Angiogenesis in multiple myeloma: correlation between in vitro endothelial colonies growth (CFU-En) and clinical–biological features

Abstract

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/10 6 BM mononucleated cells in untreated MM samples (2.07 s.d. ± 1.3) was significantly higher than in normal BM (0.28 ± 0.48), while no difference was seen between normal BM and MGUS (0.28 ± 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 ± 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 ± 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo. Leukemia (2001) 15, 171-176.

In vitro assessment of bone marrow endothelial colonies (CFU-En) in non-Hodgkin's lymphoma patients undergoing peripheral blood stem cell transplantation

“…23 In a previous study, we used an in vitro method to detect bone marrow endothelial cells in MM patients; this method was of clinical value, as it gave evidence that an increased number of endothelial colonies was correlated with disease progression and plasma cells proliferation capacity. 16 In this study, the CFU-En number in both untreated and DAV (Dexamethasone, Adryamycin, Vincristine)-treated MM patients was significantly higher than in normal bone marrow, in the melphalan group, and in monoclonal gammopathy of undetermined significance (MGUS) patients, thus confirming a role for these cells in bone marrow angiogenesis. In this paper, as alternative to bone marrow biopsies analysis or angiogenesis related molecules detection, by using the previously published in vitro unselective endothelial cell culture assay, we assessed the spontaneous growth of endothelial colonies in patients with NHL with the aim of (i) evaluating the number and the distribution of CFUEn in relation to patients' clinical characteristics, and (ii) exploring the role played by endothelial cells in a pathological BM microenvironment.…”

“…Because an in vitro method for the analysis and enumeration of endothelial colonies was lacking, we have recently described a rapid and feasible assay to detect BM endothelial cell colonies (CFU-En) that spontaneously grow in the human BM of multiple myeloma (MM) patients. 16 In the same report, we provided evidence in favor of an association between in vitro CFU-En overgrowth and plasma cells proliferation, suggesting a role of these cells in MM angiogenesis process, that may be parallel to that observed in vivo. However, no data are available in the literature regarding the possible role played by BM endothelial cells in hematological disorders such as NHL.…”

“…At the 15th day, using an inverted microscope (10 Â , WilovertWill Wetzlar, Germany), CFU-En detection was carried out as previously described. 16 After 20 days of culture, in order to assess the proliferation capacity, the endothelial cells were subcloned and plated with M199 Glutamax I (Gibco BRL, Life Technology, Paisley, Scotland, UK) supplemented with 20% of fetal calf serum (StemCell Technologies Inc., Vancouver, BC, Canada), 1% penicillin/ streptomycin and 1% L-glutamine (Hyclone Europe Ltd., Cramlington, UK). Endothelial cell growth supplement (ECGS, 75 mg/ml, from bovine neural tissue) and vascular endothelial growth factor (VEGF, 30 ng/ml; both from Sigma-Chemical, St Louis, MO, USA) were also added to the medium.…”

Importance of the bone marrow microenvironment in inducing the angiogenic response in multiple myeloma

“…Emerging evidence indicate that bone marrow-derived circulating EPC can contribute to tumor angiogenesis and growth of certain tumors (Ribatti, 2004). A higher number of EPC has been demonstrated in the bone marrow of patients with active MM than in those with treated MM or with MGUS or in subjects with normal marrow (Dominici et al, 2001), which reflects the increased angiogenic potential in MM. Zhang et al, 2005 have assessed the view that EPC level and function are correlated with MM activity.…”

Vascular Endothelial Growth Factor Signaling Pathway as an Emerging Target in Hematologic Malignancies

“…While it has been known for some time that induction of tumor angiogenesis is associated with tumor volume expansion, the role of this process in hematologic malignancies has, until recently, been unrecognized. Recently, however, it has become clear that angiogenic factors play an important role in the pathophysiology of lymphocytic and myelogenous leukemias, myelodysplastic syndromes, myeloproliferative diseases, multiple myeloma, and non-Hodgkin's lymphomas (Table 1) [6][7][8][9][10]. Because of its broad relevance, neoplastic angiogenesis is now recognized as a biological target of paramount importance.…”