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 Updated on: Jan 9 2014

Tumor-host interaction: identification and quantitation of bioactive molecules

 

Subject
The marrow tropism of MM cells does not merely reflect the growth requirements of its normal counterpart, the plasma cell found in other locations like tonsils and Peyers patches of the gut; sites that are not favored growth places for MM cells.


Furthermore, human MM cells usually do not grow in immuno-compromised mice, but are able to grow inside human bone fragments implanted.


A reasonable explanation for this tropism is that MM cells are critically dependent on factors that are present only within the human bone marrow, for their prolonged survival and growth. The dependence of MM cells on the fertile soil of the bone marrow is a potential Achilles’ heel for the myeloma cell clone. Several factors are found in increased amounts when compared to specimens from control subjects, and/or are found to predict patient prognosis. Although several studies have been performed in recent years to identify levels of bioactive molecules in samples from MM patients, many factors have not yet been thoroughly examined.


Present Status

Possible candidates for factors that sustain the growth of MM cells in the bone marrow include glycoproteins in the extracellular matrix (e.g. fibronec-tin, thrombospondin-1, osteopontin) or cytokines secreted by cells in the marrow microenvironment. Several cytokines are known to support the growth of MM cells, including IL-6, IL-10, IL-15, IL-21, TNF, HGF and IGF-1, but they cannot, alone or in combinations, make primary MM-cells grow for longer periods in vitro, indicating that critical factors are yet to be discovered.


Bioactive proteins can also be secreted by the MM cells themselves (e.g. fibronectin, HGF, VEGF, TNF, IL-6) and have the potential to act in an autocrine manner. Reported evidence points to interactive loops where proteins expressed by MM cells can stimulate stromal cells to produce IL-6 and other myeloma growth factors. Furthermore, HGF and VEGF produced by myeloma cells, as well as bFGF produced by the microenvironment, are potent angiogenic cytokines. Angiogenesis is increased in the MM bone marrow and is inversely linked to prognosis.

The concentration of HGF, bFGF and VEGF in peripheral blood from MM patients at the time of diagnosis is higher than in samples from healthy controls. Several studies have shown strong correlation between HGF concentration and poor disease outcome, whereas data for VEGF and bFGF are still scarce (or yet conflicting) at this point.


MM cells also produce proteolytic enzymes (uPA, MMP-2, MMP-7, and MMP-9), molecules that can degrade matrix fibers and are likely to promote dissemination of MM cells throughout the bone marrow. Possible prognostic information in the concentration levels of proteolytic enzymes in body fluids from MM patients has not yet been thoroughly examined.

Of special interest is syndecan-1 (CD138), a heparan sulfate proteoglycan abundantly expressed on the surface of MM cells. Among several functions, syndecan-1 adheres MM cells to neighboring cells and acts as a co-receptor for several heparin-binding cytokines.

By proteolytic cleavage of its core protein, syndecan-1 is released from the cell membrane to the extracellular space. When measured in serum from peripheral blood, concentration of soluble syndecan-1 correlates strongly with a negative outcome for MM patients. In a MM mouse model, malignant cells that were genetically transfected to secrete soluble syndecan-1 led to more rapid and severe disease than cells with predominantly surface-expressed syndecan-1. The modulatory effect of syndecan-1 on heparin-binding bioactive proteins in the myeloma bone marrow is yet only partially studied.


An alternative, and not mutually exclusive, hypothesis for the above-mentioned tropism of MM cells, is production in the marrow of chemokines that attract MM cells to this location. SDF-1, IGF-1, VEGF and MIP-1a are reported candidates, but other factors are likely to work in the same manner.


Other bioactive proteins inhibit the growth and/or induce apoptosis in MM cells. These factors include members of the TNF cytokine family (TRAIL and Fas-ligand) as well as members of the TGFb family (activin and bone morphogenetic proteins (BMP)- 2, -4, -5, -6 and –7). The levels of these factors in the bone marrow as well as the levels of naturally occurring inhibitors of the same substances are largely unknown.


Bone destruction is one of the most conspicuous clinical features of MM. The destructive bone process is still not fully explained at the molecular level. There is an imbalance between bone resorption and formation, most likely caused by non-physiological concentrations of the two main local, bone-regulatory molecules, RANK-ligand and osteoprotegerin (OPG). The levels of RANK-ligand and OPG are determined by many other local and systemic factors, and several such upstream regulators have been proposed as important for the bone disease in MM (e.g. MIP-1a, HGF, VEGF, IL-11, IL-6, IL-1, TNF, and LT).


Preview of Programme Proposed
A host of different proteins have been proposed as important for the initiation and progression of MM. In general this information is based on data obtained by in vitro research on cell lines and primary myeloma cells. In order to verify the importance of candidate molecules access to tissue and blood samples from patients and controls subjects are important. The final goal of this workpackage is to define the prognostic importance of these markers;

  • biological samples (serum, plasma, bone marrow fluid, bone marrow smears and biopsies) from all patients under study, at the time of diagnosis and at various stages of disease progression
  • surface membrane molecules will be assayed by standardized immunostaining immediately after the samples are taken. Other samples will be sent from the various centres to the biobank network for storage
  • factors examined will include soluble proteins that have shown prognostic information in previous studies (IL-6, IL6-receptor, bFGF, VEGF, HGF, syndecan-1, and IGF-1) as well as proteins that have not yet been examined in large prospective studies (uPA, uPA-receptor, various MMPs, IL-10, IL-15, IL-21, osteopontin, members of the TNF family, various BMPs and others)
  • assays to be used are immunoassays (ELISA) and multiplex methodology (protein array)
  • to correlate these markers with relevant prognostic disease characteristics including response to therapy and survival.

Deliverables and Cooperations
The importance of bioactive factors that are found in abnormal levels or have prognostic value, is twofold;

  • to document current activity in this area and to establish a network of interested scientists
  • increase our knowledge about the biology of the disease, which is essential for the identification of new targets for treatment
  • the information obtained can be used to monitor treatment and to predict outcome in the future - the choice of treatment may potentially be based on such information. 

     

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