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 Updated on: Jan 9 2014

In vitro and animal models

In vitro and animal models: evaluate specific genetic and therapeutic manipulations

 

Subject
Unravelling myeloma plasma cell differentiation has suffered from lack of an in vitro system for reproducible growth of myeloma subsets. Recently the SCID-human mouse, which harbours a human microenvironment in the form of fetal bone, has allowed most myeloma cells to growth. From such studies it has been concluded that a normal human bone marrow environment will support the growth of myeloma cells. Other model systems reley upon the selective targeting of oncogenes to the plasma cell compartment using Ig gene enhancers.

The internationally accepted 5TMM and MOPC315 murine models are available in Europe for the study of multiple myeloma. For classical in vitro evaluation of specific genetic events and therapeutic manipulations a panel of myeloma cell lines are also available as an assay for B cell differentiation.

 

Present Status
The 5T2MM model best represents human MM, with a moderate growth and the development of osteolytic bone lesions.


The 5TMM models can be used for both in vitro and in vivo experiments. The specific antibodies allow the separation of MM cells by flow cytometry or with magnetic beads, generating pure MM cell populations for further in vitro investigation. The 5TMM models generate a typical MM disease and different methods are available to assess tumor load in the bone marrow, serum paraprotein concentrations, bone marrow angiogenesis (by measu-ring the microvessel density) and osteolytic bone lesions (by a combination of radiography, densitometry and histomorphometry).


The investigation of these latter parameters allow the use of the 5TMM models in a preclinical setting and study the growth and biology of the myeloma cells in a complete syngeneic microenvironment. Both, molecules targeting the MM cell themselves and molecules targeting the bone marrow microenvironment can be studied. While the 5T33MM model can be used to target both the microenvironment and the MM cells themselves, the 5T2MM model can also be used to study the myeloma associated bone disease. The bone disease is quantified by measuring the number of bone lesions by X rays, by bone density measurements, and by histological measurements of bone volume and osteoclast number.

Multiple myeloma-like disease may also be induced by injection of mineral oil in the peritoneal cavity of certain strains of mice. A number of transplantable cell lines with myeloma-like features have been established.

One of the best studied is MOPC315. Actually, the first demonstration that immunization with myeloma protein may induce resistance to a subsequent challenge with myeloma cells was performed in the MOPC315 model by Eisen et al. in 1972. The immunization with the myeloma protein induced an immune response against antigenic determinants, called Idiotopes(Id) in the variable regions of the myeloma protein. This Id-specific immune response evidently made the mice resistant against a tumor challenge.

 

It is important to understand the components of the Id-specific immunity that make the mice resistant to tumors as it may be exploited in Id-vaccination of patients with multiple myeloma. Anti-Id antibodies appear not to be important in myeloma because myeloma cells usually do not express much surface Ig and because the effect of anti-Id antibodies would be blocked by myeloma protein in the serum. Thus, researchers have focused on the role of Id-specific T cells.

An Idiotypic sequence of the light chain of the myeloma protein M315 produced by the MOPC315 tumor has been defined. This sequence is presented on MHC class II molecules to Id-specific CD4+ T cells. T cell receptor (TCR) genes from this clone were isolated and used to establish a TCR-transgenic mouse. Such TCR-transgenic mice are resistant to challenge with MOPC315 cells and the resistance can be transferred to other mice with purified Id-specific CD4+ T cells. Further experiments have demonstrated that myeloma protein secreted by the s.c. myeloma prime dendritic cells in the tumor with Id. These Id-primed dendritic cells then stimulate CD4 Id-specific CD4+ T cells that as activated cells somehow kill the myeloma cells.

In parallel, in vitro models have established mature B cells from pro-B cells and further generated membrane immunoglobulin B cells or plasma cells by additional stimuli , which may be improved by lymph node dendritic cells as well as "antigen" selected as idiotype specific peptide mimotopes.

 

 

Preview of Programme Proposed
The 5TMM models are exclusively in vivo growing models (in C57BlKaLwRij mice). In addition to the 5T model a number of different animal and in vitro models exist. We will interact with American investigators who have developed models of myeloma based on transgenic expression of oncogenes under the control of promoter specific for plasma cells. We will develop models based on SCID mice. In addition a number of investigators have described in vitro models to look at plasma cell development. Consequently, we propose to:

  • develop a European core facility for the animal model in Brussels
  • make the 5T2 and 5T33MM in vivo models available and maintain a set of standardized protocols to evaluate the myeloma disease
  • comparison of different treatment strategies within the same model with special emphasis on tumour load, angiogenesis and development of bone disesae;
  • to develop an in vitro culture system to study myeloma precursor differentiation into plasma cells in the presence of marrow stromal cells, stromal cell lines transfected by CD40L, lymph node stromal tissue, dendritic cells, idiotypic binding epitopes (mimotopes), recombinant human cytokines, chemokine receptor ligands.


It is important to find out how Id-specific CD4+ T cells become activated and how they kill myeloma cells. It is also important to develop this model so that it even more resembles multiple myeloma disease in humans. We propose to;
 

  • to develop an intravenous model of MOPC315 with myeloma growth in bone marrow like in human disease
  • track activation of Id-specific CD4+ T cells in TCR-transgenic mice challenged intravenously with MOPC315 by in vivo imaging of Id-specific CD4+ T cells. This is accomplished by using NFkB-Luciferase transgenic mice in conjunction with our TCR-transgenic mice
  • to find out the molecular mechanism by which activated Id-specific CD4+ T cells kill tumour cells.

Finally,

  • to select a panel of MM cell lines for full genomic and proteomic characterization as a standardized European resource

 

Deliverables and Cooperations

  • make the 5T2 and 5T33MM in vivo models available for other laboratories and to maintain a set of standardized protocols to evaluate new drugs
  • carry out in vivo and vitro experiments with special emphasis on oncogenesis, tumour progression, angiogenesis and bone disease
  • establish a bone marrow disease model of MOPC315 and study the role of Id-specific CD4+ T cells. Many partners in this network have expertise and interest in this project, that will deal with myeloma cells and T cells in the bone marrow environment
  • molecular mechanism of killing effected by Id-specific CD4+ T cells
  • apoptosis of myeloma cells is a central part of this application and many participants may contribute to elucidation of this question
  • document and make available a range of well-characterized cell lines.

Beyond 18 months the primary goal is to develop a coordinated strategy to define, and to initiate a coordinated effort to perform a common research program between the participants in this area.

Secr. Hans E. Johnsen | Depart. of Haematology | Aalborg University Hospital | Sdr. Skovvej 15 | DK-9000 Aalborg | Denmark | T:+45 9766 3871 | F:+45 9766 6369