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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.

The aim of our study was to investigate the production of hyaluronan (HA) by the intima-media during the sclerotic response to aortic injury with a catheter balloon in the rat. In addition we analyzed, for the first time in this model, the production of a glycoprotein (hyaluronectin, HN) which binds specifically to HA. HA and HN were analyzed in control (D0), 14 (D14) and 28 (D28) days after injury using biochemical and immunohistochemical techniques. Intima-media DNA content and wet weight increased significantly on D14 and declined on D28 (but remained significantly increased in comparison to controls). HA content (median in D0 = 448 ng) increased significantly on D14 (2P < 0.04) and on D28 (2P < 0.02). HN content (median in D0 = 920 ng) increased significantly on D14 (2P < 0.05) but decreased on D28 to return to the control level. On D0 the amount of HN was about 3 times higher than that of HA (median ratio HA/HN = 0.34). The ratio remained unchanged on D14 but significantly increased on D28 (2P < 0.02). HPLC and Western blotting showed no difference between HN extracted from normal aorta and HN extracted from injured aorta at D14. Different isoforms of HN were present in both cases, ranging from 400 to 45 kDa. The HA increase on D14 and D28 was not related to a change in hyaluronidase activity of aortic tissue. Immunohistochemical analysis showed at D0 a small amount of HA around arterial smooth muscle cells (ASMC) in media, at D14 more HA was localized around and between ASMC in media and neointima but at D28 it was localized mainly near the vessel lumen. HN formed all the time (D0, D14 and D28) a continuous layer localized near the vessel lumen. In vitro studies showed that production of HA and HN was stimulated when ASMC proliferate and HA at high concentrations (1-100 micrograms/ml) reduced, in a dose dependent manner, ASMC growth. In conclusion our results show that both neointima formation in vivo and ASMC proliferation in vitro correlated with increased HA and HN production. This suggests that HA and HN are probably involved in the formation of neointima. On the other hand, the finding that HA continued to increase in the aorta when neointima decreased and that high concentrations of HA reduce ASMC proliferation in culture suggest that HA might be involved in the regression of neointima.

Several techniques for assaying and localizing hyaluronan (HA), all based on the affinity to hyaluronan of proteins isolated from cartilage, chondrosarcoma, or brain, have been proposed. We show here that a unique reagent, alkaline phosphatase-linked hyaluronectin, can be used to assay hyaluronan in biological fluids or tissue extracts (enzyme-linked sorbent assay method) and to characterize it in cells or tissue sections in two steps: reagent incubation and staining. Results of assays in biological fluids or tissue extracts showed a good correlation with results of the previously described technique using antibodies to detect hyaluronectin bound to a plastic microtest plate (B. Delpech et al., 1985, Anal. Biochem. 149, 555-565) for both low concentrations (< 1 mg/liter, r = 0.973, P < 0.001) and high concentrations (> 1 mg/liter, r = 0.953, P < 0.001). The interassay variations were 8.5% when the assay was performed at 4 degrees C and 18.5% at 37 degrees C. The intraassay variations under those conditions were, respectively, 14.4 and 6.5%. Tissue HA could be detected easily with the reagent, as shown in fetal tissues and in tumors. Specificity of the reaction was controlled either by blocking the reagent with an excess of hyaluronan (which was not possible with other glycosaminoglycans) or by destroying tissue hyaluronan with streptomyces hyaluronidase. Alkaline phosphatase-linked hyaluronectin was also used to assay hyaluronidase activity in several biological fluids. One-hour incubation of hyaluronidase preparations on HA-coated plates made it possible to detect as low as 1 mU bovine testis hyaluronidase and 0.1 mTRU streptomyces hyaluronidase. Four-hour incubation made it possible to detect activity in a 1/12,500 dilution of human serum.

Hyaluronectin (HN), a hyaluronan (hyaluronic acid, HA)-binding glycoprotein isolated from human brain, was studied in normal and atherosclerotic human arteries. It can be detected and assayed in tissue samples by immunohistochemistry. In addition, its high and specific affinity for HA makes it possible to develop specific histological localization of HA using HN as a probe. We tested the presence of HN and HA in human carotid artery samples from adults and newborns. In atheroma-free arterial samples HN was found in the intima, between smooth muscle cells and in the adventitial extracellular matrix. In atherosclerotic lesions, HN was strongly expressed in the diffuse thickened intima and surrounding extracellular microcrystalline calcium deposits, and very little in the lipid core. HA was found in the same locations. The similar localizations of HN and HA shown by immunohistology and demonstration of HN-HA complexes by high pressure liquid chromatography (HPLC) suggest that they are associated in vivo.

The use of a hyaluronic acid-binding proteoglycan (hyaluronectin) as a probe for the detection of hyaluronic acid has facilitated the development of an indirect enzymo-immunological assay for hyaluronidase. Plastic microtest ELISA plates were coated with hyaluronic acid. Incubation with hyaluronidase led to the destruction of insolubilized hyaluronic acid in proportion to the hyaluronidase concentration of samples. Residual hyaluronic acid was assayed by its capacity to bind immune complexes made up of hyaluronectin supplemented with alkaline phosphatase-conjugated anti-hyaluronectin antibodies. The technique was very sensitive and permitted the detection of as little as 10(-10) NFU of bovine testicular hyaluronidase. Hyaluronidase was detected by this technique in human sera, bee venom and culture medium of human hepatoma cell lines.

Three established cell lines derived from three human astrocytomas were grafted into nude mice. Hyaluronectin, a marker of loose connective tissue, was detected in the grafts. The histopathology and the hyaluronectin staining were similar to those of the tumours of origin. Absorption experiments demonstrate that one of the three human grafted tumours (CB 109) contains a human type hyaluronectin, in contrast to the other two. In these two grafts, hyaluronectin is a constituent of the host stromal reaction, whereas in the CB 109 graft hyaluronectin apparently originates from the human tumour cells. This tumour could be used to test the production of human hyaluronectin in vitro and in vivo.