Hyaluronectin modulation of lung metastasis in nude mice.

Hyaluronectin (HN) is a glycoprotein with a high affinity to hyaluronic acid (HA) and known to be a component of the extracellular matrix of tumours. Clinical studies have shown that a low level of HN correlates to tumours with poor prognosis, whereas a high level of HN correlates to tumours with good prognosis. We previously demonstrated in vitro that hyaluronidase activity, which promotes tumour progression and metastatic spread by degradation of HA into angiogenic oligosaccharides, was inhibited or promoted by HN, according to the level of HN-expression. This raises the question of the role played by HN in cancer, and particularly if high and low levels of HN-expression could trigger opposite effects on tumour growth and/or metastatic spread. To address this issue, we used a model of spontaneous lung fluorescent metastases that we characterised previously. We stably transfected the human HN cDNA into fluorescent H460MGFP cells and selected two clones characterised by different levels of HN-expression: HN110 and HN704, with a high and a low level of HN-expression, respectively. In vitro, we demonstrated that HN704 cell migration was significantly increased. Inoculation of clones to nude mice had no significant effect on tumour growth, but clearly revealed opposite effects on metastatic spread: HN110 significantly decreased the number of fluorescent metastases whereas HN704 significantly increased it. We also analysed HN, HA and hyaluronidase contents in sera and tumours. These results demonstrate that HN can play a role as either a suppressor or promoter of metastatic spread.

Reticulated hyaluronan hydrogels: a model for examining cancer cell invasion in 3D.

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. The roles of HA cell surface receptors and hyaluronidases (HAses) in this process are still controversial. In order to investigate their involvement in cancer pathogenesis, we developed a reticulated HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied thirteen cell lines, from primary tumors or metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. The number of colonies, which reflects tumor cell invasiveness, ranged from 7 to 193 after 5 days of culture. Invasion was dependent on the production of HAse as well as other factors. Optimal colonization occurred when cells released HAse, lacked HA-binding sites and did not secrete HA. Moreover, we describe for the first time a HAse activity at physiological pH that may be responding to the confinement of the enzyme in a three-dimensional structure. We show here that this reticulated matrix provides a three-dimensional model for investigating mechanisms involved in malignant invasion.

Biodistribution of injected tritiated hyaluronic acid in mice: a comparison between macromolecules and hyaluronic acid-derived oligosaccharides.

BACKGROUND: Hyaluronan (HA) has been reported to bind specifically and with high affinity to various cell types and to directly modify cell behaviour. In a previous report we demonstrated that both high molecular weight molecules (HA(H)) and HA-derived oligosaccharides were efficient at triggering terminal differentiation of acute myeloid leukemia (AML) blasts, in vitro, through CD44 ligation. MATERIALS AND METHODS: To explore the possibility of using HA for a differentiation therapy in AML, we investigated whether intravenous injection of tritiated HA(H) and/or HA-derived oligosaccharides (HA10-20) into mice accumulated in bone marrow, the main site of AML cell proliferation. RESULTS: The present work showed that the level of HA in bone marrow: 1) was maximum 5 hours after injection of either HA(H) or HA10-20; 2) was about 40 times higher after HA(H) than after HA10-20 injection. The amount of HA in bone marrow (5.8% ID/g) was two-fold higher than in serum, indicating that it was not due to circulating blood. Finally, using chromatographic analysis, we showed that about 34% of tritiated HA present in bone marrow 5 hours after HA(H) injection displayed a size higher or equal to HA10. CONCLUSION: After a single injection of macromolecular hyaluronan in mouse bone marrow we obtained a concentration of oligosaccharides close to the one shown to trigger AML cell differentiation in vitro. A part of the oligosaccharides had a size higher than or equal to the minimal one required to interact with HA receptors.

Inhibition of tumor growth and metastatic spreading by overexpression of inter-alpha-trypsin inhibitor family chains.

The inter-alpha trypsin inhibitor (ITI) family is a group of proteins built up from different combinations of I light chain (ITI-L) and 3 highly homologous heavy chains (ITI-HI, -H2 and -H3). To investigate a potential role of the ITI family chains in cancer and metastasis spreading, we engineered human H460M cell lines expressing both the green fluorescent protein (GFP) and one of these chains. These clones were subcutaneously injected in athymic nude mice, and lung metastasis number and primary tumor weight were determined after 28 days. Expression of the ITI-L chain considerably decreased tumor weight and fluorescent lung metastasis number. ITI-HI and ITI-H3 chain expression induced a significant decrease of metastasis number, whereas no decrease of tumor weight could be detected. In vitro, ITI-L expression significantly decreased chemotaxis and ITI-HI and ITI-H3 expression increased cell attachment. These results argue for the antitumoral or antimetastatic properties of ITI-L, -HI and -H3 chains.