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Summer 1999 Volume 3, Issue 5:
Study Of Homing Mechanisms Involved In The Biology Of Multiple Myeloma
By Ivan Van Riet, PhD
07.01.99
Overview by Prof. Brian G. M. Durie

This study from the myeloma group at the Free University in Brussels focuses on why myeloma cells grow mostly in the bone marrow and rarely spread to other tissues and organs in the body. Clearly an understanding of this bone marrow "homing" can lead to block the homing and new and better treatment for myeloma. Like all research of this type there will be a painstaking path from the laboratory to the clinic. Only time will tell if important clues can lead to innovative therapy. As always, we applaud the efforts of this dedicated group and wish them success.

Study Of Homing Mechanisms Involved In The Biology Of Multiple Myeloma
by Ivan Van Reit, PHD, Free University,Brussels, Belgium

A striking feature of myeloma plasma cells concerns their tendency to reside in the bone marrow compartment during the main course of the disease evolution. This micro-environment provides the appropriate signals for growth and survival of the tumor cells. Since small amounts of myeloma cells are also detectable in the peripheral blood, it can be assumed that these cells represent lymph node- and/or bone marrow-derived tumor cells that directly contribute to disease spreading. The detection of such myeloma-related cells in the circulation implies that they must be equipped with the appropriate surface molecules that mediate binding to endothelium, responsiveness to chemokines, transendothelial migration and extravasation. The specificity of this migration process might be a second important factor that determines the selective homing of myeloma cells in the marrow microenvironment. During the end phase of the disease, increasing numbers of myeloma cells become detectable in the blood circulation and also extramedulary tumor localization at other sites (liver, lung, ascites and pleural fluid) can occur. Molecular evolution towards stroma-independency in association with an enhanced extravasation potential are likely to be the key mechanisms that underlie this disturbed homing behavior.
So far very little information is available about the molecular mechanisms that are involved in the directed migration and homing of myeloma cells. With this project, we would like to clarify how myeloma cells interact with and migrate through bone marrow endothelium and how changes in the migration and motility capacity of the tumor cells relates to disease evolution. As experimental tools, both human material as well as an in vivo mouse model will be used. This last model was recently introduced in our laboratory and includes a tumor line (5T2) that causes restricted tumor growth (bone marrow and spleen) and a cell line (5T33) express a more leukemic-like homing behavior.

In the first instance, we would like to examine which adhesion molecules are involved in myeloma cell binding to bone marrow endothelial cells. Therefore, adhesion assays will be performed using bone marrow derived myeloma cell lines in combination with a recently established bone marrow endothelial cell line (all of human origin). Myeloma cell binding will be evaluated before and after blocking with specific antibodies against adhesion molecules. The same experiments will be repeated with 5T2 mouse myeloma cells on endothelial cell layers, cultured from bone marrow of syngeneic animals. Next, we will investigate whether the directed migration of myeloma cells is influenced by specific bone marrow derived chemotactic factors. Therefore chemokine-receptor expression on myeloma cells will be investigated. Migration assays will be performed in the presence of various stromal factors with known chemotactic potential (chemokines was well as extracellular matrix proteins). If relevant factors are identified, it will be determined whether these factors can also trigger transendothelial migration of myeloma cells. The role of these factors in the in vivo migration of myeloma cells will be evaluated in the 5T2-mouse model by injecting tumor cells pre-coated with blocking antibodies against the corresponding receptors.

To determine whether more generalized disease localization reflects changes in responsiveness to chemotactic factors or enhanced automotility, both spontaneous and chemokine-induced migration will be compared between the mouse 5T2 and 5T33 cell lines. At this point of our project we will also focus on the involvement of the motility-related protein (MRP-1/CD9) in the motility and migration of myeloma cells. Using Represential Difference Analysis and PCR selected cDNA Subtraction, we could recently identify this molecule as being downregulated in the stroma-independent variant (MM5.2) of a human stroma-dependent myeloma cell line (MM5.1). Other groups related recently, the absence of this molecule to the metastatic capacity of lung and breast carcinoma cells. By transfection of MM5.2 cells with MRP-1/CD9 cDNA, we hope to determine its possible effect on the spontaneous migration of the tumor cells. Finally, we will also evaluate whether myeloma cell lines can produce factors themselves which trigger the automotility of the tumor cells. With this project, we hope to contribute to the further unraveling of the complex biology of multiple myeloma, which might ultimately lead to the development of new therapeutic strategies for this still incurable malignancy.


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