Tumor-Associated Macrophages: toward a better understanding of their cancer driver capability in Malignant Pleural Mesothelioma

Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the serosal cavities, characterized by a non-resolving, long-lasting inflammation driven by the presence of non-degradable asbestos fibers. MPM is strongly related to professional or environmental exposure to airborne asbestos. About 1400 new cases/year are observed in Italy, and its peak incidence is estimated around 2020- 2025. Chronic inflammation, eventually causing irreversible DNA damage, has been established as the main pathogenic cause. MPMs are known to be rich in Tumor-Associated Macrophages (TAMs), cells of the innate immunity abundantly present at cancer sites, and major producers of reactive mediators which trigger, perpetuate and amplify the inflammatory cascade. Furthermore, the presence of high TAM numbers in MPM was associated in some studies with poor patient prognosis. In this thesis, we wanted to better characterize the inflammatory pathways that are mostly expressed in MPM tissues. We performed a gene expression analysis on human mesothelioma tumor surgical samples; among classical mediators of inflammation (cytokines, chemokines) we identified two highly upregulated genes: GPNMB, coding for glycoprotein non metastatic B (GPNMB) and SPP1, coding for osteopontin (OPN). Both proteins were found by our group upregulated also in tumorconditioned macrophages (Solinas et al, 2010), raising the question whether TAMs or cancer cells, or both, are the producers. The circulating levels of OPN and GPNMB (measured by ELISA) were significantly higher in MPM patients than in healthy donors and, interestingly, were not downregulated by chemotherapy treatment, unlike other inflammatory mediators. Although OPN has long been known to be expressed in MPMs, its biological role is still not completely defined; on the other hand, GPNMB has never been investigated in this disease. In order to explore if OPN and GPNMB influence tumor growth, we used three murine mesothelioma cell lines with different histotypes as observed in human MPMs (sarcomatoid, biphasic, epithelioid): AB1, AB12, and AB22. We have set up a novel mouse model of mesothelioma injecting the tumor cells directly in the thorax, close to the pleural surface. In vivo experiments confirmed that murine mesothelioma are highly infiltrated by immune cells, especially macrophages. OPN was strongly expressed by the three cell lines, while GPNMB levels were very low, unlike human MPMs. We therefore silenced OPN and over-expressed GPNMB in murine mesothelioma cells to study the role of these proteins in vivo. We observed that silencing of OPN strongly decreased tumor growth, while over-expression of GPNMB promoted tumor progression. To study the role of GPNMB when produced by the microenvironment, we injected non-engineered AB22 mesothelioma cells in mice that are deficient for this protein. The results indicated that tumor incidence and tumor volume were higher in GPNMB WT mice, suggesting a pro-tumoral role of this protein when produced by host cells in the microenvironment. Overall the results indicate that OPN and GPNMB have a pro-tumoral role in MPM and may be considered as molecular targets for therapeutic interventions.