Abstract
The extracellular matrix (ECM) research has become fundamental to understand cancer. This thesis focuses on the exploration of ECM composition and organization in canine mammary tumors, with a special interest in the large chondroitin-sulfate proteoglycan (PG), versican.
Chapter 1 gives an overview of the recent concepts in matrix biology, particularly
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in tumors, and introduces versican and other ECM molecules investigated in the following chapters.
In Chapter 2 we show that versican protein expression is increased in tumors. Versican accumulates at three main locations in tumors: (1) in periductal tissues; (2) in peripheral invasive areas; (3) and the G1-domain of versican in myoepithelial-like spindle cell proliferations and myxoid tissues of complex and mixed tumors.
In Chapter 3 we show up-regulated mRNA expression of versican, collagen type II and aggrecan in tumors, especially in complex and mixed tumors. Expression of cartilage biomarkers was noted not only in the cartilagenous tissues of mixed tumors, but in myoepitheliomas and in myoepithelial-like cell proliferations and myxoid tissues of complex and mixed tumors. Therefore, the latter tissues appear to be the precursor tissues of the ectopic cartilage in mixed tumors. In addition, co-localization of hyaluronan (HA) and link protein (LP) with versican surrounding invading tumor cells was demonstrated. The three molecules may form complexes in canine mammary tumors and provide a viscoelastic microenvironment that promotes tumor cell proliferation and migration.
Chapter 4 shows that versican is present in multiple forms, including splice variants and catabolic products. V1, V0 and V3 versican mRNA and protein have been identified, with a prevalence of V1/V0 variants in canine mammary tumors and cell lines. The tumor cell lines expressed increased mRNA and protein of V0 and V1 versican compared to the normal mammary fibroblasts and the myoepithelial-like tumor cells expressed the highest levels of all mRNA splice variants. The high expression of the hydrophilic V0/V1 variants may contribute towards the formation of a highly hydrated tumor microenvironment. Furthermore, a broad range of versican fragments are present in canine mammary tumors, among which ADAMTS generated G1-bearing V1/V0 fragments were identified. These fragments are abundant in the myxoid tissues of complex and mixed tumors, indicating regulated versican degradation and a distinct biological role for the truncated versican forms in canine mammary tumors.
In Chapter 5 immunohistochemistry, gelatine/casein zymography, in situ zymography with quenched fluorogenic DQ-gelatin/substrate and quenched fluorescent substrate assay was used to measure the in vivo activity of MMPs and cathepsin B. MMPs were secreted by both tumor cells and stromal fibroblasts; however, in situ zymography showed that gelatinases exert their activity primarily in the tumor stroma, particularly surrounding ductal structures. All methods indicated elevated enzyme expression and activity in tumors and their metastases, confirming the role of these proteases in tumor development and progression. Gelatinases seem to play an important role in converting in situ carcinomas to invasive lesions.
In Chapter 6 we give an overview of the major findings and conclusions of this thesis and discuss the future directions of research.
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