Study of the role of tumour: associated macrophages in the tumoural microenvironment of canine mammary tumours: development of an anti-CCR2 antibody as a new therapeutic strategy

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Currently, progress in biotechnology has made available a great collection of genomic, transcriptomic, metabolomic and proteomic information on the tumour microenvironment, where cytokine and chemokine networks support interactions between cancer cells and their surrounding inflammatory landscape. Tumour-Associated Macrophages (TAMs) and Regulatory T-Lymphocytes (Tregs) have been identified as major participants in the tumour microenvironment, playing a dynamic role in the modulation of immunosuppression and supporting evasion of tumour immune surveillance. The tumour-promoting inflammatory environment is now part of the major cancer hallmarks. Our aim is to explore pathways of macrophage interaction with mammary cancer cells and seek to understand the mechanisms involved. In the first chapter we observed an association between tumour-associated macrophages and the expression of VEGF, a marker of angiogenesis, and also with several clinicopathological variables in malignant canine mammary tumours (CMT). We proceeded to further explore the interactions between macrophages and a mammary carcinoma cell line, REM 134, by performing in vitro studies of the role of CCL2/CCR2 axis in cell proliferation, and in the polarization of RAW 264.7 macrophages. Modelling 3D cultures of REM 134 cells, supplemented with conditioned media from macrophages, has allowed observation of several alterations in the expression of E-cadherin, CCR2, CSF1R, Twist1 and Tribbles1. By targeting Twist1 in RAW 264.7 macrophages, a parallel decrease was observed in STAT3 expression. Our results suggest a role for Twist1 as a mediator of interactions between mammary cancer cells and macrophages and also highlight the role of the CCL2/CCR2 axis in conditioning responses to extracellular cytokines and possibly in altering the secretome of macrophages. The composition of the inflammatory microenvironment warranted further research in models of cancer-related inflammation, therefore, on the second chapter we decided to focus on canine inflammatory mammary carcinomas (CIMC), where inflammation is seemingly implicated in the neoplastic disease. Human inflammatory breast cancer (IBC) and CIMC are the most aggressive forms of mammary cancer. The clinical course is rapid and characterized by early metastasis and short survival times. Current research aims to establish new therapeutic targets to provide better treatment options. Here, we investigated protein and gene expression levels associated with the inflammatory microenvironment, comparing with non-inflammatory CMT. RNA was extracted from 32 formalin-fixed paraffin-embedded samples of canine mammary tumours (CIMC=26; tubulopapillary carcinoma=6). The same tumour samples were processed and analysed for clinicopathological characteristics. cDNA was obtained by and qPCR performed to measure relative gene expression levels of COX-2, Tribbles1, Synuclein Gamma (SNCG), Vimentin, CCL2, VEGF, CCR2 and CSF1R. Immunohistochemical (IHC) analysis of CCR2, CSF1R and MMP-9 expression was also performed. Statistical analysis was carried out using the software SPSS (Statistical Package for the Social Sciences 17.0). Gene expression differences between CIMC and non-CIMC types were obtained for COX-2, SNCG, Tribbles1, VEGF and CSF1R. Among these biomarkers correlations were found, particularly between SNCG and Tribbles1 (R=0.512, P=0.001), but this correlation could not be observed in vitro in REM134 CMT and Lilly CIMC cell lines. In the IHC analysis, CCR2 expression demonstrated nuclear subcellular localization which was observed predominantly in CIMC tumours. Upregulation and correlation of SNCG and Tribbles1 in CIMC might be the result of cooperation between the microenvironment and cancer cells, promoting efficient metastasis formation. However, further investigation is required to support this hypothesis. In order to create a solution to target the tumour microenvironment, in the final chapter, we aimed to develop a monoclonal antibody targeting canine CCR2, a receptor involved in TAMs recruitment to the tumour microenvironment. Recently, a focus in cancer immunotherapies has been observed, whilst trying to present precision medicine alternatives to conventional cancer therapeutics. The development of monoclonal antibodies against molecular receptors involved in the main cancer pathways, led to a new generation of promising cancer treatments. Studies performed in canines, in a pre-clinical setting could significantly reduce the length of human clinical trials, owing to the availability of biologic material, a shorter life-span and the spontaneity of neoplastic lesions. TAMs were appointed as a novel therapeutic target in both human and canine cancers, due to their role in assisting angiogenesis, metastasis and invasion, and having been verified the association with a worse prognosis and clinicopathological variables of tumour aggressiveness. The possibility of hampering the recruitment of TAMs to the tumour core through the development of a monoclonal antibody against the receptor of monocyte chemotactic protein-1 (CCR2) is explored. Hereby we discussed factors in favour and against the use of adjuvant therapy with a monoclonal antibody against the CCR2 which we developed using a canine model of human breast cancer.
Tese de Doutoramento em Ciências Veterinárias
Macrófagos , Neoplasias das mamas animais , Recetores CCR2 , Uso terapêutico