Ph.D Thesis

Ph.D StudentAmit Cohen Bat-Chen
SubjectHypoxia and Co-culture Regulate MMP-9 and VEGF via EMMPRIN
Effect of Tumor Cell-Monocyte Interactions on
Tumor Progression and
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Haim Bitterman
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Tumor development is regulated by the interaction between tumor cells and stroma cells, including fibroblasts, T cells, endothelial cells and particularly macrophages that can take up to half of the tumor mass. These cells secrete factors to the microenvironment, which inhibit anti tumoral immune response; up regulates angiogenesis and support tumor development. Two key molecules in these processes are VEGF (which is a strong angiogenic factor) and MMP-9 (which is crucial for tumor growth, invasiveness and angiogenesis as it degrade the ECM). Additionally, as the tumor expands its mass and increases its metabolic demands, it lacks blood and oxygen supply, which can lead to formation of areas with low oxygen tension (hypoxia) that may affect macrophage activation state. In many solid tumors, expression of VEGF and MMP-9 is up-regulated by EMMPRIN, a protein expressed on the surface of tumor and stroma cells, including macrophages. EMMPRIN is a membranal protein that can also be secreted, but the precise mechanisms leading to the induction of either MMP-9 or VEGF by EMMPRIN are largely unknown. In this work we studied the effect of tumor cell- macrophage interactions on the expression of EMMPRIN, VEGF and MMP-9.

To simulate the tumoral microevironment in vitro we used two human cancer cell lines (MCF-7 and A498) and co-cultured them with human monocyte cell line (U937). Monocultures and co-cultures were incubated in the presence or absence of TNFa, which is an important cytokine expressed at low concentrations in tumors, and in a hypoxic chamber to induce low oxygen concentration.

We found that co-culture, but not hypoxia, increases both soluble and membranal EMMPRIN in macrophages and tumor cells. In addition we found that EMMPRIN is necessary for VEGF and MMP-9 expression in co-culture since EMMPRIN inhibition by a neutralizing antibody and by siRNA led to a decreased secretion of both proteins. We also found that EMMPRIN soluble form led to the induction of these two proteins and that it is generated by shedding (by a serine protease). No difference was found in EMMPRIN mRNA levels between co-cultured and monocultured cells therefore we concluded that EMMPRIN is not transcriptionally regulated. Preliminary results suggest a post transcriptional regulation by, at least in part, miR146a, which its inhibition led to a decrease in EMMPRIN levels.  These results suggest that interactions between tumor cells and macrophages are of great importance to the progression of the tumor.