Accumulation of small hyaluronan oligosaccharides in tumour interstitial fluid correlates with lymphatic invasion and lymph node metastasis.

BACKGROUND: Association studies have implicated the glycosaminoglycan hyaluronan (hyaluronic acid, HA) and its degrading enzymes the hyaluronidases in tumour progression and metastasis. Oligosaccharides of degraded HA have been ascribed a number of biological functions that are not exerted by high-molecular-weight HA (HMW-HA). However, whether these small HA oligosaccharides (sHA) have a role in tumour progression currently remains uncertain due to an inability to analyse their concentration in tumours. METHODS: We report a novel method to determine the concentration of sHA ranging from 6 to 25 disaccharides in tumour interstitial fluid (TIF). Levels of sHA were measured in TIF from experimental rat tumours and human colorectal tumours. RESULTS: While the majority of HA in TIF is HMW-HA, concentrations of sHA up to 6 µg ml(-1) were detected in a subset of tumours, but not in interstitial fluid from healthy tissues. In a cohort of 72 colorectal cancer patients we found that increased sHA concentrations in TIF are associated with lymphatic vessel invasion by tumour cells and the formation of lymph node metastasis. CONCLUSIONS: These data document for the first time the pathophysiological concentration of sHA in tumours, and provide evidence of a role for sHA in tumour progression.

Flow cytometry-based isolation of dermal lymphatic endothelial cells from newborn rats.

The lymphatic system plays a key role in tissue homeostasis, fatty acid transport, and immune surveillance. Pathologically, dysfunction of the lymphatic system results in edema, and increased lymphangiogenesis can contribute to tumor metastasis. Lymphatic vessels are composed of lymphatic endothelial cells (LECs) that can be identified by distinct marker molecules such as Prox-1, podoplanin, VEGFR-3 and LYVE-1. Primary LECs represent a valuable tool for the study of basic functions of the lymphatic system. However, their isolation remains a challenge, particularly if rodent tissues are used as a source. We developed a method for the isolation of rat dermal LECs from the skin of newborn rats based on sequential enzymatic digestion with trypsin and Liberase followed by flow cytometric sorting using LYVE-1 specific antibodies. Cells isolated according to this protocol expressed the lymphatic markers Prox-1, podoplanin, LYVE-1 and VEGFR-3, and displayed an endothelial-like morphology when taken into culture. These primary cells can be used for studying lymphatic biology in rat models, and the protocol we describe here therefore represents an important extension of the experimental repertoire available for rats and for modeling the human lymphatic system.

ASAP1 promotes tumor cell motility and invasiveness, stimulates metastasis formation in vivo, and correlates with poor survival in colorectal cancer patients.

We have previously performed an unbiased screen to identify genes whose expression is associated with the metastatic phenotype. Secondary screening of these genes using custom microarray chips identified ASAP1, a multi-domain adaptor protein with ADP-ribosylation factor-GAP activity, as being potentially involved in tumor progression. Here, we show that at least three different splice forms of ASAP1 are upregulated in rodent tumor models in a manner that correlates with metastatic potential. In human cancers, we found that ASAP1 expression is strongly upregulated in a variety of tumors in comparison with normal tissue and that this expression correlates with poor metastasis-free survival and prognosis in colorectal cancer patients. Using loss and gain of function approaches, we were able to show that ASAP1 promotes metastasis formation in vivo and stimulates tumor cell motility, invasiveness, and adhesiveness in vitro. Furthermore, we show that ASAP1 interacts with the metastasis-promoting protein h-prune and stimulates its phosphodiesterase activity. In addition, ASAP1 binds to the SH3 domains of several proteins, including SLK with which it co-immunoprecipitates. These data support the notion that ASAP1 can contribute to the dissemination of a variety of tumor types and represent a potential target for cancer therapy.