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Radiation Sensitization of Sarcoma by Trabectedin
E598
International Journal of Radiation Oncology Biology Physics
novel 3D flow-perfusion bioreactor system as an ex vivo model for NE cancer and for personalized therapeutic testing. Materials/Methods: Polydimethylsiloxane bioreactors were designed to house 3D extracellular matrix (ECM) made of bovine collagen I and Matrigel in various ratios to determine the optimal composition. TT (RFP/ Luc) cell line and freshly collected tumor cell suspensions originating from mouse or patients were suspended in the ECM. The bioreactors were connected to a peristaltic pump to circulate media through the NE cancer surrogates. Growth was determined by non-invasive fluorescence and bioluminescence imaging using the IVIS Lumina system. H&E stained histologic sections were prepared for morphologic analysis by a pathologist. Finally, the growth of the surrogates were assessed after 6gy radiation and after perfusion with potential drug candidate Thailandepsin-A (TDPA). Results: The optimal ECM composition for NE cancer cell growth was determined to be 50% collagen I and 50% Matrigel. Linear growth of cell line (RFP) surrogates were observed up to 14 days (RFP 7.1 fold increase at 14 days compared to 0 days). Similarly, imaging of the human NE tumor surrogates with near infrared dye showed an increased signal from days 3 to 9 (1.4 fold increase). Histologic sections showed epithelial clustering characteristic of NE cancers. Surrogates originating from subcutaneous xenograft were used for therapeutic testing. While the bioluminescence signal of surrogate perfused with DMSO control increased 2.2 fold in 2 days, perfusion with 25nM TDP-A lead to 1.2 fold increase and 6gy radiation showed no growth after 2 days. Conclusion: We demonstrated that the 3D surrogates system can be used to simulate physiologically relevant stromal context for NE cancer cells and grow patient derived tumors for therapeutic testing. The surrogates were compatible with standard histological techniques. Further testing should be done with additional patient tumors, various radiation dose regimens, FDA approved drugs and drug candidates to test cytotoxic and anti-proliferative effects. Author Disclosure: S. Jang: Graduate fellow; Howard Hughes Medical Institute. K. Goliwas: Trainee; University of Alabama at Birmingham. J. Berry: None. E.S. Yang: Research Grant; Aacr, Susan G Komen Foundation, Eli Lilly. Advisory Board; Bayer Pharmaceutical. Z. Abujania: None. H. Chen: University of Alabama at Birmingham. A. Frost: None. R. Jaskula-Sztul: None.
24 hours with 300pg/mL trabectedin, followed by 6 Gy, then fixed after 48 hours. Statistical significance was calculated using the student’s t-test. Results: A dose titration was first performed and the optimal dose of trabectedin for clonogenic survival assays was found to be 100pg/mL. Clonogenic assay of sarcoma cells demonstrated that trabectedin sensitized SK-LMS-1 cells to radiation (DEFZ1.53), but did not sensitize SW872 cells. Cell cycle analysis demonstrated an increase in S phase and G2/M phase cell cycle distribution in the SK-LMS-1 cells (p<0.01), however, the cell cycle distribution was not affected in SW872 cells up to a concentration of 2.4ng/mL for over 48 hours. There was also a further increase in G2/M phase cell cycle distribution after 6 Gy which did not occur in the SW872 cells (p<0.05). There was an increase in clonogenic cell death by mitotic catastrophe in SK-LMS-1 cells treated with trabectedin and radiation (p<0.001), which was not replicated in the SW872 cells. Conclusion: Trabectedin effectively sensitized the leiomyosarcoma cell line SK-LMS-1 to radiation, however, it did not sensitize the liposarcoma cell line SW872. This may have been a result of synchronization of SKLMS-1 cells in the more radiosensitive G2/M phase of the cell cycle, which led to an increase in clonogenic cell death by mitotic catastrophe. Efforts are underway to more fully characterize this mechanism of radiation sensitization as well as to determine the mechanism of resistance to further improve this therapeutic strategy. Author Disclosure: J. Jarboe: None. S. Zhang: None. A. Paz Mejia: None. R. Yechieli: None. B. Marples: Honoraria; ASTRO. Travel Expenses; ASTRO. J. Trent: Lead the sarcoma research program; University of Miami.
3423 Radiation Sensitization of Sarcoma by Trabectedin J. Jarboe,1 S. Zhang,2 A. Paz Mejia,2 R. Yechieli,3 B. Marples,4 and J. Trent2; 1Jackson Memorial Hospital/Jackson Health System, Miami, FL, 2University of Miami, Miami, FL, 3Department of Radiation Oncology, University of Miami / Sylvester Comprehensive Cancer Center, Miami, FL, 4Beaumont Health, Royal Oak, MI Purpose/Objective(s): The natural marine product trabectedin is FDA approved for the treatment of advanced soft tissue sarcoma and for relapsed ovarian cancer. Recently, our center and others have begun treating patients with un-resectable sarcoma concurrently with trabectedin and radiation in the hopes of improving cure rates. There are, however, limited pre-clinical data on trabectedin’s effect on radiation sensitivity. Those that do exist suggest that trabectedin can synchronize cells in the G2/M phase of the cell cycle and can also sensitize cells to ionizing radiation. We thus aimed to study the effect of trabectedin on the radiation sensitivity of sarcoma cell lines. We hypothesized that trabectedin would sensitize sarcoma cells to radiation by synchronizing cells in the G2/M phase. Materials/Methods: The liposarcoma cell line SW872 and the leiomyosarcoma cell line SK-LMS-1 were used for these studies. A clonogenic assay was used to determine radiation sensitization by trabectedin using standard protocols. Cells were treated with 100pg/mL trabectedin for 24 hours followed by 2, 4 or 6 Gy. Cell cycle analysis was conducted by propidium iodide staining flow cytometry. Cells were treated for 24 hours with 300pg/mL trabectedin, followed by 6 Gy, and then harvested 24 hours later. Clonogenic cell death by mitotic catastrophe was assessed by immunofluorescence quantification of DAPI stained nuclei. Cells were treated for
3424 Combination Radiation Therapy and Imipridone ONC201 for the Treatment of Solid Tumors S.R. Jhawar,1 S. Goyal,2 A. Thandoni,1 H. Wu,1 S. Hassan,3 D.S. Schiff,1 J. Allen,4 M. Stogniew,4 R. Tarapore,4 M. Stein,1 J. Bertino,1 B.G. Haffty Jr,1 and A. Zloza1,5; 1Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 2Rutgers Cancer Institute of New Jersey Department of Radiation Oncology, New Brunswick, NJ, 3Rutgers University, New Brunswick, NJ, 4Oncoceutics, Inc., Philadelphia, PA, 5Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ Purpose/Objective(s): Imipridones (IMIPs) represent a new class of anticancer agents that target specific G protein-coupled receptors (GPCRs) controlling critical cancer cell signaling pathways. We used a first-in-class IMIP that works via activation of ER stress pathways, upregulation of TRAIL, and activation of death receptors 4 and 5 (DR4/DR5), leading to tumor cell death. The DR5 pathway is also required for radiation (RT)induced apoptosis. We, thus, hypothesized that the use of an imipridone could increase the effectiveness of RT. Materials/Methods: Human cancer cells (2-5x103 per well) were treated in 6-8 replicates with radiation, IMIP, or the combination. Eight melanoma and 7 breast cancer cell lines were utilized. Radiation (0-8 Gy) was delivered via a Gammacell 40 exactor. IMIP was given at varying does 0-10 mM. Delivery was varied with either IMIP first, radiation first, or concurrent administration. AlamarBlue cell viability assay was performed 48-120 hours after initial treatment. Cell lines found to respond to either therapy were used in clonogenic assays. Mechanisms were elucidated using western blots of cleaved PARP (cPARP) and cleaved caspase 3 (cC3). In vivostudies were conducted utilizing mouse models of melanoma. Results: Multiple lines were found to exhibit decreased cell viability after combination therapy, particularly when IMIP was given 12 h prior to RT. In particular, IMIP (1 mM) followed by RT (8 Gy) resulted in a significant decrease in MB468 breast cancer cell viability compared to no treatment (36.9%, P<0.0001), RT alone (30.5%, p<0.0001) and IMIP alone (26.9%, p<0.0001). No significant decrease was achieved with either treatment alone compared to no treatment. A clonogenic assay using MB468 revealed a decreased surviving fraction at every RT dose (2, 4, 6, and 8 Gy). Western blot analysis revealed increasing cPARP with increasing IMIP at 2 Gy and increasing cC3 with increasing doses of IMIP at 4 and 8 Gy. In vivostudies utilizing B16 melanoma demonstrated that combination RT (6 Gy) and
International Journal of Radiation Oncology Biology Physics
novel 3D flow-perfusion bioreactor system as an ex vivo model for NE cancer and for personalized therapeutic testing. Materials/Methods: Polydimethylsiloxane bioreactors were designed to house 3D extracellular matrix (ECM) made of bovine collagen I and Matrigel in various ratios to determine the optimal composition. TT (RFP/ Luc) cell line and freshly collected tumor cell suspensions originating from mouse or patients were suspended in the ECM. The bioreactors were connected to a peristaltic pump to circulate media through the NE cancer surrogates. Growth was determined by non-invasive fluorescence and bioluminescence imaging using the IVIS Lumina system. H&E stained histologic sections were prepared for morphologic analysis by a pathologist. Finally, the growth of the surrogates were assessed after 6gy radiation and after perfusion with potential drug candidate Thailandepsin-A (TDPA). Results: The optimal ECM composition for NE cancer cell growth was determined to be 50% collagen I and 50% Matrigel. Linear growth of cell line (RFP) surrogates were observed up to 14 days (RFP 7.1 fold increase at 14 days compared to 0 days). Similarly, imaging of the human NE tumor surrogates with near infrared dye showed an increased signal from days 3 to 9 (1.4 fold increase). Histologic sections showed epithelial clustering characteristic of NE cancers. Surrogates originating from subcutaneous xenograft were used for therapeutic testing. While the bioluminescence signal of surrogate perfused with DMSO control increased 2.2 fold in 2 days, perfusion with 25nM TDP-A lead to 1.2 fold increase and 6gy radiation showed no growth after 2 days. Conclusion: We demonstrated that the 3D surrogates system can be used to simulate physiologically relevant stromal context for NE cancer cells and grow patient derived tumors for therapeutic testing. The surrogates were compatible with standard histological techniques. Further testing should be done with additional patient tumors, various radiation dose regimens, FDA approved drugs and drug candidates to test cytotoxic and anti-proliferative effects. Author Disclosure: S. Jang: Graduate fellow; Howard Hughes Medical Institute. K. Goliwas: Trainee; University of Alabama at Birmingham. J. Berry: None. E.S. Yang: Research Grant; Aacr, Susan G Komen Foundation, Eli Lilly. Advisory Board; Bayer Pharmaceutical. Z. Abujania: None. H. Chen: University of Alabama at Birmingham. A. Frost: None. R. Jaskula-Sztul: None.
24 hours with 300pg/mL trabectedin, followed by 6 Gy, then fixed after 48 hours. Statistical significance was calculated using the student’s t-test. Results: A dose titration was first performed and the optimal dose of trabectedin for clonogenic survival assays was found to be 100pg/mL. Clonogenic assay of sarcoma cells demonstrated that trabectedin sensitized SK-LMS-1 cells to radiation (DEFZ1.53), but did not sensitize SW872 cells. Cell cycle analysis demonstrated an increase in S phase and G2/M phase cell cycle distribution in the SK-LMS-1 cells (p<0.01), however, the cell cycle distribution was not affected in SW872 cells up to a concentration of 2.4ng/mL for over 48 hours. There was also a further increase in G2/M phase cell cycle distribution after 6 Gy which did not occur in the SW872 cells (p<0.05). There was an increase in clonogenic cell death by mitotic catastrophe in SK-LMS-1 cells treated with trabectedin and radiation (p<0.001), which was not replicated in the SW872 cells. Conclusion: Trabectedin effectively sensitized the leiomyosarcoma cell line SK-LMS-1 to radiation, however, it did not sensitize the liposarcoma cell line SW872. This may have been a result of synchronization of SKLMS-1 cells in the more radiosensitive G2/M phase of the cell cycle, which led to an increase in clonogenic cell death by mitotic catastrophe. Efforts are underway to more fully characterize this mechanism of radiation sensitization as well as to determine the mechanism of resistance to further improve this therapeutic strategy. Author Disclosure: J. Jarboe: None. S. Zhang: None. A. Paz Mejia: None. R. Yechieli: None. B. Marples: Honoraria; ASTRO. Travel Expenses; ASTRO. J. Trent: Lead the sarcoma research program; University of Miami.
3423 Radiation Sensitization of Sarcoma by Trabectedin J. Jarboe,1 S. Zhang,2 A. Paz Mejia,2 R. Yechieli,3 B. Marples,4 and J. Trent2; 1Jackson Memorial Hospital/Jackson Health System, Miami, FL, 2University of Miami, Miami, FL, 3Department of Radiation Oncology, University of Miami / Sylvester Comprehensive Cancer Center, Miami, FL, 4Beaumont Health, Royal Oak, MI Purpose/Objective(s): The natural marine product trabectedin is FDA approved for the treatment of advanced soft tissue sarcoma and for relapsed ovarian cancer. Recently, our center and others have begun treating patients with un-resectable sarcoma concurrently with trabectedin and radiation in the hopes of improving cure rates. There are, however, limited pre-clinical data on trabectedin’s effect on radiation sensitivity. Those that do exist suggest that trabectedin can synchronize cells in the G2/M phase of the cell cycle and can also sensitize cells to ionizing radiation. We thus aimed to study the effect of trabectedin on the radiation sensitivity of sarcoma cell lines. We hypothesized that trabectedin would sensitize sarcoma cells to radiation by synchronizing cells in the G2/M phase. Materials/Methods: The liposarcoma cell line SW872 and the leiomyosarcoma cell line SK-LMS-1 were used for these studies. A clonogenic assay was used to determine radiation sensitization by trabectedin using standard protocols. Cells were treated with 100pg/mL trabectedin for 24 hours followed by 2, 4 or 6 Gy. Cell cycle analysis was conducted by propidium iodide staining flow cytometry. Cells were treated for 24 hours with 300pg/mL trabectedin, followed by 6 Gy, and then harvested 24 hours later. Clonogenic cell death by mitotic catastrophe was assessed by immunofluorescence quantification of DAPI stained nuclei. Cells were treated for
3424 Combination Radiation Therapy and Imipridone ONC201 for the Treatment of Solid Tumors S.R. Jhawar,1 S. Goyal,2 A. Thandoni,1 H. Wu,1 S. Hassan,3 D.S. Schiff,1 J. Allen,4 M. Stogniew,4 R. Tarapore,4 M. Stein,1 J. Bertino,1 B.G. Haffty Jr,1 and A. Zloza1,5; 1Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 2Rutgers Cancer Institute of New Jersey Department of Radiation Oncology, New Brunswick, NJ, 3Rutgers University, New Brunswick, NJ, 4Oncoceutics, Inc., Philadelphia, PA, 5Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ Purpose/Objective(s): Imipridones (IMIPs) represent a new class of anticancer agents that target specific G protein-coupled receptors (GPCRs) controlling critical cancer cell signaling pathways. We used a first-in-class IMIP that works via activation of ER stress pathways, upregulation of TRAIL, and activation of death receptors 4 and 5 (DR4/DR5), leading to tumor cell death. The DR5 pathway is also required for radiation (RT)induced apoptosis. We, thus, hypothesized that the use of an imipridone could increase the effectiveness of RT. Materials/Methods: Human cancer cells (2-5x103 per well) were treated in 6-8 replicates with radiation, IMIP, or the combination. Eight melanoma and 7 breast cancer cell lines were utilized. Radiation (0-8 Gy) was delivered via a Gammacell 40 exactor. IMIP was given at varying does 0-10 mM. Delivery was varied with either IMIP first, radiation first, or concurrent administration. AlamarBlue cell viability assay was performed 48-120 hours after initial treatment. Cell lines found to respond to either therapy were used in clonogenic assays. Mechanisms were elucidated using western blots of cleaved PARP (cPARP) and cleaved caspase 3 (cC3). In vivostudies were conducted utilizing mouse models of melanoma. Results: Multiple lines were found to exhibit decreased cell viability after combination therapy, particularly when IMIP was given 12 h prior to RT. In particular, IMIP (1 mM) followed by RT (8 Gy) resulted in a significant decrease in MB468 breast cancer cell viability compared to no treatment (36.9%, P<0.0001), RT alone (30.5%, p<0.0001) and IMIP alone (26.9%, p<0.0001). No significant decrease was achieved with either treatment alone compared to no treatment. A clonogenic assay using MB468 revealed a decreased surviving fraction at every RT dose (2, 4, 6, and 8 Gy). Western blot analysis revealed increasing cPARP with increasing IMIP at 2 Gy and increasing cC3 with increasing doses of IMIP at 4 and 8 Gy. In vivostudies utilizing B16 melanoma demonstrated that combination RT (6 Gy) and