REVERSAL BY HYALURONIDASE OF ADHESION-DEPENDENT MULTICELLULAR DRUG-RESISTANCE IN MAMMARY-CARCINOMA CELLS

Background: De novo or acquired resistance to chemotherapeutic drugs c ontinues to be one of the most important obstacles hindering the succe ssful treatment of cancer patients, Consequently, enhancing the effica cy of conventional chemotherapeutic drugs has become an important rese arch goal, Our previous studies using the mouse EMT-6 mammary carcinom a selected for resistance to various alkylating agents in vivo demonst rated that such acquired drug resistance may be manifested in vitro on ly in cells growing in a three-dimensional configuration but not in co nventional monolayer culture, We also found that this phenomenon, whic h we refer to as ''acquired multicellular resistance,'' is associated with an increase in intercellular adhesion or compaction of the alkyla ting agent-resistant cell lines grown as aggregates in three-dimension al culture. Purpose: The present study further investigates the impact of three-dimensional architecture on acquired multicellular drug resi stance and its influence on cell cycle kinetics, cell cycle arrest, an d cell survival, Methods: To test the hypothesis that an increase in t hree-dimensional compaction is related to the drug resistance properti es of the cells, ave did the following: 1) selected clones of the EMT- 6 cell line that spontaneously formed tightly or loosely adherent aggr egates and assessed their respective drug resistance properties in vit ro; 2) assayed tumorigenic potential of the tight and loose clones aft er exposure to defined concentrations of the activated form of cycloph osphamide, 4-hydroperoxycyclophosphamide (4-HC) in vitro; and 3) treat ed the tight clones with hyaluronidase, an agent capable of disrupting EMT-6 spheroids, and assayed what effect this treatment had on chemos ensitivity, We used fluorescence-activated cell sorter analysis to mon itor any potential alterations in cell cycle kinetics, Results: The in crease in compaction in three-dimensional culture was sufficient to co nfer resistance to 4-HC, This increase in intercellular adhesion was a lso associated with a lower proliferating fraction of tumor cells and with an almost completely diminished ability of the cells to arrest in the G(2)/M phase of the cell cycle after drug exposure, Furthermore, these changes were detectable only in three-dimensional culture, not i n conventional monolayer culture, In conventional monolayer culture, a ll cell types consistently showed a high level of proliferation and ar rested in G(2)/M after exposure to 4-HC. Moreover, hyaluronidase was a ble to disrupt intercellular adhesion and chemosensitize tumor cells b oth in vitro and in vivo in an ascites model, Conclusion: Earlier stud ies have demonstrated that hyaluronidase is able to sensitize tumor ce lls to various anticancer agents, Our studies now demonstrate that thi s sensitization can occur by a mechanism independent of increased drug penetration, This mechanism is likely to be related to the ''anti-adh esive'' effect of hyaluronidase, which overrides cell contact-dependen t growth inhibition, recruits cells into the cycling pool, and renders tumor cells more sensitive to cytotoxic agents that preferentially ki ll rapidly dividing cells, Implications: Other tumor-specific ''anti-a dhesives'' should be explored that can be effective chemosensitizers w hen used in combination with cell cycle-specific drugs for the treatme nt of small, solid tumors.