K. J. Hachey1, R. Liu1, M. Grinstaff2, Y. Colson1 1Brigham And Women’s Hospital,Thoracic Surgery,Boston, MA, USA 2Boston University,Departments Of Biomedical Engineering And Chemistry,Boston, MA, USA
Introduction: Curative resection for the treatment of sarcoma is a significant challenge due to tumor size and anatomic complexity, resulting in 5-year locoregional recurrence (LRR) rates of over 50% and relative resistance to systemic chemotherapy and radiation. We previously demonstrated that local treatment with paclitaxel (Pax)-loaded polymer films after partial tumor resection in a murine xenograft model of human chondrosarcoma resulted in improved LRR and survival compared to adjuvant systemic Pax. This study aims to characterize mechanisms of cytotoxicity induced by Pax films that may explain improved outcomes with local drug delivery.
Methods: Pax drug effect was assessed in the human chondrosarcoma cell line CS-1 at 1000nM and 10nM Pax, which approximate in vivo concentrations previously measured in local tissues one day after treatment with Pax films and systemic Pax, respectively. In vitro incubation with Pax for 30hr continuous or 4hr pulse dose (with additional 26hr incubation in fresh media) was perfomed to evaluate response to Pax as a function of dose and duration. CS-1 monolayers treated with these regimens were harvested at 30hrs for cell cycle analysis or inoculation into Nu/J mice (n=7/group) to assess differences in tumor volume and disease-free survival (DFS).
Results: Treatment with 1000nM pax for both 4hr pulse dose and 30hr continuous incubation in vitro induced a predominant G2/M peak on flow cytometric analysis by 30hrs, with a decline in p53 expression below control levels. In contrast, cell cycle profiles and p53 levels in the 10nM group remained similar to controls even after 30hrs. When treated monolayers were harvested at 30hrs and injected in vivo, treatment with 1000nM Pax resulted in improved DFS with both treatment durations (fig). The 4hr and 30hr 1000nM Pax-treated groups showed statistically significant reductions in measured tumor volume at 28 days post-treatment compared to 10nM groups treated for the equivalent duration (p=0.01 and p=0.007, respectively).
Conclusion: CS-1 monolayers exposed to 1000nM Pax, as demonstrated during Pax film treatment in vivo, undergo a G2/M arrest that correlates with significantly reduced in vivo tumor growth and improved DFS. In contrast, monolayers treated with 10nM Pax, as demonstrated following systemic Pax in vivo, appear relatively resistant to G2/M accumulation, or ‘escape’ from mitotic arrest, resulting in inferior DFS. Cell death during a p53-independent G2/M arrest, with microtubule damage and mitochondrial collapse, is a hallmark of cytotoxicity at high Pax concentrations but both dose and duration appear important for improved efficacy as achieved by polymer films in vivo compared to equivalent bolus dose Pax given systemically.
