Organ involvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells in the C57BL/KaLwRij mouse

Abstract

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...

Murine 5T multiple myeloma cells induce angiogenesis in vitro and in vivo

“…Since the lumen of the sinusoids belongs to the extramedullary compartment this finding confirms the presence of circulating tumour cells. As previously observed (Vanderkerken et al, 1997), the 5T33MM cells were more blastic (centrally located nuclei, distinctive nucleoli) compared to 5T2MM cells in which the plasmacytic differentiation was more readily observed (eccentric location of the nucleus, clumped chromatin).…”

“…Immunostaining for the CD31 antigen of the blood vessels in the BM of 5T2MM and 5T33MM diseased mice revealed a significantly higher MVD in the diseased animals compared to the control animals. The higher angiogenic potential of the 5T33MM model can be due to the more aggressive growth of the 5T33MM cells compared to the 5T2MM cells (Vanderkerken et al, 1997).…”

“…The tumour take was followed up by protein electrophoresis of the serum samples (Vanderkerken et al, 1997). Mice were killed when a serum paraprotein concentration of 10 mg ml 71 was reached.…”

“…Subsequently, the cells were further purified on a Percoll (Pharmacia, Uppsala, Sweden) 60% (for 5T2) and 70% (for 5T8); gradient centrifugation at 450 g for 25 min. Cells were stained with anti-idiotype antibodies and purity was assessed by flow cytometry (Vanderkerken et al, 1997). The 5TMM cells (1610 6 cells ml 71 ) were cultured in serum-free MCDB131 medium (an optimised medium for microvascular endothelial cell growth) or were immediately snap-frozen for RNA isolation.…”

“…By this way, several 5TMM models were developed (Radl et al, 1979) with similar characteristics as the human disease: the disease occurs spontaneously at older age, the MM cells are localised in the bone marrow, tumour load can be assessed by paraproteinaemia, osteolytic lesions develop and the molecular mechanisms are like in humans (Vanderkerken et al, 1996;Radl et al, 1988;Asosingh et al, 2000). As previously reported, the 5T2MM model more closely represents human MM, characterized by a moderate progressive course of the disease and induction of osteolytic lesions, while the 5T33MM model represents an aggressive, rapidly progressive variant (Vanderkerken et al, 1997;Radl et al, 1988). Therefore, the 5T2MM and 5T33MM models are good in vivo murine models of myeloma which mimic different aspects of the human disease .…”