Safuan, Sabreena
(2012)
Lymphovascular invasion in melanoma and breast cancer.
PhD thesis, University of Nottingham.
Abstract
The theory of metastatic cascade suggests that vasculature plays a central role in the metastatic processes by being the major route of spreading. Two main circulatory systems in the body are responsible for cancer cell dissemination; the blood vascular system and the lymphatic system. However, comparing between these circulatory systems, much less is known about lymphatic vessels, with few studies being conducted about the initial steps of metastasis.
In the first part of this project, a series of 202 formalin fixed paraffin embedded (FFPE) cutaneous melanoma sections were stained with D2-40, CD34 and CD68 to identify lymphatics, blood vessels and macrophages respectively, to examine vessel distribution and the involvement of inflammatory infiltrate in mediating vascular invasion (VI). Sections were also stained by conventional haematoxylin and eosin (H&E), to assess VI, and results compared against those obtained by immunohistochemistry (IHC) that allow discrimination of lymphatic and blood vessel invasion. It was found that lymphatics are mainly located at the peritumoural area of the tumour but intratumoural lymphatics are present and appeared to be functional based on the presence of tumour emboli in the vessels. In addition, vascular invasion in melanoma is mainly lymphatic vessel invasion with H&E assessment underestimating its incidence. Lymphatic vessel invasion were significantly associated with markers of aggressive disease which suggest their importance in melanoma. Lymphatic vessel invasion was also associated with a high macrophage count, suggesting a role for macrophage in mediating the process of metastatic via lymphatic vessels.
In the second part of this project, the adhesion pattern of melanoma and breast cancer cell lines to blood and lymphatic endothelial cell models; large vessel versus microvessel and primary versus immortalised cells were compared. In addition, the effect of macrophage secreted cytokines; TNF-α and IL-1β, tumour conditioned media and macrophage conditioned media on the adhesive process were also studied. Both melanoma and breast cancer cells exhibited a higher level of adhesion to blood compared to the lymphatic endothelial cells. IL-1β stimulation of endothelial cells, tumour cells or both together showed a significant increased in the percentage of adhered tumour cells to the endothelial cell models with a higher increased to the lymphatic endothelial cells. A significant increased tumour cell adhesion was also observed with macrophage conditioned media and this effect seemed to be associated with the amount of IL-1β present. Interestingly, the increased adhesion effect observed with this supernatant was removed with the use of interleukin-1 converting enzyme (ICE) inhibitor.
Expression of adhesion molecules; CLEVER-1, ICAM-1 and VCAM-1 were examined to study which adhesion molecules might regulate the process of tumour-endothelial interactions. Stimulation of endothelial cell models with IL-1β did not show any significant altered CLEVER-1 expression suggesting that although CLEVER-1 is an important lymphatic specific adhesion molecule, it may not be the principle regulator of tumour-endothelial interactions. This process may be regulated by ICAM-1 and VCAM-1 in which the expression increased significantly upon stimulation with IL-1β.
In the third part of this study, the effect of TNF-α, IL-1β, tumour conditioned media and macrophage conditioned media stimulation on melanoma and breast cancer cell migration were investigated using wound healing assays. Following exposure to TNF-α and IL-1β, a significant increase in the percentage of wound closure was observed and the increase was higher in IL-1β stimulated cells. Similarly, when tumour cells were exposed to macrophage conditioned media, there was an increase in the percentage of wound closure compared to control cells. The effect of IL-1β and macrophage conditioned media on breast cancer cell migration across blood and lymphatic endothelial cells were also studied using Boyden chamber transmigration assay. Significant increased in tumour cells transmigration was observed with IL-1β stimulation, with similar affinity across both endothelial cell types. However, when cells were stimulated with macrophage supernatant from lipopolysaccharide (LPS) stimulated macrophages, an increase transmigratory effect was notably observed to the lymphatic endothelial cells. Interestingly, the increased adhesion effect was removed with the used of ICE inhibitors.
The last part of this study dealt with IL-1β expression in breast tissue samples. 1511 early stage breast cancer tissue microarray samples were stained with commercially available IL-1β antibody to examine the association with lymphatic vessel invasion, clinicopathological variables and clinical outcome. High IL-1β expression in tumour cells was significantly associated with the absence of both intra-tumoural and peri-tumoural lymphatic vessel invasion. A significant association was also observed between low IL-1β expression in tumour cells with breast cancer specific survival and disease free interval.
In conclusion, lymphatic vessels have been found to play a significant role in breast cancer and melanoma cells progression by being the major route for vascular dissemination. In the in-vitro settings, this study has shown that IL-1β, with macrophages as the main producer, could regulate tumour cell invasion especially to the lymphatic circulation. This project has yielded some important results towards understanding of the lymphatic vasculature and modulation of lymphatic vessel invasion. However, more studies are needed to enable translation of research into clinical management of cancer.
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