Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

Abstract

IntroductionWith the establishment of more effective treatments for patients with chronic lymphocytic leukemia (CLL) over the past decade, complete remissions are no longer the exception. 1 Despite these major improvements in CLL treatment, we still consider CLL an incurable disease, because patients generally relapse from minimal residual disease (MRD). 2 There is growing evidence suggesting that CLL cells are protected from conventional drugs in tissue microenvironments, such as the bone marrow and secondary lymphoid organs, with facilitation of residual disease that is drug resistant and ultimately paving the way to clonal evolution and relapses. The complex cellular and molecular contexts in the tissues, collectively referred to as the CLL microenvironment, provide signals for the expansion of the CLL clone and for primary drug resistance. This is largely dependent on direct contact between the malignant B cells and stromal cells, 3 and therefore has been designated as cell adhesion-mediated drug resistance. 4 Disrupting cross talk between leukemia cells and their milieu is an attractive novel but yet incompletely tested strategy for treating CLL. Appropriately, there is growing interest in understanding the biology of CLL-stroma cross talk to find ways to eliminate residual CLL cells that are "hiding" in stromal niches within the marrow and the lymphatic tissues.Importantly, once CLL cells are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma, they undergo spontaneous apoptosis, highlighting the importance of external signals from accessory cells. 5 Previous studies have shown that CLL cell cocultures with different adherent cell types, collectively referred to as stromal cells, induce leukemia cell survival, migration, and drug resistance. These stromal cells include mesenchymal marrow stromal cells (MSCs), 3,6,7 CD68 ϩ nurselike cells derived from monocytes, 7-10 and follicular dendritic cells. 11 Immunohistochemistry showed that in situ, ␣SMA ϩ mesenchymal stromal cells, 12 the in vivo counterpart of MSCs, are a dominant stromal cell population in the CLL microenvironment, which is in contrast to other B-cell lymphomas, particularly high-grade lymphomas, which harbor larger numbers of CD68 ϩ hemangiogenic cells. 12 MSCs regulate normal hematopoiesis by providing attachment sites and secreted or surface-bound growth factors that constitute the marrow microenvironment. 13 During B-cell development in the marrow, programmed cell death regulates B-cell homeostasis by diverting a large fraction of immature B cells into an apoptotic death pathway to eliminate functionless or potentially harmful cells. 14,15 Critical factors for the survival of selected B cells are interactions with MSCs in the marrow microenvironment, [16][17][18] expression of surface immunoglobulin molecules, and expression of apoptosis-regulatory proteins, such as Bcl-2. 19 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the For personal use only. on May 7,...

Stromal cells modulate TCL1 expression, interacting AP-1 components and TCL1-targeting micro-RNAs in chronic lymphocytic leukemia

“…41,42 Previous studies to model the in vivo marrow microenvironment employed co-culture assays with various BMSC of murine 6,43 and human 11,44 origins, and we recently demonstrated that murine and human BMSC do not significantly differ in their capacity to support CLL cell viability, protection from cytotoxic drugs and maintenance of antiapoptotic proteins, such as MCL1. 12 We previously reported that BMSC induce activation and/or increased expression of molecules involved in CLL survival, such as MCL1. 45 This study, however, is the first to systematically analyze GE changes induced by BMSC, and to uncover BMSC-induced TCL1 expression.…”

“…The bone marrow stroma cell (BMSC) lines KUSA-H1 (murine) and StromaNKtert (human) 23 were purchased from RIKEN (Ibaraki, Japan) and cultured as described. 12 CLL cell viability was studied by the analysis of mitochondrial transmembrane potential using 3,3 0 -dihexyloxacarbocyanine iodide (DioC6; Molecular Probes, Life Technologies, Grand Island, NY, USA) and by cell membrane permeability to propidium iodide (SigmaAldrich, St Louis, MO, USA), and measured by FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).…”

“…9 Co-culture of CLL cells with BMSC results in the rapid, spontaneous migration of a fraction of CLL cells beneath and underneath the BMSC (pseudoemperipolesis) 10 and protects CLL cells from spontaneous or drug-induced apoptosis. 6,11,12 The prototypic TCL1 gene family member TCL1 encodes for a 114 amino acid, 14 kDa proto-oncogene that initially was identified as a target of chromosomal inversions and translocations at the 14q32.1 chromosome breakpoint region in T-cell prolymphocytic leukemia. 13 TCL1 is differentially expressed during normal B-cell development, with robust expression levels from pre-B cells through peripheral naïve B cells, repression in germinal center B cells and silencing in post-germinal center memory B and plasma cells.…”

Actinomycin D induces p53-independent cell death and prolongs survival in high-risk chronic lymphocytic leukemia

“…SDF-1-CXCR4 and VCAM-1-VLA-4 interactions, delivering a major part of cell-adhesion-based protection to CLL cells, are highly conserved between the human and murine system, and thus CLL cell co-culture with murine M2-10B4 has been shown to be a reliable in vitro model to study complex microenvironmental interactions. 31,32 The viability of fludarabine-treated CLL cells was enhanced by co-culturing with M2-10B4 (from 52 ± 23% to 83±38%; P40.001). In contrast, the viability after actinomycin D treatment was similar in the absence (26±21%) or presence (25 ± 22%) of M2-10B4 stromal cells (P ¼ 0.367, Figure 2b).…”

Study of the Quantitative, Functional, Cytogenetic, and Immunoregulatory Properties of Bone Marrow Mesenchymal Stem Cells in Patients with B-Cell Chronic Lymphocytic Leukemia

“…The dependence of leukemic Bcells on microenvironment signals is further highlighted by the observation that despite their prolonged survival in vivo [2], they undergo spontaneous apoptosis when cultured ex vivo [2]. Interestingly, a variety of stromal cell lines have been shown to enhance the survival of CLL cells in co-culture experiments [3][4][5][6][7][8][9][10] and even protect them from drug-induced apoptosis [9,[11][12][13][14].…”