Posters— Basic Science/Biology

ECM INTERACTIONS AND PRODUCTION OF PROTEOLYTIC ENZYMES IN MALIGNANT FIBROUS HISTIOCYTOMAS

Spessotto P, Mucignat MT, Wasserman B, Burino I, Bogetto L, Cigana A, Frustaci S, Perris R, Colombatti A. Centro di Riferimento Oncologico – National Cancer Institute – Aviano, Italy (33081)

We have established several novel cell lines from surgical specimens of recurrent metastases of different type of soft tissue sarcomas, focusing our attention on malignant fibrous histiocytomas (MFH), which were found to undergo spontaneous growth behaviour-conversion in vitro, loosing progressively their anchorage-dependence without incurring into apoptotic processes. From each of two of this type of sarcoma three sub-lines different for morphological criteria were obtained: we defined large and big spindle cells as “sub-line A”, smaller and less adherent fibroblastic cells as “sub-line B” and cells growing in suspension as “sub-line C”. The malignancy of these cells, evaluated by a colony forming unit assay, was higher for sub-lines B and C than A in both cell lines. In order to verify whether these sarcoma cells could represent an useful model to study three different steps in the process of metastasis, we looked for their binding activity to extracellular matrix (ECM) molecules, capacity to invade and to produce proteolytic enzymes. As regards ECM static and dynamic interactions, we found that the three sub-lines differed for the ability to adhere to a variety of ECM substrates and for migratory/invasive behaviour but these differences were not the same for the two MFHs. The distinct behaviour of the sub-lines was also observed for the production of degradative enzymes. Zymographic analyses were performed for the production of the metalloproteinases -2 and –9 (MMP-2 and MMP-9), and of the urokinase-type activator of plasminogen (uPA), which are highly correlated with malignancy in carcinomas. The sub-lines of the two sarcomas displayed a production pattern different either under normal culture conditions or after activation; it seemed that the induced production of MMP-9 and the increase in uPA secretion were correlated with a more aggressive phenotype. Extending our study on other proteolytic enzymes with a RT-PCR approach, we observed that the MMP expression of MFH sub-lines was identical for MT1-MMP, MMP-3 and MMP-13 but completely different for MMP-7 and MMP-11. In conclusion, our results suggest that interactions with ECM and MMP expression and production could be consistent “markers” to distinguish apparently similar sarcomas in different categories of biological aggressiveness/malignancy.