We study the complex interplay between cells of the immune system and the extracellular matrix at site of tissue transformation. The molecules involved in such cross-talk are expected to be early markers of tumorigenesis as well as responsible of tumor progression and perhaps potential target for new therapies.

Group expertise

The Molecular Immunology Unit has a long lasting expertise in Tumor Immunology also using transplantable and spontaneous mouse tumors modeling the human counterpart. The group pioneered the study of immune infiltration and its modulation by cytokine gene transduction and unveiled the role of the extracellular matrix (ECM) protein SPARC (secreted protein acidic and rich in cysteine) in such modulation. Studying the cross-talk between neoplastic cells and the surrounding tumor microenvironment, the focus is on the immune cellular components endowed of immunosuppressive activities (myeloid cells, mast cells and regulatory T cells) and on the ECM, particularly SPARC and osteopontin (OPN), that, apparently not redundant in functions, have important role in tissue homeostasis, attempt to heal and therefore in cancer progression.  The availability of several transgenic and knock-out mouse models allows of investigating the role of these matricellular proteins in solid tumor development, progression and metastasis, in particular in mammary and prostate cancers, distinguishing the distinct role of the proteins when produced by cancer or host cells. In addition, we study the role of SPARC and OPN in the specialized environments of primary (Bone Marrow) and secondary lymphoid organs (SLOs) in relation to cancer-induced myeloproliferation and autoimmunity-lymphomagenesis transition.

Extension project

We study the complex interplay among cells of the immune system, the extracellular matrix, and tissues undergoing transformation. Transforming cells require permissive interaction with the surrounding environment composed of cellular stromal elements and extracellular matrix. We are focused on cells of the innate immune system that sense environmental changes and on extracellular matrix proteins that, involved in tissue remodeling, try to activate, unsuccessfully, healing functions. Particularly, we are studying mast cells, neutrophils, macrophages and other myeloid cells and their production or sensing of the matricellular proteins SPARC and osteopontin.

Role in the project, experimental design

Works from the group generated the idea that stroma cell components sense early transformation and provide or amplify local signals that through circulation can reach and alert the bone marrow in demand of new myeloid cells.

The combined experimental approaches and effort of the units involved in WP1 allowed to demonstrate that BM is a true sensor of early malignant transformation occurring at distant sites.

The well-characterized MMTV-HER2/NeuT transgenic model of luminal B breast carcinoma has been used to follow the early phases of tumor development and its progression that well recapitulates the human breast cancer counterpart.

A throughout characterization of the BM hematopoietic compartment, the peripheral blood and of primary mammary lesions has been performed combining flow cytometry, histopathological analysis, gene expression and circulating miRNA profiles on every single mouse of different cohorts.

Results identified profound modifications in composition and spatial arrangement of hematopoietic and mesenchymal cells compartments, as well as differences in gene expression beginning at early time points.  Also, a different circulating miRNA profile between tumor-bearers and control sibling mice has been identified. This throughout characterization of mouse "patients" provides a global picture of the BM hematopoietic changes and of relative gene expression profile associated to a defined stage of mammary primary lesion and of circulating miRNA signature. The Extension Program would allow of using such information to develop a multi-parametric classifier for every stage of disease that taking into account all types of BM and peripheral modifications should be able to classify each mouse patient according to such characteristics.

In the EDERA Project we have defined miRNAs that are differentially expressed between transgenic and control animals at early and late disease stages (identified as specific time points: 12 and 24 weeks of age). Because of the variability in disease severity among mice collected at selected time points (which in some way reflects the individual variability of patients with stage-matched cancers), we plan to refine this miRNA signature in a co-clinical study in which a new large cohort of MMTV-NeuT and WT siblings will be blood-sampled blindly, at different time points, independently from their NeuT genotype. Testing the expression of each miRNA of our profile during effective disease progression versus age progression (physiological breast development/remodeling), we should be able to assess how miRNAs perform at every stage of disease. In this respect, we plan to create clusters of circulating miRNAs specific for different disease severity that might predict the disease stage rather than detecting the sole presence or absence of a neoplastic lesion.

Such signatures will be also tested in a second luminal breast cancer model, the MMTV-PyMT, for which we have already collected BM, plasma and primary lesions.

To test the translational relevance of our signatures, this program extension includes our testing of samples from the Babe clinical study that enrolls women who have had mammography detection of a nodular mass of less than 20 mm either benign or neoplastic at pathology screening. The plasma collected from these patients for paralleled studies (see Tagliabue's Group) will be tested for presence of our stage-related miRNA signatures/clusters such to assess whether the stage of malignant lesions can be identified directly in blood.

Staff

Mario Paolo Colombo

Unit director, staff scientist

Claudia Chiodoni

Staff scientist

Tiziana Ada Renzi

Post-doc

Sabina Sangaletti

Contract scientist

Laura Botti

Contract biologist

Mariella Parenza

Senior staff technician

Barbara Cappetti

Staff technician

Ivano Arioli

Senior staff technician

Our research group provides the expertise in translational pathology that is fundamental to integrate microRNA expression profile data with phenotypic analyses, evaluation of the tumor progression stage and of the composition of the immunological microenvironment, in the contest of murine models of spontaneous carcinogenesis. Our final aim is to define molecular and microenvironment risk signatures, specific for each disease stage.

Our research activity is focused on studying the interactions between neoplastic cells, the surrounding stromal microenvironment, and the systemic hematopoietic macroenvironment.

Our group contributed to the idea that the elements of the stromal microenvironment, such as mesenchymal elements and the extracellular matrix can actively drive the transition from a pre-malignant state of altered tissue homeostasis toward a full-blown malignancy. Moreover, our group has focused on dissecting the mechanisms underlying the reciprocal influence between hematopoietic responses and peripheral tissue perturbations.

The expertise of Prof. Tripodo in translational and experimental pathology allowed the development of novel lines of research that integrate perfectly within the general frame of the “Programma AIRC 5 per mille”, complementing the expertises of the unit led by Dr. Colombo. They combine immunolocalization and quantitative analyses of the stromal modifications (associated to the different stages of peripheral cancerogenesis) with the study of molecular and immunophenotypic profiles of systemic hematopoiesis.

Our group has specific skills in histopathological, immunophenotypic, cytogenetic and molecular in situ analyses, and it is actively involved in developing novel informatic instruments to perform morphometric and quantitative analyses.

Role in the project, experimental design

During the first 5 years of the “Programma AIRC 5 per Mille”, we demonstrated an adaptation of the hematopoiesis within the bone marrow in response to the development of a malignant transformation of the mammary gland.

Since its early stages, the neoplastic clone influences the bone marrow, driving a tumor-reprogrammed hematopoiesis.

The murine models of spontaneous mammary gland carcinogenesis represent an invaluable tool for studying the tumoral progression through its discrete stages and to perform an in depth characterization of the associated changes occurring in the central hematopoiesis.

The task of our group is to perform hystopatological, immunophenotypic and molecular analyses in order to characterize the mammary gland neoplasias and the composition of their associated stromal and immunological microenvironment. Moreover, our group will quantitatively evaluate the progression of the foci of malignant transformation (i.e. the tumoral burden) and correlate the data with the extent and quality of central modifications in the bone marrow hematopoiesis. Furthermore, we will correlate these quantifications with the expression of specific signatures of circulating microRNAs, with the aim of stratifying the disease in “murine patient” cohorts.

The histomorphological and ancillary analyses, together with miRNA and GEP profiles obtained for each “murine patient”, will allow to create a multiparametric classifier aimed at identifying novel relevant biological and co-clinic correlations.

Staff

Claudio Tripodo

MD, Group Leader

Walter Arancio

Biologist, PhD, Research Fellow

Alessandro Gulino

Biologist, PhD, Research Fellow

Beatrice Belmonte

MD, Pathologist, PhD

Valeria Cancila

Biologist, PhD student

The research activity of our group is focused on breast carcinoma through experimental and translational studies aimed to define the role of the oncoprotein HER2 and extracellular matrix (ECM) molecules in tumor onset and progression and in early diagnosis and response to therapy.

Role in the project, exsperimental design

Within the AIRC-funded special program 5xMille, the research group, in strict collaboration with radiologists, clinicians, pathologists, biostatisticians and with the Institutional Biobank, acquired the necessary skills to study tumor microenvironment-related molecules as biomarkers for tumor progression and early diagnosis of breast cancer.

While standard screening tests (mammography and breast ultrasound scan) have reduced the probability of death from breast cancer, biopsy is still required to prove that abnormal areas by diagnostic imaging are malignant or benign in a large number of women. Obtaining a tissue biopsy is an invasive procedure and expensive for the health system, especially in high-risk women, who require frequent monitoring. A simpler and valid alternative might reside in monitoring circulating molecular markers in blood able to distinguish benign from malignant breast disease. In this context we investigated the possibility that tumor-driven remodeling of the surrounding microenvironment during cancer development may lead to ECM molecule release into the bloodstream. In silico analyses and the use of in vitro preclinical models revealed the release of soluble ECM proteins in culture supernatant from normal fibroblasts conditioned by breast cancer cells. Some of these molecules were found in plasma of women with breast cancer and their levels discriminate tumor patients from patients with benign breast disease.

In addition, a panel of circulating microRNAs and small peptides able to identify women with malignant breast disease were detected through the analysis of plasma by OpenArray technology and liquid chromatography mass spectrometry, respectively.

The next step is to validate the diagnostic performance and the clinical relevance of identified molecular classifiers in a prospective clinical study. The study BABE (BreAst Blood Early diagnosis) is enrolling women with imaging suggestive of breast malignant tumor by ultrasound scanning or mammography, which underwent biopsy for diagnostic assessment in our Institute. Plasma samples obtained from enrolled patients, upon informed consent, will be used to detect the relevant biomarkers. For patients surgically treated in our Institute upon tumor histopathological diagnosis, collection of plasma samples after surgery and during follow-up is also ongoing. According to routine biopsies in our Institute, 40% of collected samples are expected from women with breast cancer and 60% with benign breast disease.

If BABE study confirms the diagnostic performance of these circulating molecular classifiers a new multicentre clinical study will be designed in order to demonstrate the diagnostic advantage to combine liquid biopsy and conventional imaging in early breast cancer diagnosis.

Staff

Elda Tagliabue

Biologist, group leader

Rosaria Orlandi

Biologist staff member, responsible for peptide analysis

Marta Giussani

Post-doc FIRC fellowship, responsible for BABE study

Viola Regondi

Biotecnologist fellowship, responsible for plasma sample preparation

Gianfranco Scaperrotta

Radiologist, responsible for patient enrollment in BABE study

Patrizia Casalini

Tecnician, responsible for reagent preparation

Giulia Saba

Research Nurse, responsible for patient blood sampling during follow-up

We study the complex interplay between cells of the immune system and the extracellular matrix at site of tissue transformation. The molecules involved in such cross-talk are expected to be early markers of tumorigenesis as well as responsible of tumor progression and perhaps potential target for new therapies.

Group expertise

The Molecular Immunology Unit has a long lasting expertise in Tumor Immunology also using transplantable and spontaneous mouse tumors modeling the human counterpart. The group pioneered the study of immune infiltration and its modulation by cytokine gene transduction and unveiled the role of the extracellular matrix (ECM) protein SPARC (secreted protein acidic and rich in cysteine) in such modulation. Studying the cross-talk between neoplastic cells and the surrounding tumor microenvironment, the focus is on the immune cellular components endowed of immunosuppressive activities (myeloid cells, mast cells and regulatory T cells) and on the ECM, particularly SPARC and osteopontin (OPN), that, apparently not redundant in functions, have important role in tissue homeostasis, attempt to heal and therefore in cancer progression.  The availability of several transgenic and knock-out mouse models allows of investigating the role of these matricellular proteins in solid tumor development, progression and metastasis, in particular in mammary and prostate cancers, distinguishing the distinct role of the proteins when produced by cancer or host cells. In addition, we study the role of SPARC and OPN in the specialized environments of primary (Bone Marrow) and secondary lymphoid organs (SLOs) in relation to cancer-induced myeloproliferation and autoimmunity-lymphomagenesis transition.

Extension project

We study the complex interplay among cells of the immune system, the extracellular matrix, and tissues undergoing transformation. Transforming cells require permissive interaction with the surrounding environment composed of cellular stromal elements and extracellular matrix. We are focused on cells of the innate immune system that sense environmental changes and on extracellular matrix proteins that, involved in tissue remodeling, try to activate, unsuccessfully, healing functions. Particularly, we are studying mast cells, neutrophils, macrophages and other myeloid cells and their production or sensing of the matricellular proteins SPARC and osteopontin.

Role in the project, experimental design

Works from the group generated the idea that stroma cell components sense early transformation and provide or amplify local signals that through circulation can reach and alert the bone marrow in demand of new myeloid cells. The combined experimental approaches and effort of the units involved in WP1 allowed to demonstrate that BM is a true sensor of early malignant transformation occurring at distant sites. The well-characterized MMTV-HER2/NeuT transgenic model of luminal B breast carcinoma has been used to follow the early phases of tumor development and its progression that well recapitulates the human breast cancer counterpart.

A throughout characterization of the BM hematopoietic compartment, the peripheral blood and of primary mammary lesions has been performed combining flow cytometry, histopathological analysis, gene expression and circulating miRNA profiles on every single mouse of different cohorts.

Results identified profound modifications in composition and spatial arrangement of hematopoietic and mesenchymal cells compartments, as well as differences in gene expression beginning at early time points.  Also, a different circulating miRNA profile between tumor-bearers and control sibling mice has been identified.

This throughout characterization of mouse "patients" provides a global picture of the BM hematopoietic changes and of relative gene expression profile associated to a defined stage of mammary primary lesion and of circulating miRNA signature. The Extension Program would allow of using such information to develop a multi-parametric classifier for every stage of disease that taking into account all types of BM and peripheral modifications should be able to classify each mouse patient according to such characteristics.

In the EDERA Project we have defined miRNAs that are differentially expressed between transgenic and control animals at early and late disease stages (identified as specific time points: 12 and 24 weeks of age). Because of the variability in disease severity among mice collected at selected time points (which in some way reflects the individual variability of patients with stage-matched cancers), we plan to refine this miRNA signature in a co-clinical study in which a new large cohort of MMTV-NeuT and WT siblings will be blood-sampled blindly, at different time points, independently from their NeuT genotype. Testing the expression of each miRNA of our profile during effective disease progression versus age progression (physiological breast development/remodeling), we should be able to assess how miRNAs perform at every stage of disease. In this respect, we plan to create clusters of circulating miRNAs specific for different disease severity that might predict the disease stage rather than detecting the sole presence or absence of a neoplastic lesion.

Such signatures will be also tested in a second luminal breast cancer model, the MMTV-PyMT, for which we have already collected BM, plasma and primary lesions.

To test the translational relevance of our signatures, this program extension includes our testing of samples from the Babe clinical study that enrolls women who have had mammography detection of a nodular mass of less than 20 mm either benign or neoplastic at pathology screening. The plasma collected from these patients for paralleled studies (see Tagliabue's Group) will be tested for presence of our stage-related miRNA signatures/clusters such to assess whether the stage of malignant lesions can be identified directly in blood.

Staff

Mario Paolo Colombo

Unit director, staff scientist

Claudia Chiodoni

Staff scientist

Tiziana Ada Renzi

Post-doc

Sabina Sangaletti

Contract scientist

Laura Botti

Contract biologist

Mariella Parenza

Senior staff technician

Barbara Cappetti

Staff technician

Ivano Arioli

Senior staff technician

The research activity of our group is focused on breast carcinoma through experimental and translational studies aimed to define the role of the oncoprotein HER2 and extracellular matrix (ECM) molecules in tumor onset and progression and in early diagnosis and response to therapy.

Role in the project, experimental design

Within the AIRC-funded special program 5xMille, the research group, in strict collaboration with radiologists, clinicians, pathologists, biostatisticians and with the Institutional Biobank, acquired the necessary skills to study tumor microenvironment-related molecules as biomarkers for tumor progression and early diagnosis of breast cancer.

While standard screening tests (mammography and breast ultrasound scan) have reduced the probability of death from breast cancer, biopsy is still required to prove that abnormal areas by diagnostic imaging are malignant or benign in a large number of women. Obtaining a tissue biopsy is an invasive procedure and expensive for the health system, especially in high-risk women, who require frequent monitoring. A simpler and valid alternative might reside in monitoring circulating molecular markers in blood able to distinguish benign from malignant breast disease. In this context we investigated the possibility that tumor-driven remodeling of the surrounding microenvironment during cancer development may lead to ECM molecule release into the bloodstream. In silico analyses and the use of in vitro preclinical models revealed the release of soluble ECM proteins in culture supernatant from normal fibroblasts conditioned by breast cancer cells. Some of these molecules were found in plasma of women with breast cancer and their levels discriminate tumor patients from patients with benign breast disease.

In addition, a panel of circulating microRNAs and small peptides able to identify women with malignant breast disease were detected through the analysis of plasma by OpenArray technology and liquid chromatography mass spectrometry, respectively.

The next step is to validate the diagnostic performance and the clinical relevance of identified molecular classifiers in a prospective clinical study. The study BABE (BreAst Blood Early diagnosis) is enrolling women with imaging suggestive of breast malignant tumor by ultrasound scanning or mammography, which underwent biopsy for diagnostic assessment in our Institute. Plasma samples obtained from enrolled patients, upon informed consent, will be used to detect the relevant biomarkers. For patients surgically treated in our Institute upon tumor histopathological diagnosis, collection of plasma samples after surgery and during follow-up is also ongoing. According to routine biopsies in our Institute, 40% of collected samples are expected from women with breast cancer and 60% with benign breast disease.

If BABE study confirms the diagnostic performance of these circulating molecular classifiers a new multicentre clinical study will be designed in order to demonstrate the diagnostic advantage to combine liquid biopsy and conventional imaging in early breast cancer diagnosis.

Staff

Elda Tagliabue

Biologist, group leader

Rosaria Orlandi

Biologist staff member, responsible for peptide analysis

Marta Giussani

Post-doc FIRC fellowship, responsible for BABE study

Viola Regondi

Biotecnologist fellowship, responsible for plasma sample preparation

Gianfranco Scaperrotta

Radiologist, responsible for patient enrollment in BABE study

Patrizia Casalini

Tecnician, responsible for reagent preparation

Giulia Saba

Research Nurse, responsible for patient blood sampling during follow-up