Periodic Reporting for period 2 - ENDOCAM (TARGETING ENDOTHELIAL CELL AND CANCER AMOEBOID MOVEMENT TO OVERCOME RESISTANCE TO ANTI-VEGF AND ANTI-PROTEASE THERAPIES)
Periodo di rendicontazione: 2019-12-01 al 2020-11-30
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. The experiments conducted for this research project 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”.
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. The experiments conducted for this research project 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”.
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.
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.
Until now, the existence of a protease-independent angiogenesis was unknown, as well as the stimulation of angiogenesis by a protease inhibitor mix. For years, indeed, synthetic metalloproteinase inhibitors were developed and utilized in human clinical trials but the results were disappointing. The results so far obtained help to understand the reason of the failure of synthetic metalloproteinase inhibitors as cancer therapy providing the basis for an alternative strategy of therapy. In the next future, the molecules identified as essential for amoeboid and mesenchymal motility, 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.
The restrictions due to COVID-19 emergency caused a significant delay in the working plan, so the researcher was not able to perform properly the project activities planned and generate the publications by the expected date. Despite not being responsible for any of these violations, the researcher is making every effort to continue and complete the research even after the official conclusion of the fellowship, using beneficiary laboratory’s funding. The final aim, hopefully expected in a short time, is to publish the results of the second part of the project and afterwards a review to provide scientific community an update about the topic of the ENDOCAM project. Both will be obviously deposited in a repository and the EU funding will be acknowledged, as requested. In particular, the preparation of the final manuscript, that will be published in an international peer reviewed journal, is in progress and the researcher is just completing it while waiting for the last experiment results.
Despite the negative Covid-19 impact, ENDOCAM project, of which both the outgoing and return institutions were beneficiaries, has created a positive environment for the fellow to acquire new skills in the outgoing years, and then bring them back to Europe and implement them as part of the research lines of the return institutions during the third year of the fellowship establishing new collaborations.
The restrictions due to COVID-19 emergency caused a significant delay in the working plan, so the researcher was not able to perform properly the project activities planned and generate the publications by the expected date. Despite not being responsible for any of these violations, the researcher is making every effort to continue and complete the research even after the official conclusion of the fellowship, using beneficiary laboratory’s funding. The final aim, hopefully expected in a short time, is to publish the results of the second part of the project and afterwards a review to provide scientific community an update about the topic of the ENDOCAM project. Both will be obviously deposited in a repository and the EU funding will be acknowledged, as requested. In particular, the preparation of the final manuscript, that will be published in an international peer reviewed journal, is in progress and the researcher is just completing it while waiting for the last experiment results.
Despite the negative Covid-19 impact, ENDOCAM project, of which both the outgoing and return institutions were beneficiaries, has created a positive environment for the fellow to acquire new skills in the outgoing years, and then bring them back to Europe and implement them as part of the research lines of the return institutions during the third year of the fellowship establishing new collaborations.