During the outgoing phase the researcher provided a data set disclosing that endothelial cells can move and differentiate into vascular structures in vitro and in vivo also in the absence of proteases activity, performing a new type of neovascularization: the "amoeboid angiogenesis". The paper was published in an international peer reviewed journal.
The researcher evaluated also the in vivo presence of protease inhibitors in the serum of prostate and breast cancer patients at different stage of the disease. The UCSD Biorepository provided high quality and well-characterized cancer-related biological serum of patients at early stages of cancer progression, intermediated (II and III stages) and with high grade of malignancy. Results showed that in the breast cancer serum samples analyzed, the levels of TIMP1, TIMP2, alpha2-antiplasmin and cystatin increase significantly in the high grades compared to the low ones. In the prostate cancer serum samples only PAI-1 levels increase significantly in the low, middle and high grades compared to the healthy pool.
Invasion and capillary morphogenesis assay with physiological single protease inhibitors produced no or scarce decrease of invasion and tubular formation capacity compared to all the inhibitors mixed together (MIX).
During the returning phase, however, since some experiments showed that TIMP1, 2 and 3 resulted the most high-powered inhibitors in the MIX, the experiments conducted were done using Marimastat, a broad-spectrum matrix metalloproteinase inhibitor.
Several experiments conducted during the returning phase, such as morphological studies, collagenolytic activity, invasion assay, capillary morphogenesis assay, proliferation assay, invasion in 3D matrigel and in vivo matrigel plug assay, confirmed that Marimastat, the inhibitor of MMPs, instead of inhibiting as previously thought, promotes the invasion and tubular formation of mature endothelial cells and ECFCs, a subpopulation of Endothelial Progenitor cells.
During the third year of the fellowship, the researcher also analyzed the main intracellular signaling pathways involved during angiogenesis. The results obtained showed that Marimastat treatment induces amoeboid characteristics in terms of Rho-GTP and pMLC expression, enhances ERK and AKT pathways and increases the expression of uPAR.
The experiments with Marimastat confirmed also the “indifference” of ECs and ECFCs to VEGF stimulation that was described, under amoeboid conditions induced by the physiological protease inhibitor MIX, during the outgoing phase. This can be ascribed to the endothelial cells capacity to migrate and differentiate into tubular structures at the maximum levels under amoeboid conditions thus justifying the limited efficacy of VEGF-targeted therapies. Matrigel plug assay in mice, was performed using ECFCs for the in vivo evaluation of vessel formation in amoeboid conditions. Another un vivo experiment was performed to evaluate ECFC angiogenesis ability in matrigel plugs injected together with breast or prostate cancer cells in the presence and in the absence of Marimastat and/or VEGF and/or the monoclonal antibody against VEGF, Bevacizumab.
The results so far obtained could justify the failure of synthetic metalloproteinase inhibitors (MPIs) as cancer therapy. The failure of this treatment at the initial stages of tumor development could be ascribed also to the onset of the angiogenic transition, during which the tumor microenvironment is able to skip the attack of the MPI therapy by allowing blood vessel formation using the “amoeboid” strategy.
