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TARGETING ENDOTHELIAL CELL AND CANCER AMOEBOID MOVEMENT TO OVERCOME RESISTANCE TO ANTI-VEGF AND ANTI-PROTEASE THERAPIES

Periodic Reporting for period 1 - ENDOCAM (TARGETING ENDOTHELIAL CELL AND CANCER AMOEBOID MOVEMENT TO OVERCOME RESISTANCE TO ANTI-VEGF AND ANTI-PROTEASE THERAPIES)

Reporting period: 2017-12-01 to 2019-11-30

Cancer cells, like healthy cells, need oxygen and nutrients to survive and grow. The angiogenic factors are signals sent by cancer cells to encourage new blood vessels to grow into tumor to bring in nutrients and oxygen. Therefore, based on cumulative experimental and clinical evidences, tumor angiogenesis is a well-established therapeutic target. Growth factors secreted by the tumor create chemotactic gradients to recruit endothelial cells (EC) and pericytes from pre-existent vascular beds and endothelial progenitor cells (EPC) from the bone marrow. With the support of the cooperative tumor microenvironment, cancer continues to have the opportunity to survive, to accumulate mutations and metastasize. Understanding the tumor as an organ requires an understanding of angiogenesis.
It was known that ECs could form new blood vessels only using the mesenchymal type of motility characterized by the activity of membrane-associated proteases that open a new path allowing the invasion of endothelial cells through the extracellular matrix. We demonstrated 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”. Therefore, the main goal of this program is to identify the multiple mechanisms shared by cancer cells and endothelial cells in the regulation of amoeboid movements, with the final aim to identify common therapeutic strategies to impair vascular growth and cancer cell invasion at the same time.
"During the first 3 months of the outgoing phase the researcher worked on experiments for the resubmission of a paper that describes the preliminary results used for the writing of the Marie Curie project and strictly related to it. 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 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.
Experiments conducted for the study of Ephrins A2 and B2 showed that they are not involved in EC amoeboid movement as aspected.
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). However, since some experiments showed that TIMP1, 2 and 3 resulted the most high-powered inhibitors in the MIX, the following experiments were done using Marimastat, a broad-spectrum matrix metalloproteinase inhibitor.
Collagenolytic activity of endothelial cells under mesenchymal and amoeboid conditions induced by MIX and Marimastat treatment, confirmed that the % of collagen degradation by endothelial cells treated with Marimastat is very low compared to the cells without the treatment. Morphological studies, invasion assay, capillary morphogenesis assay, proliferation assay, biochemical evidences, 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 endothelial cells."
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.
After completing the experiments shifted from the outgoing phase to the third year, the molecules identified will be used as candidates for targeted therapy through the delivery of cargo liposomes containing inhibitors of the connection of such molecules with the cortical actin cytoskeleton, in pre-clinical experiments with the perspective of a possible application to humans. We expect that this approach will give a progress in the treatment of tumors thus improving outcomes for cancer patients.
Vessel infiltration of tumor cells, dissemination and extravasation in a different site