Abstracts of 2nd Scientific Meeting on Bone Disease in Multiple Myeloma
Homing of myeloma cells in the 5T murine model of myeloma
Karin Vanderkerken, Kewal Asosingh, Els Van Valckenborgh, Isabelle Vanden Broek, Ivan Van Riet and Ben Van Camp: BUSSELS
The 5TMM models were initially developed by Radl (1979). The myeloma (MM) cells originated spontaneously in aging (> 2 years) mice with a frequency of 0.5%. The development of the disease was characterised by the occurrence of monoclonal serum Ig and the development of osteolytic lesions. The bone marrow of the diseased animals was isolated and transplanted in naive, young, syngeneic mice. Several 5TMM lines were developed, each with its own characteristics (Asosingh et al, 2000, in press). The lines were maintained in vivo by intravenous transfer into young syngeneic mice.
In our studies we selected two lines, 5T2MM and 5T33MM. Both lines have similar characteristics, i.e. increasing concentrations of serum Ig during development of the disease and localisation of the MM cells in bone marrow and spleen (a hematopoietic organ of the mouse) (Vanderkerken et al, 1997). In the 5T33MM model, tumor cells were also localised in the liver. In the 5T2MM model, which is a less aggressive growing compared to the 5T33MM model, osteolytic lesions develop during the course of the disease and at endstage-ill mice several lesions can be detected by X-ray radiography (Vanderkerken et al, 1996).
Both 5T2 and 5T33MM lines are in vivo growing although in vitro short-term cultures are possible when cocultured on bone marrow fibroblasts. For the 5T33MM line however, an in vitro stroma-independent growing variant was established. The clonal identity of this line was proven (Asosingh et al, 2000) and when injected, similar disease developed as the original 5T33MMvivo line.
One of the topics which is dealt by our group is the investigation of the homing characteristics of these lines. Homing of cells can be defined as the entry from the vascular to the extravascular compartment. One of the main characteristics of MM cells is their predominant localisation in the bone marrow. MM cells have a post-germinal origin (Bakkus, 1992) and their selective presence in the bone marrow implies transendothelial migration for the initial entry in the bone marrow and/or for the intravasation and subsequent extravasation to distant bone marrow sites. This situation is mimicked in the murine model where the 5TMM cells are injected i.v. and where in a later stage proliferating MM cells are observed in the extravascular compartment of the bone marrow. Transendothelial migration is a process (Butcher, 1996; Foxman, 1997) known to determine the specific migration of leukocytes. It is the combination of the specificity of each step which determines the high specific process of homing.
We previously demonstrated the selective presence of 5T2MM cells in bone marrow and spleen of diseased mice. To investigate whether this could be explained by a selective homing to or a selective survival in the bone marrow or by a combination of both, immediate homing experiments were performed. Hereby 5T2MM cells were intravenously injected into naive mice and 18h later the presence of 5T2MM cells in different organs investigated. Tracing of radioactive labelled 5T2MM cells, FACS analysis for the presence of 5T2MM idiotype positive cells and specific PCR demonstrated a selective homing to bone marrow, spleen and liver. In 5T2MM diseased animals tumoral cells were only detected in bone marrow and spleen indicating a combination of a selective homing to and a selective survival/growth in the bone marrow (and spleen). To investigate which key molecules are involved in the selective homing, we investigated different processes of transendothelial migration:
- Attraction of MM cells by endothelial cells,
- Adhesion of MM cells to endothelial cells,
- Attraction of MM cells to the extravascular compartment,
- Production of matrix metalloproteinases (MMP) by MM cells, necessary to invade the basement membrane,
- Upregulation of expression of key molecules in these processes by the bone marrow microenvironment.
1. The attraction of MM cells by endothelial cells is a prerequisite for initial arrest before transendothelial migration. Endothelial cells are known to secrete several chemokines. Monocyte chemoattractant protein-1 (MCP-1) is known to be secreted by endothelial cells and to attract monocytes. We here investigated the production of MCP-1 by different bone marrow endothelial cell lines. RT-PCR demonstrated the presence of transcripts for MCP-1 by different bone marrow endothelial cells while ELISA confirmed the presence of the protein in culture supernatant. MCP-1 is known to bind with high affinity to CCR2, a chemokine receptor belonging to the b chemokine family. RNase protection assay was performed on purified 5T2 and 5T33MM cells and demonstrated the presence of transcripts for CCR2. FACS analysis on tumour idiotype positive MM cells confirmed the membranic expression of CCR2 on MM cells. Endothelial cell conditioned medium induced chemoattraction of MM cells, a phenomenon inhibited by anti-MCP-1 antibodies. In conclusion, MM cells express the CCR2 receptor and are attracted by MCP-1, secreted by bone marrow endothelial cells.
In addition to MCP-1, laminin-1 was found to be a chemoattractant for 5TMM cells. The 67kD receptor was identified as the receptor mediating the laminin-1 induced migration of the MM cells (Vande Broek I et al, submitted).
2. The specificity of adhesion of MM cells to bone marrow endothelial cells was determined by comparison of bone marrow and lung endothelial cells. A significant lower adhesion to lung endothelial cells was observed (Vanderkerken et al, 2000). We are presently investigating which molecules are involved in this differential adhesion.
3. 5TMM cells are attracted towards conditioned medium of bone marrow fibroblasts. Addition of blocking anti-IGF-1 antibodies could abolish this attraction while exogenous addition of IGF-1 could mimic this attraction. The expression of IGF-1 receptor on 5T2 and 5T33MM cells was analysed by flow cytometry. A heterogeneous expression was observed. When migrated MM cells, both in vitro and in vivo, were phenotyped, only IGF-1 receptor positive cells migrated (Vanderkerken et al, 1999).
4. The production of MMP-9 by MM cells was investigated. Gelatine zymography clearly demonstrated the production of MMP-9 by 5T2 and 5T33MMvivo cells. When Matrigel invasion assays were performed, a significant invasion was observed, which could be inhibited by the broad-spectrum inhibitor Batimastat.
5. When the expression of key molecules, as described above, was compared between the 5T33MMvivo and 5T33MMvitro cells, striking differences were observed. When 5T33MMvitro cells were injected into mice and isolated upon development of the MM disease, an upregulation of IGF-1R and CD44v6 expression (Asosingh et al, 2000) and MMP-9 was observed. This upregulation was accompanied by upregulated migration towards bone marrow conditioned medium, adhesion to bone marrow fibroblasts and invasion through Matrigel. Cocultures with bone marrow endothelial cells demonstrated that this contact was, at least in part, responsible for the observed upregulation.
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