טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentValter Miriam
SubjectEMMPRIN Vaccination: Effects on Tumor Progression and
Angiogenesis via Inhibition of MMP-9 and VEGF
DepartmentDepartment of Medicine
Supervisors Professor Nitza Lahat
Dr. Michal Rahat
Full Thesis textFull thesis text - English Version


Abstract

Tumor mass consists of up to 50% macrophages, that instead of eradicating tumor cells, support them by secreting different factors that promote tumor growth, angiogenesis and invasion, and suppress the anti-tumoral immune response. Among these factors, MMPs and VEGF have been strongly associated with tumor progression. MMPs, particularly MMP-9, degrade the interstitial ECM allowing cell migration and invasion, release and activate pro-angiogenic factors, including VEGF. VEGF is potent pro-angiogenic factor that also act as strong chemoattractant for macrophages, and upregulates the expression of MMP-9. In many solid tumors, expression of MMP-9 and VEGF is enhanced by EMMPRIN, a transmembranal protein that is expressed in both tumor cells and macrophages. The mechanisms regulating its effects on MMPs and VEGF are still unclear.

We hypothesized that the MMP-9 and VEGF induction by EMMPRIN can be disrupted by antibodies generated against EMMPRIN. We proposed to map the specific EMMPRIN epitopes responsible for VEGF and/or MMP-9 induction, by immunizing against specific, highly antigenic peptide sequences within the protein molecule. Thus, we synthesized specific peptides that we designed, and raised polyclonal antibodies directed against them. We established an in vitro system of four mouse of four mouse tumor cell lines: TRAMP-C1, TRAMP-C2, CT26, and RENCA that were co-incubated in normoxia or hypoxia, with the macrophage cell line (RAW 264.7), or with primary thioglycollate (TG)-elicited peritoneal macrophages, and compared the secretion of MMP-9 and VEGF to each of the single cultures. Next, we screened for the antibody that could best inhibit induction of MMP-9 and VEGF in the in vitro co-culture system established. We serially diluted the immune and pre-immune sera and evaluated the secreted amounts of MMP-9 and VEGF in the co-cultures. Out of the 8 different peptides we designed, only one antibody raised against a specific epitope significantly inhibited MMP-9 and VEGF secretion CT26 and TRAMP-C2 co-cultures with RAW 264.7 macrophages. We examined the effects of this antibody on s.c. RENCA or CT26 tumors, an showed a 50-76% (p<0.05) inhibition of tumor size, in comparison to the control group that received only saline injections. Moreover, when we used the peptide sequence that elicited this polyclonal antibody, synthesized as MAP peptide, we could show about 50% reduction in tumor size in the RENCA tumors. In the CT26 tumors, the higher amount resulted in complete regression of the tumors.