Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells.

De novo or acquired resistance to chemotherapeutic drugs continues to be one of the most important obstacles hindering the successful treatment of cancer patients. Consequently, enhancing the efficacy of conventional chemotherapeutic drugs has become an important research goal. Our previous studies using the mouse EMT-6 mammary carcinoma selected for resistance to various alkylating agents in vivo demonstrated that such acquired drug resistance may be manifested in vitro only in cells growing in a three-dimensional configuration but not in conventional monolayer culture. We also found that this phenomenon, which we refer to as "acquired multicellular resistance," is associated with an increase in intercellular adhesion or compaction of the alkylating agent-resistant cell lines grown as aggregates in three-dimensional culture. The present study further investigates the impact of three-dimensional architecture on acquired multicellular drug resistance and its influence on cell cycle kinetics, cell cycle arrest, and cell survival. To test the hypothesis that an increase in three-dimensional compaction is related to the drug resistance properties of the cells, we did the following: 1) selected clones of the EMT-6 cell line that spontaneously formed tightly or loosely adherent aggregates and assessed their respective drug resistance properties in vitro; 2) assayed tumorigenic potential of the tight and loose clones after exposure to defined concentrations of the activated form of cyclophosphamide, 4-hydroperoxycyclophosphamide (4-HC) in vitro; and 3) treated the tight clones with hyaluronidase, an agent capable of disrupting EMT-6 spheroids, and assayed what effect this treatment had on chemosensitivity. We used fluorescence-activated cell sorter analysis to monitor any potential alterations in cell cycle kinetics. The increase in compaction in three-dimensional culture was sufficient to confer resistance to 4-HC. This increase in intercellular adhesion was also associated with a lower proliferating fraction of tumor cells and with an almost completely diminished ability of the cells to arrest in the G2/M phase of the cell cycle after drug exposure. Furthermore, these changes were detectable only in three-dimensional culture, not in conventional monolayer culture. In conventional monolayer culture, all cell types consistently showed a high level of proliferation and arrested in G2/M after exposure to 4-HC. Moreover, hyaluronidase was able to disrupt intercellular adhesion and chemosensitize tumor cells both in vitro and in vivo in an ascites model. Earlier studies have demonstrated that hyaluronidase is able to sensitize tumor cells to various anticancer agents. Our studies now demonstrate that this sensitization can occur by a mechanism independent of increased drug penetration. This mechanism is likely to be related to the "anti-adhesive" effect of hyaluronidase, which overrides cell contact-dependent growth inhibition, recruits cells into the cycling pool, and renders tumor cells more sensitive to cytotoxic agents that preferentially kill rapidly dividing cells. Other tumor-specific "anti-adhesives" should be explored that can be effective chemosensitizers when used in combination with cell cycle-specific drugs for the treatment of small, solid tumors.