Project Title:

V.I.T.A.S. – Evaluation of Immunotherapy Toxicity Associated with Statins

Total 5×1000 allocation: 37.304,66 €
Project start date: 01/01/2025
Project end date: 31/12/2025

Project Summary – Abstract:

Despite numerous clinical and epidemiological studies, the effectiveness of statins in cancer prevention remains controversial. A 2008 analysis in post–myocardial infarction patients suggested a potential antitumor effect of lipophilic statins, such as simvastatin, due to the inhibition of neoangiogenesis, i.e., the formation of new blood vessels that nourish tumors. Subsequently, other studies have investigated how metabolism influences cancer and the immune system. It has emerged that metabolic changes, caused by drugs or genetic modifications, can:

1. reactivate the immune system against cancer, particularly by acting on cells within the tumor microenvironment;

2. enhance the efficacy of certain immunotherapeutic drugs, helping the immune system to better recognize and combat cancer, thereby improving patient survival.

Simvastatin, in particular, may enhance the activity of immune checkpoint inhibitors (CPI), improving recognition and elimination of tumor cells by immune cell populations. Furthermore, cancer and cardiovascular diseases share common mechanisms, such as immune, metabolic, and vascular alterations; moreover, cancer therapies themselves can cause cardiovascular toxicity.

Our study aims, on one hand, to evaluate the effectiveness of combining simvastatin with CPIs (specifically anti–PD-1) against tumors, and on the other, to assess whether this combination may induce potential toxic effects on the cardiovascular system. We will focus on two tumor types in which immunotherapy has shown very promising results: melanoma (a highly aggressive skin cancer) and lung cancer.

In vitro models will be generated to study both the antitumor effects of the simvastatin + anti–PD-1 combination and, at the same time, its impact on endothelial cells and cardiomyocytes, which represent the main “healthy” cells potentially at risk of damage from the combination under investigation.

Melanoma and/or lung cancer cell lines will be cultured in the laboratory together with commercial lines of human monocytes/macrophages and Natural Killer (NK) cells, i.e., effector cells of our immune system, to test the effects of immunotherapy with and without simvastatin. We will assess whether simvastatin stimulates activation of immune cells and the production of inflammatory and cytotoxic cytokines, in order to identify a possible mechanism that enhances the efficacy of immunotherapy.

We will also evaluate whether the proposed combination can trigger excessive and uncontrolled activation of the immune system, as a potential cause of cardiovascular damage mechanisms. To address this critical issue, conditioned media collected from the co-culture systems will be used to treat, in vitro, human endothelial cells (HUVECs), human cardiomyocytes, and/or human cardiac fibroblasts. Induction of cardiovascular toxicity will be assessed through assays of cell proliferation, activation of molecular pathways associated with endothelial and cardiac damage, using qPCR, flow cytometry, secretome analysis, and western blotting.

Based on the results obtained from the in vitro models described above, and subject to the availability of clinical samples, we will perform an experimental validation using a cohort of melanoma patients before and after receiving anti–PD-1 therapy. For these experiments, sera from these patients will be tested on cardiomyocyte, endothelial cell, and PBMC cultures, which will be analyzed using the same experimental approaches described for the in vitro models above. Patient sera will also be analyzed to assess the presence of circulating biomarkers of cardiotoxicity induced by immunotherapy, through biochemical assays.

Date of receipt of 5 x 1000 funds: 01/10/2024

Budget

 Status updated as of July 2025

Project Title:

Development of a 3D in vitro model of bone marrow vasculature for the study of cellular and molecular determinants shared by cardiovascular and neoplastic diseases

Total 5 x thousand allocation: 36,918.44 €
Project Start: 01/01/2024
Project End: 31/12/2024

Project Summary – Abstract:

Among chronic-degenerative diseases, cardiovascular and oncological diseases represent the leading cause of mortality. Although considered separate nosological entities, with distinct preventive, diagnostic, and therapeutic pathways, a strong connection between the two has emerged over the past decade. Indeed, on the one hand, antitumor treatments are associated with the development of cardiovascular diseases, which cardio-oncology seeks to investigate in terms of causes and consequences, in order to develop targeted preventive approaches. Conversely, patients with cardiovascular diseases have a higher risk of developing tumors compared to the general population, a topic addressed by the emerging field of ‘reverse cardio-oncology.’ It is important to note that cardiovascular and oncological diseases not only share risk factors such as obesity, diabetes mellitus, alcoholism, and smoking, but also multiple molecular mechanisms. In light of this evidence, the Scientific Directorate of IRCCS MultiMedica has decided to allocate the relevant 5×1000 donation funds to conduct new translational, clinical, and population studies aimed at understanding the mechanisms underlying this association. The capacity of IRCCS MultiMedica to operate actively in both clinical and translational research in the cardiovascular and oncological fields will enable the development of innovative strategies, based on the results of these studies, that can improve the primary prevention of both diseases, refine early diagnostic mechanisms, and identify new and precise therapeutic pathways.

Among the mechanisms shared by cardiovascular diseases and neoplastic conditions, the understanding of the biological processes that regulate cell trafficking in the bone marrow is of great interest. In particular, it is essential to understand the determinants of the release from the bone marrow of circulating CD34⁺ progenitor cells, which are protective for the cardiovascular system (a process altered in both diabetes and atherosclerotic disease, known as mobilopathy), as well as the entry of tumor cells (bone marrow metastasis), in order to guide shared precision therapies.

Sophisticated in vitro systems composed of human cells cultured in a physiologically similar environment represent a recent tool that offers a valid alternative to the variability observed with traditional animal models. In this project, we therefore intend to develop a 3D model of human bone marrow in vitro for the study of cell migration through the bone marrow vasculature. To this end, patients with atherosclerotic disease, with or without diabetes, as well as controls, will be enrolled. Bone marrow samples will be collected from waste material obtained during hip prosthesis surgery and/or from peripheral blood. In the first phase of the project, vascular cells (pericytes) and CD34⁺ cells collected from controls, together with commercial tumor cells, will be used to optimize the migration model in vitro. In parallel, using a single-cell sequencing approach, the gene expression profile of CD34⁺ cells associated with the disease will be assessed to identify molecular targets for intervention with targeted therapies. Subsequently, various compounds or drugs known for their ability to promote or inhibit the migration of CD34⁺ and tumor cells, respectively, will be tested in the new model.

In conclusion, this project will primarily enable the identification of new disease biomarkers and targets for innovative therapies. Furthermore, it will lead to the development of an innovative model useful for studying the mechanisms of tumor metastasis and diabetic mobilopathy, thereby enabling the development of effective and personalized therapies.

Date of receipt of 5 x 1000 funds: 22/09/2023

Budget

 Status updated as of July 2024

Project Title:

Project Title: “MATISSE—A Model of Artificial Organ of the Human Bone Marrow Vasculature”

Total 5 x 1000 allocation: €35,900.39
Project Start date: 01/01/2023
Project End date: 31/12/2023

Project Summary – Abstract:
The identification of the molecular mechanisms regulating cellular function could lead to the development of innovative therapies. In this context, the study of the interaction between cells of the cardiovascular system (both mature and progenitor) and the immune system is of fundamental importance in order to prevent and counteract the progression of cardiovascular diseases.
Central regulation of vascular homeostasis is exercised by the bone marrow, the primary source of leukocyte cells that provide protective action for the cardiovascular system. The bone marrow is the principal organ responsible for the synthesis of hematopoietic stem cells, which enable the continuous renewal of erythrocytes and leukocytes (granulocytes, monocytes, and lymphocytes) present in the blood, and also give rise to mononuclear cells specialized in reparative angiogenesis. The evidence collected through the study of altered mechanisms in cardiometabolic diseases has led us to hypothesize that functional deterioration of the bone marrow represents one of the main mechanisms responsible for the disease, through the dysfunction of the regenerative cells it contains.

Such deterioration may have consequences for peripheral organs, with impairment of the cardiovascular system even at early stages of geriatric frailty. To date, there are no available in vitro experimental models suitable for the cellular and molecular characterization of alterations in the bone marrow niche, and in particular of the vasculature, using primary human endothelial cells from bone marrow.
This project therefore aims to further investigate the mechanisms of crosstalk between hematopoietic/inflammatory cells and human bone marrow vascular cells (endothelial cells and pericytes), utilizing innovative in vitro models of functional interaction.

A model of artificial organ composed of primary human bone marrow vascular cells is currently under development in the laboratories of the project’s external collaborator, Dr. Camilla Cerutti at IEO, whose main research interest is the study of bone marrow metastases. The aim is therefore to combine the expertise of researchers at IRCCS MultiMedica with that of IEO to develop and test a new artificial organ model of human bone marrow vasculature.
In particular, models of cell co-culture in 2D and 3D will be developed. At MultiMedica, endothelial cells and pericytes from human bone marrow will be isolated and characterized; these cells will then be cultured at IEO on dedicated microfluidic supports (chips) in 2D and 3D, with the application of a controlled flow containing circulating cells to mimic the physiological conditions of human bone marrow. Upon completion of an initial phase of collection and characterization of vascular cells from the bone marrow, these cells will be analyzed and characterized within the in vitro artificial organ model in co-culture with circulating cells.

Initially, with funding provided by Dr. Cerutti, leukemic and breast cancer cells will be analyzed. These analyses will enable us to assess the bone marrow capillary network within the context of disease (tumor metastasis), which will serve as our prototype for subsequently transferring the acquired information to the non-oncological field for the study of aging and frailty.

Subsequently, we will examine the interaction between the bone marrow vasculature and progenitor or mature immune system cells with a protective function for the cardiovascular system. In addition to the functional aspects of circulating and vascular cells studied in the in vitro organ model—such as proliferation, adhesion, and migration—the molecular mechanisms involved in cellular interaction will also be analyzed.

Date of receipt of 5 x 1000 funds: 22/09/2022

Budget

 Status updates as of July 2023

Project Title: Molecular identification in individuals at cardiovascular risk during aging and as a consequence of COVID-19.

Total 5 x 1000 allocation: 38,041.68
Project Start date: 01/01/2022
Project End date: 31/12/2022

Project Summary – Abstract:

Based on the results achieved in the 2018-2019 financial years, we intend to continue a study on early markers of cardiovascular damage in elderly individuals, with particular focus to the role of frailty and COVID-19 in susceptibility to adverse cardiovascular events.

Frailty is a syndrome affecting approximately 20% of the elderly population, in which musculoskeletal and cognitive decline is associated with an increased risk of cardiovascular disease and mortality. The characterization of this syndrome and the identification of early markers of its onset and progression are of crucial importance for the clinical management of the elderly population, particularly during the COVID-19 health emergency. Indeed, COVID-19 affects older adults more frequently and, although the primary clinical manifestations involve the respiratory system, the implications for the cardiovascular system occur at various levels, with both acute and chronic harmful consequences.

At IRCCS MultiMedica, clinical research data (frailty index, lung capacity, and cardiac function) and basic research data (abundance of CD34+ cardiovascular regenerative cells and their gene profile) were collected from 35 pre-frail subjects over the age of 65. Even at early stages of frailty, alterations in cardiac and respiratory function have been observed, as well as a pro-inflammatory shift in CD34+ cells expressing high levels of the alarmins S100A8 and S100A9, two proteins with negative effects on the heart. Furthermore, an association has been identified between the inflammatory profile (cytokines and cells) in circulation and parameters of diastolic dysfunction. In collaboration with the University of Padua, the levels of CD34+ cells and alarmins were studied in a cohort of more frail subjects, demonstrating an association with the risk of adverse cardiovascular events (MACE). These results have been published in the journal Aging Cell (Bonora & Spinetti, Hematopoietic progenitor cell liabilities and alarmins S100A8/A9–related inflammaging associate with frailty and predict poor cardiovascular outcomes in older adults).

Furthermore, in order to assess the risk of cardiovascular damage associated with COVID-19, a biobank of biological material (peripheral blood) from patients hospitalized at the MultiMedica hospital was established (protocol approved by the MultiMedica Ethics Committee, code: CE-111-2020, entitled COVID-19: Creation of a biobank to study circulating predictors of disease progression. CANADA Study).

The current objectives are as follows:
1) To confirm inflammatory markers of risk for cardiovascular events in frail elderly individuals by enrolling a new cohort of subjects, on whom an in-depth in vitro analysis of the molecular mechanisms of interaction between circulating hematopoietic/inflammatory cells and vascular cells will also be conducted;
2) Conduct a study to identify molecular markers of severe progression and cardiovascular damage from COVID-19 through follow-up of subjects for whom a biobank was established in previous years (protocol CE-80-200271, title: Identification of Circulating Indicators of Severe Progression and Cardiovascular Damage in Patients Affected by COVID-19 – TORONTO Study).

Date of receipt of 5 x 1000 funds: 10/21/2021

Budget

 Status updated as of July 2022