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

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

**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

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

cells to promote viability and chemoresistance of leukemia cells [67].

physical separation in control experiment (24 h: transwell system).

32 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

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 on T cells suppresses their effector function.

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.

proliferation. Labeling of responder cells with CFSE allowed to track their proliferation after coculture with tTreg, which have been preincubated with either CML-derived or control extracellular vesicles. This in turn allowed to assess differences in tTreg suppressive activity due to influence of EVs. Combination with additional staining of surface receptors such as CD4 or CD8 enabled to distinguish between suppression towards helper (CD4+) or cytotoxic (CD8+) T cells (**Figure 8C**).

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

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

35

Many studies already confirmed that leukemia cells behave differently whether they are cultured alone or in co-culture with stromal cells. This mimics some elements of the bone marrow microenvironment and represents more physiological conditions. Also, the cross talk between leukemia and immune cells is an example showing importance of the interactions within the leukemia microenvironment. Thus, in our opinion, analysis of cell signaling in the co-culture conditions is highly informative and might have some therapeutic implications.

Studies of leukemia cells *in vitro/ex vivo*, including analysis of primary cells and stem/progenitor subpopulations, represent an important step in development of novel therapeutic strategies. Different parameters, such as viability, proliferation, DNA damage, clonogenic potential, and others, are investigated after treatment with drugs or drug candidates. Pretreatment with investigated drugs can also be followed by mice Xenograft *in vivo* studies to investigate their therapeutic potential. All these studies are critically important as a first step in analysis of

Nevertheless, we propose that in the next step or simultaneously, some of these studies might be performed also upon co-culture conditions to add an essential element of the cross-talk with stroma components, which normally exist *in vivo*. These conditions allow to verify whether the treatment is still effective even upon protective impact of stroma. This fulfills, at

In this chapter, we described and discussed flow cytometry applications, which can be used to perform co-culture studies to analyze some signaling elements within the leukemia microenvironment. This strategy enables to distinguish between the cell types and to investigate the cross talk between cancer and surrounding cells, signaling pathways regulated by the cell-cell interactions, as well as sensitivity to treatment with anticancer drugs in the stroma-mimicking conditions. Utilizing modern flow cytometry and a broad spectrum of currently available dyes/trackers allows to perform highly informative studies not only because of the use of multiparameter cytometry but also because of more complex cellular context, which is taken under consideration. Moreover, even if the described flow cytometry applications rely on the leukemia microenvironment studies, they are uniform and can be broadly applied into another biological context.

This work was supported by the National Science Centre research grants: UMO-2013/10/E/

NZ3/00673 to K.P. and UMO-2014/15/D/NZ3/05187 to P. P.-B.

least partially, the big gap between *in vitro* experiments and *in vivo* conditions.

**6. Summary**

potential therapeutic strategies.

**Acknowledgements**

**Figure 8.** Flow cytometry analysis of T regulatory (Treg) cells differentiation and function. (A) Analysis of cell viability and proliferation (AmCyan channel—fixable viability stain eFluor 506, V450—violet proliferation dye 450). V450 staining allows to track thymocyte proliferation and distinguish them from JAWS II cells (V450-). (B) Identification of T regulatory cells by sequential gating and analysis of CTLA-4 expression. (C) CFSE staining allows to track proliferation of responder cells in an *in vitro* suppression test. An example of combination with CD4-APC staining, which helps eliminate remaining Treg cells (CFSE-, CD4+) in analysis, is presented.

To explore the CML-derived extracellular vesicles impact on T regulatory cell development and activity, we adjust two *in vitro* assays, both based on the flow cytometry.
