Summary The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease.Keywords: multiple myeloma; adhesion molecules; organ involvement; 5T2; 5T33 Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal expansion of malignant plasma cells secreting a monoclonal immunoglobulin (Ig). The disease is mainly localized in the bone marrow. In this microenvironment the myeloma plasma cells receive signals necessary for their proliferation, terminal differentiation and for the secretion of osteoclast-activating factors. The osteoclast-activating factors recruit osteoclasts, which induce in situ osteolytic bone lesions (Bataille et al, 1989;Alsina et al, 1996); this is one of the major characteristics of the disease. It has been suggested that both cytokines and adhesion molecules are involved in this complex network of signals (Van Riet and Van Camp, 1993).To elucidate the exact mechanisms described above, an in vivo MM model is necessary. Radl et al (1979) found that 0.5% of ageing C57BL/KaLwRij mice spontanously developed a disease reminiscent of MM. The MM cells isolated from the bone marrow of different mice (5T MM) did not grow in vitro but could be transplanted by intravenous injection into young recipients of the same strain. This transplantable model resembles the human disease in several aspects (Radl et al, 1988): myeloma occurred spontaneously, the frequency of development of the disease is age related, tumour load can be assessed by paraproteinaemia and the (Radl et al, 1988).In order to understand the homing mechanisms of the 5T MM cells to the bone marrow, it was essential to determine accurately the...

MM is a B-cell malignancy mainly characterized by monoclonal expansion of plasma cells in the BM, presence of paraprotein in serum and occurrence of osteolytic bone lesions. MMPs are a family of proteolytic enzymes that can contribute to cancer growth, invasion, angiogenesis, bone degradation and other processes important in the pathogenesis of MM. We investigated MMP-9 production in the 5T33MM murine model. Expression of MMP-9 protein in supernatant and cell extracts was analyzed by gelatin zymography. The in vitro, stroma-independent variant 5T33MMvt showed no protein expression of MMP-9 in contrast to in vivo growing MM cells, 5T33MMvv. However, when 5T33MMvt cells were injected into naive mice and isolated after tumor take (5T33MMvt-vv), they secreted a significant amount of MMP-9. These results were confirmed by specific staining of cytospins with an anti-MMP-9 antibody. The MMP-9 production by 5T33MMvt-vv cells disappeared when the cells were recultured in vitro. These data demonstrated that upregulation of MMP-9 occurs in vivo and that this process is dependent on the microenvironment. Key words: multiple myeloma; matrix metalloproteinase-9; bone marrow; stromal cell; endothelial cell MMPs are a family of zinc-dependent endopeptidases involved in the degradation of many components of the ECM and the basement membrane. 1,2 Depending on their substrate specificity and structure, members of the MMP family can be divided into subgroups of collagenases (MMP-1, -8, -13), stromelysins (MMP-3, -10, -11, -7), gelatinases (MMP-2, -9), membrane-type MMPs and other MMPs. 2,3 MMPs are secreted as inactive proenzymes and activated extracellularly by proteolytic cleavage. MMP production is regulated at the level of transcription, secretion and/or activation. 3,4 All MMPs are inhibited by specific TIMPs, including TIMP-1, -2, -3 and -4. TIMP-1 and TIMP-2 bind tigthly with, respectively, pro-MMP-9 and pro-MMP-2 to regulate their activity. 5 The balance between MMP and TIMP levels determines the net proteolytic activity. This equilibrium is highly regulated in physiologic conditions, e.g., wound healing and embryogenesis, 3 but is disturbed in pathologic circumstances, e.g., rheumatoid arthritis, multiple sclerosis and cancer. 6 -9 MM is a B-cell cancer mainly characterized by proliferation of malignant plasma cells in the BM, presence of a monoclonal serum immunoglobulin and occurence of osteolytic lesions. The disease occurs at older ages and remains incurable despite progress in treatment. Therefore, new approaches for therapy are necessary. The hypothesis is that MMPs are involved in a number of events underlying MM progression, e.g., transendothelial migration, invasion, osteolytic lesions and angiogenesis. 10 Barillé et al. 11 demonstrated MMP-9 production by human MM cells. MMP-9 is involved in transendothelial migration and invasion. 12,13 This suggests a role of the protease in the homing of MM cells to the BM, which implicates transendothelial migration and invasion of MM cells in the BM. Coculture of malig...

Multiple myeloma (MM) is a B-cell neoplasm, mainly characterized by the monoclonal expansion of plasma cells, the presence of monoclonal serum immunoglobulin and the occurrence of osteolysis. The malignant cell corresponds to a long-lived plasma cell located in the bone marrow (BM) carrying somatically rearranged Ig genes with clonally fixed hypervariable regions (Bakkus et al, 1992;Hallek et al, 1998). This implies a post-germinal centre origin of the MM cells (Bakkus et al, 1992) having a specific migration to the extravascular compartment of the BM. This process of migration of MM cells to the extravascular compartment of the BM, and referred to as 'homing', is believed to be highly specific since no elevated quantity of malignant B-cells are observed in the peripheral blood or in other organs. After their specific homing to the BM, these cells proliferate and differentiate into mature plasma cells. It is only at the endstage of the disease of some patients that extramedullar spread is observed.In our previous work we have reported the 5T2MM model in the C57BL/KaLwRij mice (Radl et al, 1979) as a good model to study the homing of human myeloma cells in the BM (Vanderkerken et al, 1996(Vanderkerken et al, , 1997. In this model, MM cells are isolated from diseased animals and are transplanted into young syngeneic recipients by intravenous injection. From 9 weeks on after injection we observe a specific localization of the MM cells in the BM and partly in the spleen of the mice. This study did not, however, reveal whether this selective localization of MM cells in the BM is due to a selective initial entry of the cells through the endothelial barrier of the BM, or whether this homing is more random, and survival and growth factors, only present in BM, determine the unique presence of MM cells.The migration of lymphocytes in general (Butcher and Picker, 1996) is known to include a multistep cascade of processes mainly involving the endothelium. This interaction with the endothelium requires at least four independent steps: initial tethering, arrest, adhesion and transendothelial migration. It is the combination of the selectivity of each independent step which makes the process highly specific. In vivo, BM endothelial cells (BM EC) act as gate-keepers separating the BM compartment from the sinusoidal lumen. These cells may therefore play an important role in the selective entry of the MM cells.We conclude here that MM cells have a selective homing behaviour which is the result of the combination of a selective adhesion to the BM EC followed by the entry in the extravascular compartment and a selective survival and growth, making the BM and spleen microenvironment unique. Summary One of the main characteristics of multiple myeloma cells is their predominant localization in the bone marrow. It is, however, unclear whether this is due to a selective initial entry, or whether this entry is more random and other processes like survival and/or growth stimulation, only present in the medullar microenvironment, are uniqu...

Summarv The evolution of bone lesions in transplantable C57BL KaLwxRij 5T mouse myeloma (MM) has been folloxed in vivo. Mice were anaesthetised and a radiograph of the pelVis and hind legs was performed by a radiograph dedicated for mammography. This is the first description of an in vivo technique under experimental conditions w-hereby the development of bone lesions owing to the MM growth was demonstrated.Keywords: multiple my eloma: osteolysis 51T2The ST multiple myeloma (MM) lines in C57BL KaLwRij mice w-ere originallx developed by Radl et al. (1979. 1988

Patients with multiple myeloma commonly develop focal osteolytic bone disease, as well as generalised osteoporosis. The mechanisms underlying the development of osteoporosis in patients with myeloma are poorly understood. Although disruption of the RANKL/OPG pathway has been shown to underlie formation of focal osteolytic lesions, its role in the development of osteoporosis in myeloma remains unclear. Increased soluble RANKL in serum from patients with myeloma raises the possibility that this molecule plays a key role. The aim of the present study was to establish whether sRANKL produced by myeloma cells contributes directly to osteoporosis. C57BL/KaLwRij mice were injected with either 5T2MM or 5T33MM murine myeloma cells. 5T2MM-bearing mice developed osteolytic bone lesions (p<0.05) with increased osteoclast surface (p<0.01) and reduced trabecular bone volume (p<0.05). Bone volume was also reduced at sites where 5T2MM cells were not present (p<0.05). In 5T2MM-bearing mice soluble mRANKL was increased (p<0.05), whereas OPG was not altered. In contrast, 5T33MM-bearing mice had no changes in osteoclast surface or trabecular bone volume and did not develop osteolytic lesions. Soluble mRANKL was undetectable in serum from 5T33MM-bearing mice. In separate experiments, RPMI-8226 human myeloma cells were transduced with an human RANKL/eGFP construct, or eGFP alone. RPMI-8226/hRANKL/eGFP cells, but not RPMI-8226/eGFP cells, stimulated osteoclastic bone resorption (p<0.05) in vitro. Sub-cutaneous injection of NOD/SCID mice with RPMI-8226/hRANKL/eGFP or RPMI-8226/eGFP cells resulted in tumour development in all mice. RPMI-8226/hRANKL/eGFP-bearing mice exhibited increased serum soluble hRANKL (p<0.05) and a three-fold increase in osteoclast number (p<0.05) compared to RPMI-8226/eGFP-bearing mice. This was associated with reduced trabecular bone volume (27%, p<0.05), decreased trabecular number (29%, p<0.05) and increased trabecular thickness (8%, p<0.05). Our findings demonstrate that soluble RANKL produced by myeloma cells causes generalised bone loss, suggesting that targeting RANKL may prevent osteoporosis in patients with myeloma.