The characterization of macrophages in melanoma and the effect of electroporation on melanoma conditioned macrophages

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Tremble, Liam Friel
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University College Cork
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Approximately 11,000 people are diagnosed with skin cancer in Ireland every year and approximately 1,000 of these present with malignant melanoma. Due to lifestyle changes and an increase in UV exposure, incidences are expected to continue to rise despite the presence of health campaigns. Immune cells, called macrophages, have been documented to represent up to 50% of the tumour mass in some melanomas. However, we have only a nascent understanding of the role of these cells in tumour biology and treatment responses. One treatment offered to advanced melanoma patients is electrochemotherapy, which has exceptionally high complete local response rates of up to 80%, and is under investigation with the replacement of chemotherapy with non-cytotoxic drugs such as calcium. Calcium electroporation has shown the ability to induce comparable response rates and is under increased scrutiny due to the presence of a case report in which a systemic anti-melanoma response was seen following treatment. While the effect of electroporation, and increasingly, calcium electroporation on tumour cells has been well documented. The effect of these treatments on bystander cells in the treatment area, such as tumour-associated macrophages, has not been investigated. Here we present clinical findings of the presence of distinct macrophage populations recruited to melanoma tissue. Using their inflammatory phenotype, effect on gene expression within the tumour, and correlation with survival outcomes we give compelling evidence that melanomas contain distinct populations of both active, and relatively inactive macrophages, which can vary depending on the pathological features of the tumour, such as Breslow depth and BRAF mutational status. Given the presence of an inflammatory population of macrophages in the tumour, we sought to develop an in vitro model in which we could examine the effect of electroporation on melanoma conditioned macrophages. Using an adapted model of bone marrow-derived monocyte development followed by melanoma conditioning, we were able to generate an immunologically active model of melanoma conditioned monocytes, which upregulated M2-associated surface receptors, similar to the predominant population of intratumoural macrophages. These conditioned cells showed no major increase in inducible nitric oxide synthase or arginase expression, as was seen clinically, but were able to affect T cell proliferation and polarization, indicating an influential immunological phenotype. In chapter 5, we investigate the effect of electroporation on these cells, and show that, similarly to tumour cells, their membranes do become reversibly electroporated. Using the parameters investigated in chapter 4 we show that calcium electroporation does impact their phenotype and functionality, and critically, influences their ability to subsequently activate and polarize T cells. Our data indicate that electroporation does not deplete intratumoural macrophages or inhibit their ability to drive cytotoxic T cell responses, suggesting that, from a macrophage perspective, calcium electroporation may be complementary to other immunogenic treatments.
Cancer , Melanoma , Immunology , Macrophage
Tremble, L. F. 2019. The characterization of macrophages in melanoma and the effect of electroporation on melanoma conditioned macrophages. PhD Thesis, University College Cork.
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