- Email: [email protected]
Noninvasive Bioluminescence Imaging of Caspase-3 Activity in Breast Cancer
Proceedings of the 51st Annual ASTRO Meeting Conclusions: Our results demonstrate the feasibility of using off-line molecular optical imaging to complement the SARRP for focal irradiation of molecular targets. This early experience quickly points to the potential of integrating optical-CBCT imaging on-board the SARRP to greatly enhance optical CT reconstruction and facilitate exciting mechanistic studies of radiation response in an animal model. Author Disclosure: J.W. Wong, Technology Transfer Agreement with Gulmay Medical Inc, G. Other; D. Artemov, None; E. Tryggestad, None; P. Winnard, None.
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Noninvasive Bioluminescence Imaging of Caspase-3 Activity in Breast Cancer
C. C. Olsen, F. Li, H. Zhimin, W. Li, C. Li University of Colorado Health Science Center, Denver, CO Purpose/Objective(s): Apoptosis is a major form of tumor cells death during cytotoxic therapy. Understanding the kinetics of apoptosis would greatly facilitate our understanding of this process in cancer treatment and development of more effective therapeutic approaches. In order to monitor apoptosis activities in vivo, we developed a novel bioluminescence-based reporter gene to detect caspase-3 activities, which are elevated at the execution phase of apoptosis. We conducted experiments to validate our reporter both in tissue-cultured cells and in murine tumor models. Materials/Methods: A caspase-3 reporter system was constructed by combining two different reporter proteins; green fluorescent protein (GFP) and firefly luciferase (FL) linked through multiple polyubiquitin domains with a caspase-3 recognition site. Under normal circumstances, the reporter proteins are rapidly degraded by the proteasome system. During apoptosis, activated caspase-3 cleaves off the multi-ubiquitin domain from the reporter protein. This enables the GFP and luciferase fusion reporter to be stabilized and achieve a significant gain in GFP protein and luciferase activities, which in turn could be monitored both in vitro and in vivo. The 4T1 cells transduced with CMV-luc or Caspase-3 reporter xenografts were treated with both chemotherapy and radiation therapy and monitored for apoptosis activity. Results: In vitro experiments demonstrated increased luciferase with increasing radiation dose reflective of apoptosis with background levels nearly undetectable. Taxol was associated with a time-dependent increase from 24 to 72 hours after drug exposure, indicating that apoptosis is a gradual, heterogeneous process. The EGFP signal increased from 1.85% in controls to 80.6% in cells treated with 1 uM Taxol. Xenografts showed nearly undetectable luciferase background with Cytoxan therapy resulting in a 90-fold increase, 10 Gy a 24-fold increase and fractionated RT (5 Gy 3) with a 46-fold increase. Conclusions: We developed a novel in vivo caspase reporter based on the ubiquitous proteosome system of protein degradation and bioluminescence imaging. This allowed us to assess activation of apoptosis in response to chemoradiation therapy in tissue culture and breast cancer xenografts over the course of 2–3 weeks, which has not been possible with other technologies. Author Disclosure: C.C. Olsen, None; F. Li, None; H. Zhimin, None; W. Li, None; C. Li, None.
91
MRI Assessment of Angiogenesis Inhibitor Sunitinib’s Influence on Tumor Oxygenation to Identify an Optimal Chemoradiotherapeutic Window
S. Batra1,2, S. Matsumoto3, F. Hyodo2, J. Mitchell2, M. C. Krishna2 Stanford University, Palo Alto, CA, 2National Institutes of Health, Bethesda, MD, 3National Institutes of Health, Bethesda, CA
1
Purpose/Objective(s): Radiobiologically hypoxic tumors (pO2 \10 mm Hg) associate with poor outcomes. Antiangiogenic therapies deprive proliferating cells of their blood supply and inhibit growth. Reports claim, however, that early after treatment antiangiogenic therapy induces transient ‘normalization’ of tumor vasculature making it more efficient at oxygen and drug delivery. We recently developed a novel noninvasive technique to image tissue oxygen based on electron paramagnetic resonance (EPR) imaging. The objective of this study was to use our technique to quantitate oxygen changes in murine squamous cell tumors after antiangiogenic therapy and to determine if a window to maximize combined radiotherapy exists. The agent used in this study was sunitinib, a small molecule that inhibits tyrosine kinase receptors including PDGF-R and VEGF-R. Materials/Methods: The C3H mice were subcutaneously inoculated with SCCVII murine tumor cells and treated with sunitinib (50 mg/kg) or methylcellulose/Tween80 as control by oral gavage 6 days after tumor implantation. The EPR imaging system which monitors oxygen dependent spectral broadening of an injectable paramagnetic tracer, Ox-63, was used to map pO2 levels noninvasively. Data acquisition time for 3D oxygen mapping was under 10 minutes. Immediately after oxygen imaging, mice were transferred to a 7T MRI for T2-weighted anatomic and blood volume imaging. Blood volume calculations were based on the T2* shortening effects and signal loss by USPIO injection. The EPR/MRI measurements were performed every 2 days before and during treatment. Results: A growth delay of 2 days was observed in mice treated with 4 days of sunitinib starting Day 6 after implantation while a growth delay of 1 day seen in tumors treated with 10 Gy radiation at 10 days postimplantation. A combination of these two treatments yielded a growth delay of 8 days, reflecting marked synergy between the treatment modalities. Median tumor pO2 decreased with increase in tumor size in the control group. The pO2 of the sunitinib group was 4-5 mm Hg higher at Day 4, though results from blood volume imaging by MRI and CD31 staining showed a 30% and 70% reduction in microvascular density at Day 4 and 6, respectively. No significant difference in tumor pO2 between the two groups at Day 6 was observed. Conclusions: By noninvasively and longitudinally monitoring tumor oxygen status during antiangiogenic therapy, we revealed underlying mechanisms of synergistic effects of combined antiangiogenic therapy and radiotherapy. Our preliminary results suggest that the antiangiogenic drug sunitinib transiently increases tumor pO2 with vascular normalization as a possible cause, conferring tumor radiosensitization. Author Disclosure: S. Batra, None; S. Matsumoto, None; F. Hyodo, None; J. Mitchell, None; M.C. Krishna, None.
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Noninvasive Bioluminescence Imaging of Caspase-3 Activity in Breast Cancer
C. C. Olsen, F. Li, H. Zhimin, W. Li, C. Li University of Colorado Health Science Center, Denver, CO Purpose/Objective(s): Apoptosis is a major form of tumor cells death during cytotoxic therapy. Understanding the kinetics of apoptosis would greatly facilitate our understanding of this process in cancer treatment and development of more effective therapeutic approaches. In order to monitor apoptosis activities in vivo, we developed a novel bioluminescence-based reporter gene to detect caspase-3 activities, which are elevated at the execution phase of apoptosis. We conducted experiments to validate our reporter both in tissue-cultured cells and in murine tumor models. Materials/Methods: A caspase-3 reporter system was constructed by combining two different reporter proteins; green fluorescent protein (GFP) and firefly luciferase (FL) linked through multiple polyubiquitin domains with a caspase-3 recognition site. Under normal circumstances, the reporter proteins are rapidly degraded by the proteasome system. During apoptosis, activated caspase-3 cleaves off the multi-ubiquitin domain from the reporter protein. This enables the GFP and luciferase fusion reporter to be stabilized and achieve a significant gain in GFP protein and luciferase activities, which in turn could be monitored both in vitro and in vivo. The 4T1 cells transduced with CMV-luc or Caspase-3 reporter xenografts were treated with both chemotherapy and radiation therapy and monitored for apoptosis activity. Results: In vitro experiments demonstrated increased luciferase with increasing radiation dose reflective of apoptosis with background levels nearly undetectable. Taxol was associated with a time-dependent increase from 24 to 72 hours after drug exposure, indicating that apoptosis is a gradual, heterogeneous process. The EGFP signal increased from 1.85% in controls to 80.6% in cells treated with 1 uM Taxol. Xenografts showed nearly undetectable luciferase background with Cytoxan therapy resulting in a 90-fold increase, 10 Gy a 24-fold increase and fractionated RT (5 Gy 3) with a 46-fold increase. Conclusions: We developed a novel in vivo caspase reporter based on the ubiquitous proteosome system of protein degradation and bioluminescence imaging. This allowed us to assess activation of apoptosis in response to chemoradiation therapy in tissue culture and breast cancer xenografts over the course of 2–3 weeks, which has not been possible with other technologies. Author Disclosure: C.C. Olsen, None; F. Li, None; H. Zhimin, None; W. Li, None; C. Li, None.
91
MRI Assessment of Angiogenesis Inhibitor Sunitinib’s Influence on Tumor Oxygenation to Identify an Optimal Chemoradiotherapeutic Window
S. Batra1,2, S. Matsumoto3, F. Hyodo2, J. Mitchell2, M. C. Krishna2 Stanford University, Palo Alto, CA, 2National Institutes of Health, Bethesda, MD, 3National Institutes of Health, Bethesda, CA
1
Purpose/Objective(s): Radiobiologically hypoxic tumors (pO2 \10 mm Hg) associate with poor outcomes. Antiangiogenic therapies deprive proliferating cells of their blood supply and inhibit growth. Reports claim, however, that early after treatment antiangiogenic therapy induces transient ‘normalization’ of tumor vasculature making it more efficient at oxygen and drug delivery. We recently developed a novel noninvasive technique to image tissue oxygen based on electron paramagnetic resonance (EPR) imaging. The objective of this study was to use our technique to quantitate oxygen changes in murine squamous cell tumors after antiangiogenic therapy and to determine if a window to maximize combined radiotherapy exists. The agent used in this study was sunitinib, a small molecule that inhibits tyrosine kinase receptors including PDGF-R and VEGF-R. Materials/Methods: The C3H mice were subcutaneously inoculated with SCCVII murine tumor cells and treated with sunitinib (50 mg/kg) or methylcellulose/Tween80 as control by oral gavage 6 days after tumor implantation. The EPR imaging system which monitors oxygen dependent spectral broadening of an injectable paramagnetic tracer, Ox-63, was used to map pO2 levels noninvasively. Data acquisition time for 3D oxygen mapping was under 10 minutes. Immediately after oxygen imaging, mice were transferred to a 7T MRI for T2-weighted anatomic and blood volume imaging. Blood volume calculations were based on the T2* shortening effects and signal loss by USPIO injection. The EPR/MRI measurements were performed every 2 days before and during treatment. Results: A growth delay of 2 days was observed in mice treated with 4 days of sunitinib starting Day 6 after implantation while a growth delay of 1 day seen in tumors treated with 10 Gy radiation at 10 days postimplantation. A combination of these two treatments yielded a growth delay of 8 days, reflecting marked synergy between the treatment modalities. Median tumor pO2 decreased with increase in tumor size in the control group. The pO2 of the sunitinib group was 4-5 mm Hg higher at Day 4, though results from blood volume imaging by MRI and CD31 staining showed a 30% and 70% reduction in microvascular density at Day 4 and 6, respectively. No significant difference in tumor pO2 between the two groups at Day 6 was observed. Conclusions: By noninvasively and longitudinally monitoring tumor oxygen status during antiangiogenic therapy, we revealed underlying mechanisms of synergistic effects of combined antiangiogenic therapy and radiotherapy. Our preliminary results suggest that the antiangiogenic drug sunitinib transiently increases tumor pO2 with vascular normalization as a possible cause, conferring tumor radiosensitization. Author Disclosure: S. Batra, None; S. Matsumoto, None; F. Hyodo, None; J. Mitchell, None; M.C. Krishna, None.
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