are recognized as one of the most promising targets for therapy, which could reverse the unresponsiveness of the immune system during malignancy [73]. Treg cells are essential for maintaining homeostasis of the immune system during the steady state and inflammation. The balance between immune activation and tolerance mediated by Treg cells is crucial for maintaining proper responses. The suppression exerted by Treg cells to different types of immune cells has been extensively studied. Activity of Treg cells is beneficial for maintaining self-tolerance, during resolution of inflammation, and for limiting inadequate immune responses. However, their ability to suppress different types of immune cells may also restrain beneficial responses by limiting the anti-tumor immunity. Treg cells constitute a major component of the tumor-infiltrating lymphocytes in human malignancies as well as in mouse experimental models of cancer. Moreover, the increased number of Treg cells among tumor-infiltrating lymphocytes correlates with a poor prognosis and an increased risk of recurrence in majority of solid tumors. In CML patients, the increased number of Treg cells is linked to limited anti-tumor immune effector responses [78, 79]. Data suggest that a balance between the effector and suppressor arms of the immune system could be important in mediating a successful and treatment-free remission (TFR). However, a major goal in CML treatment is to identify the uppermost target to maximize beneficial immune response and promote TFR success [74]. In this context, the study of T regulatory cells is critical.

cellular systems [60, 69, 71] to analyze direct transfer of vesicles between cells.

**5. Interactions of leukemia cells with immunosuppressive** 

**regulatory T cells**

on T cells suppresses their effector function.

**Figure 7.**(A) Overlay of confocal images presenting formation of tunneling nanotube (TNT); actin—green, microtubules—red, nuclei—blue); (B) SEM micrograph of a TNT formed between two distant cells; (C) flow cytometry dot plots showing the gating strategy to separate the donor and acceptor populations upon labeling of vesicular cargo by DiD (0 h), the uptake of vesicular cargo visible as appearance of DiD+ acceptor cells (24 h), and the absence of uptake upon donor and acceptor populations physical separation in control experiment (24 h: transwell system).

and enable long-range, direct communication between distant cells [59]. Formation of this structure has been observed between different types of cells by different groups including ours [60–63] and **Figure 7A** and **B**. They have been shown to mediate the cell-to-cell transfer of vesicles, organelles, electrical stimuli, and small molecules [64–66]. The proposed functions of TNTs and mechanism are still not well understood; however, there is growing evidence that cell-cell communication *via* TNTs might play an important role in the cancerogenesis. They are actively formed between leukemia cells and bone marrow-derived mesenchymal stromal cells to promote viability and chemoresistance of leukemia cells [67].

Vesicular cargo can be actively transferred by TNTs between two distinct cells. For now, the flow cytometry is one of the best methodologies to track and analyze cells, which obtained vesicles from donor cells [68–70]. For this, methods to label vesicles has been adapted (**Table 2**) and combined with cell type tracking to distinguish donor and acceptor cells. First, vesicles in donor cells are labeled with lipophilic dyes, such as DiD, DiO, and others, and mixed with acceptor cell population. Flow cytometry allows to separate both subpopulations—positive population of donor cells (DiD+) and negative population of acceptor cells (DiD-), together with additional combination with cell-type tracking with GFP (**Figure 7C**, time 0 h). After incubation, the fluorescence signal appears in the acceptor cells (GFP+, DiD+), indicating uptake of the vesicular cargo transferred from donor cells (**Figure 7C**, time 24 h). To confirm that vesicles were transferred by direct cell-cell contact, not secreted into the medium, the trans-well control has been performed. In this case, the vesicle uptake has not been observed (**Figure 7C**, transwell). Such methodology has been already successfully used in different cellular systems [60, 69, 71] to analyze direct transfer of vesicles between cells.
