Ahmad, N.S.
(2016)
Role of macrophage and associated cytokines in the regulation of breast tumour lymphovascular invasion.
PhD thesis, University of Nottingham.
Abstract
Lymphovascular invasion, including both blood and lymphatic vessel invasion, is an important step in tumourigenesis and is a prerequisite event in the complex process of metastasis. In breast cancer, data suggest that lymphatic vessels plays a significant role by being the major route for lymphatic vessel invasion (LVI) and that inflammatory cells, may be involved in its regulation. Macrophage, a major component of inflammatory infiltrate, has been shown to be importance in tumour growth and metastasis. The biomolecular mechanisms by which macrophage can mediate dissemination of tumour cells through the lymphatic compartment are, however, not yet fully understood.
In the first part of this project, CD68 (total) and CD163 (M2 subtype) macrophages were examined in two consecutive sections of 89 full-face invasive breast cancer samples. The density and localisation of macrophages were assessed using the Chalkley-grid counting method and their association with clinicopathological variables and clinical outcome was identified. A high count of CD68 macrophages was associated with the presence of lymphatic vessel invasion (LVI) and a high microvessel density (MVD), and tumour with high microvessel densities had high expression of CD163 macrophages, confirming a role for macrophage in mediating the process of angiogenesis and metastasis via lymphatic vessels. CD68 and CD163 macrophages were not associated with disease-free or breast cancer-specific survivals.
In order to elucidate previous conflicting data by our lab, suggesting that recombinant, and macrophage-induced IL-1β can mediate in vitro LVI, with patient based IHC data showing that high expression of tumour IL-1β was associated with improved disease specific survival and was not associated with LVI, it was of interest to study the expression of IL-1β signalling-related proteins including caspase-1, IL-1R I, IL-1R II, IL-1RacP, and IL-1RA. This was conducted in 1902 early stage invasive breast cancer patients, with long-term follow-up, using IHC. In addition, serum IL-1β concentration was measured in a matched subset of patients using enzyme-linked immunosorbent assay (ELISA) and expression levels examined for associations with the above proteins as well as clinicopathological criteria and patient prognosis. Although pre-requisites for IL-1 signalling pathways are present in breast tumours, tumour IL-1β expression was not strongly correlated with the expression of caspase-1, IL-1R I, IL-1R II, IL-1RacP and IL-1RA. Caspase-1, IL-1R I, IL-1R II, IL-1RacP and IL-1RA were not independent prognostic factors. The analysis of the markers stained in the current study could not provide any further elucidation of the conflict between previous in vitro and IHC studies regarding the role of IL-1β in regulating LVI.
The third part of the study dealt with IHC and in vitro investigations of macrophage-associated cytokines (IL-6 and IL-10) to assess their role in regulating LVI in breast cancer. Scratch wound migration assays were conducted to examine the influence of L-6 and IL-10 on breast cancer cell migration with results showing that low concentrations of IL-6 induced migration, whereas high concentrations inhibited it. Similarly, a high concentration of IL-10 also inhibited breast cancer cell migration. In addition, the effect of these cytokines, IL-6 and IL-10, on the tumour- endothelial (blood and lymphatic) adhesive process was also studied. Static adhesion assays showed that the adhesion patterns of breast cancer cell lines to the endothelial cells did not change following pre-stimulation of either blood or lymphatic endothelial cells with either IL-6 or IL-10. Thus, in vitro data suggest that both cytokines may not play a significant role in regulating LVI other than their effect on migration. The important of IHC expression of IL-6 and IL-10 were examined in a large cohort of early stage invasive breast cancer patients. Data showed that high expressions of IL-6 and IL-10 in breast cancer tissues were not associated with the presence of LVI. A significant association was found between high expression of IL-6, and longer disease-free interval, but it was not associated with improved disease-free survival. However, high IL-10 expression was not associated with improved disease-free survival, and breast cancer-specific survival.
In the final part of the project, the correlation between macrophage-associated cytokines including IL-1β, IL-6, and IL-10 and their downstream signalling elements, and target genes was assessed using Spearman’s rank correlation test. Expression data for downstream signalling elements was provided via collaboration with the Breast Pathology group. No strong correlations were observed between these cytokines. In addition, their expressions in breast cancer tissues were not strongly correlated with downstream signalling compartments, or target genes.
In conclusion, macrophages seem to play an important role in regulating LVI, with such LVI being almost entirely invasion of lymphatic vessels. Macrophage and macrophage-associated cytokines (IL-6 and IL-10) have been found to potential play role in breast cancer progression, with preliminary in vitro data suggesting that this may be via tumour cell migration rather than influencing tumour-endothelial cell interaction and LVI. This project has shed some light on the role of macrophage-associated cytokines in regulating LVI however more studies are needed to determine the mechanisms whereby IL-1β, IL-6, and IL-10 regulate the progression and prognosis of breast cancer.
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