C. S. Digesu1, K. J. Hachey1, R. Liu1, C. P. Raut1, M. W. Grinstaff2, Y. L. Colson1 1Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA 2Boston University,Department Of Biomedical Engineering,Boston, MA, USA
Introduction: Soft tissue sarcoma is primarily treated with surgical resection, yet despite macroscopically complete resection, locoregional recurrence (LRR) remains high. Traditional radiation and systemic chemotherapy have yielded relatively little benefit. However, our group previously demonstrated that local delivery of paclitaxel (Pax) via drug-eluting polymer films drastically reduces LRR and improves survival in murine models. We investigate the mechanisms of sustained delivery of high concentration Pax leading to these improved outcomes in murine models of human sarcoma.
Methods: To simulate in vivo drug levels obtained via systemic therapy or Pax-eluting polymer films, CS-1 cells derived from human chondrosarcoma, were treated with 10nM or 1000nM Pax via 4hr pulse dose (and subsequent culture in media alone for 26 hours) or 30hr sustained dose. Cells were harvested and analyzed for cell cycle, incorporation of pulse BrdU, and expression of the p53/p21 pathway. To confirm results, Nu/J mice were inoculated with CS-1. Tumors were sliced at 250um intervals using a vibratome and ex vivo tissue slices were cultured at 37o in the above Pax treatment groups. Immunohistochemistry (IHC) was carried out for expression of caspase-3, p21, and incorporation of BrdU.
Results: High concentration 1000nM Pax resulted in suspension of cells in G2/M of the cell cycle with both the 4hr pulse and sustained 30hr treatment; however, only cells treated with 1000nM Pax for 30hrs demonstrated upregulation of p21. Upstream p53 was constitutively expressed. A significant proportion of p21+ cells co-localized to cells in G2/M or a post-mitotic (4N) state (6.4% in control vs. 32.4% 1000nM for 30hrs, Fig 1). Incorporation of BrdU was similar between controls and 10nM Pax, whereas <10% of cells incorporated BrdU with 1000nM 30hr treatment. IHC on cultured ex vivo tissue slices revealed similar rates of caspase-3 and p21 expression among controls, 10nM, and 1000nM Pax at 4hrs; however there was an increase in p21 expression with 1000nM Pax at 30hrs (<10% for controls vs. 25%). A majority of p21+ cells were post-mitotic, multinucleated cells.
Conclusion: Paclitaxel-eluting polymer films locally deliver high-concentration doses of Pax not clinically feasible through traditional systemic chemotherapy. Our work demonstrates that sustained duration of drug exposure and concentration of drug are both required for the increased efficacy of these Pax-eluting polymer films likely via multifactorial mechanisms including upregulation of caspases as well as p21. The difference is striking and suggestive of a new mechanism of cell death secondary to local Pax delivery that could potentially lead to overall prolonged in vivo survival in sarcoma.
