Functional Characterization In vivo

Workpackage 7

Objectives

In the previous workpackages a multiple myeloma stem cell population will be characterized based on a combination of phenotypical markers. In this workpackage we will investigate the presence of a similar population in the 5T2 and 5T33MM murine model.

Description of work

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 generates a typical MM disease and different methods are available to assess tumor load in the bone marrow, serum paraprotein concentrations, bone marrow angiogenesis (by measuring the microvessel density) and osteolytic bone lesions (by a combination of radiogaphy, densitometry and histomorphometry). The investigation of these latter parameters allow the use of the 5TMM models in the study of the biology of myeloma in a complete syngeneic microenvironment.

In the present application we will investigate whether a murine homologue of the human myeloma stem cell can be identified and isolated (using performant FACS systems). These cells can then be transplanted (repeatedly if necessary) in naive mice and the development of the myeloma disease assessed (as described above), with special emphasis on tumor development, induction of angiogenesis and ostolysis. In vitro assays are also established allowing the analysis of induction of angiogenesis (rat aortic ring assay) and activation of osteoclasts (osteoclast assay). Based on the data obtained in the previous WP, 5TMM cells will be sorted. The clonogenic capacity and the resistance to different chemotherapies will be assessed. Subsequently the isolated myeloma stem cells will be injected in young syngeneic animals (C57BlKaLwRij mice). The development of the myeloma disease will be analysed by assessment of tumor load in the bone marrow, serum paraprotein concentration, microvessel density (after CD31 immunostaining), assessment of development of bone disease (number of lesions, density, trabecular bone area and number of TRAP positive osteoclasts).

This development of the myeloma disease will be compared to animals injected with the total (unsorted) myeloma populations. If differences in tumor load, angiogenesis and bone disease are observed, in vitro assays will be performed assessing the proliferative capacity (3H thymidine incorporation), the induction of angiogenesis (rat aortic ring assay) and activation of osteoclasts (bone resorption assay).

Deliverables

• A core facility for the 5TMM model and necessary technologies.
• Characterization of the MSC of the 5TMM model in clonogenic in vitro assays
• Transplantation experiments and in vivo characterization of MSC in C57BlKaLwRij mice

Milestones

• Isolation of a MM stem cell population from the 5TMM models (from 6-12 months depending on previous WP identifying the human MSC population).
• In vitro clonogenic assay of the selected population (12-18 months).
• In vivo transfer of the isolated MM stem cell population in C57BlKaLwRij mice (18-36months).
• Analysis of the MM development in vivo with special emphasis on tumor burden, angiogenesis and bone lesions (18-36 months).