**2. Bone marrow niche in leukemia**

The concept of "niche" has been introduced in 1978 by Schofield to describe the possibility of association of hematopoietic stem cell (HSC) with its surroundings that regulates and determines HSC fate, such as self-renewal, differentiation, proliferation and survival [5]. Since then, many evidences appeared showing its composition and function. The bone marrow microenvironment (BMM) is complex and consists of the extracellular matrix and multiple cell populations, including different types of endothelial cells, osteolineage cells, osteoclasts, adipocytes, sympathetic neurons, non-myelinating Schwann cells, mesenchymal cells and macrophages [1, 6]. Interactions between leukemia cells and their niche are bi-directional. They represent either direct interactions by cell-cell contact or indirect interactions by secreted soluble factors and extracellular vesicles.

Over the years, our understanding of the BMM role in leukemogenesis has greatly increased. Bone marrow microenvironment supports proleukemic features of cells. Regulation of leukemia stem/progenitor cells (LS/PC) strongly depends on the signals from the bone marrow microenvironment [3, 7]. They promote leukemia progression as well as development of resistance to therapy. Bone marrow microenvironment is a key player in the pathogenesis and leukemia development by creating conditions which promote survival and proliferation of leukemic blasts [8].

**1. Introduction**

within the microenvironment.

**2. Bone marrow niche in leukemia**

soluble factors and extracellular vesicles.

Cancer cells, including leukemia cells, reside in a microenvironment, which influences biology and activity of these cells. The importance of microenvironment in leukemia cells biology is widely accepted. Leukemia microenvironment is a complex and dynamic network, comprising different types of cells [1]. There is a direct and indirect cross talk between these cells, which modulates cell function, signal transduction and response to therapy. This intercellular interactions can contribute to leukemogenesis [2]. Thus, unraveling of interplay between all types of cells and the microenvironment is crucial for understanding the biology of leukemia. Because of its key regulatory role, the cross talk between leukemia and bystander cells is of interest as possible therapeutic target [3]. It is critically important to take such cell-cell interactions into account in the studies of potential therapeutic strategies, novel therapeutic targets and novel treatment regimes. To partially mimic the physiological conditions, an increasing number of the basic research as well as preclinical studies in the cancer biology area use the *in vitro* cell co-culture systems, in which two or more types of cells are cultured together [4]. This allows natural interactions to occur between different types of cells, which normally coexist

20 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

Therefore, it becomes crucial to improve analytical methods of investigation of co-cultured cells to study their interactions and so to understand biology of leukemia in order to offer novel therapeutic strategies. The principle of flow cytometry is separation of cells/events based on their fluorescence signals. Using different types of cell tracking techniques, combined with modern, multiparameter flow cytometry methods, it has become possible to analyze interactions between different types of cells within the leukemia microenvironment. Flow cytometry allows for gating cells of interest from the whole cell mixture based on the fluorescence parameter to study different cell features, cellular processes and signaling in separated subpopulations.

The concept of "niche" has been introduced in 1978 by Schofield to describe the possibility of association of hematopoietic stem cell (HSC) with its surroundings that regulates and determines HSC fate, such as self-renewal, differentiation, proliferation and survival [5]. Since then, many evidences appeared showing its composition and function. The bone marrow microenvironment (BMM) is complex and consists of the extracellular matrix and multiple cell populations, including different types of endothelial cells, osteolineage cells, osteoclasts, adipocytes, sympathetic neurons, non-myelinating Schwann cells, mesenchymal cells and macrophages [1, 6]. Interactions between leukemia cells and their niche are bi-directional. They represent either direct interactions by cell-cell contact or indirect interactions by secreted

Over the years, our understanding of the BMM role in leukemogenesis has greatly increased. Bone marrow microenvironment supports proleukemic features of cells. Regulation of leukemia stem/progenitor cells (LS/PC) strongly depends on the signals from the bone marrow microenvironment [3, 7]. They promote leukemia progression as well as development of resistance There are many evidence confirming protective role of the bone marrow stroma. Several mechanisms leading to chemoresistance in leukemia cells mediated by BMM have been described. Among these, stroma fibroblasts represent one of the main population within the bone marrow niche, strongly supporting leukemogenesis. Interaction of leukemia cells with stroma fibroblasts results in the increased resistance to cell death in response to drug treatment, changes in metabolism and decreased clonogenic potential [9, 10]. Leukemia cells can also shape the bone marrow microenvironment. We have observed that leukemia cells participate in the remodeling of stroma extracellular matrix [11]. Due to secretion of stromamodifying enzymes, leukemia cells support disarrangement in the leukemic bone marrow. Another aspect of the leukemia microenvironment is development and promotion of the inflammatory microenvironment [12, 13]. Activation of intrinsic factors like NF-κB as well as extrinsic components including cytokines, chemokines and adhesion molecules leads to complex responses promoting proinflammatory state. In general, this supports the process of transformation, survival and proliferation of leukemia cells.

Altogether, due to their importance and key role, targeting the leukemia-stroma interactions has gained big interest as a novel and attractive strategy for anti-leukemia treatment. Currently, our understanding of the function and importance of the bone marrow niche in hematological neoplasms has increased due to improved murine models and development of imaging tools [14–16]. Nevertheless, the *in vitro* studies are still necessary to investigate mechanisms and signaling between leukemia and stromal cells as well as a proof of concept experiments in the drug designing and testing of novel therapeutic strategies.

Our group is involved in the studies of leukemia-stroma interactions and remodeling of stroma cell functions mediated by leukemia cells. We have used experimental setups to investigate leukemia cells *in vitro*, either cell lines or primary cells from patients, in the conditions mimicking the bone marrow millieu, thus hypoxia and co-culture with stroma fibroblasts. Herein we present and discuss some of the experimental systems utilized to study the role of the cross talk between leukemia and stromal cells to which multiparameter flow cytometry can be adapted. Importantly, these experimental approaches can be transferred to other cell types and different biological systems.
