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315 TUMOR MICROENVIRONMENTAL REACTIONS INFLUENCING RESPONSE TO RADIOTHERAPY
S167 probability of a given side effect (NTCP-value). One of the output parameters of this step are the most relevant Dose Volume Histogram (DVH) parameters that can be used to optimize radiation treatment. Step 2: In silico planning comparative studies. In this phase protons are compared with photons with regard to their ability to reduce the most relevant DVHparameters resulting from step 1. Step 3: Integration step 1 and 2. By integrating the results of the individual in silico planning comparison into the validated NTCP-models, the differences in dose can be translated into a difference in NTCP-value in each individual patient. This 3 step-methodology will be illustrated with some clinical examples. Finally, the potential benefits of protons should be clinically validated in step 4. 314 GENERATION OF A MULTI-MODALITY ANATOMICAL ATLAS OF PRECLINICAL ANIMAL MODELS R. Hentkowski, D. McGrath, W. Foltz, N. Samavati, P. Lindsay, L. Dawson, K. Brock Princess Margaret Hospital, Toronto Purpose: The use of preclinical mice and rat models for studying radiation therapy is increasing, as is their complexity, in part due to the need for better understanding of mechanism of normal tissue and tumor response to hypofractionation and SBRT, as well as combinations of radiation therapy with novel targeted therapies. Pre-clinical image-guided radiation therapy systems designed for conformal, targeted treatments are becoming more widely available. Multimodality serial preclinical imaging studies can be used to study the response of tumors and normal tissues to therapies. Information to educate the investigator on normal, preclinical anatomy on multi-modality images is limited and often makes discerning small tumors or abnormal anatomy on images challenging. In addition, the availability of “reference” mouse and rat anatomy to enhance pre-treatment kV CBCT images available on many pre-clinical image-guided irradiators could be beneficial when quantification of normal tissue irradiation is desired. The purpose of this research is to develop a comprehensive, multi-modality image-based atlas of a male and female mouse and rat using highresolution full body axial MR and CT, combined with gross section imaging, and selected histological evaluation. The MR and CT images enabled the generation of an image-based anatomical atlas. The histology provides a cellular-based correspondence to the high-resolution imaging. Methods and Materials: Two healthy, mature mice (1 female, 1 male) and two healthy, mature rats (1 female, 1 male) were sacrificed under an approved animal care protocol. All animals were then immediately imbedded in a gel composed of agarose and gelatin and solidified for immobilization prior to image acquisition. Each animal was imaged using a micro CT and 7T micro MR. The CT images were acquired at a 100 micron resolution. The mice MR imaging was performed using a B-GA12 gradient coil and 7.2 cm I.D. quadrature cylindrical volume resonator. A 3D spin-echo acquisition sequence was used with an echo time of 10 ms and a repetition time of 500 ms. The MR images were acquired with a 150 micron isotropic resolution, a
ICTR-PHE 2012 field of view (FOV) of 100 x 60 x 32 mm, with 2 averages, resulting in a scan time of 23 hours and 46 minutes. The rat MR imaging was performed using a BGA20S gradient coil and a 15.5 cm I.D. quadrature cylindrical RF resonator. A 3D spin-echo acquisition sequence was used with an echo time of 10 ms and a repetition time of 625 ms. The MR images were acquired with a 350 micron isotropic resolution, a FOV of 170 x 134.4 x 62.2 mm, with 2 averages, resulting in a scan time of 22 hours and 56 minutes. Following imaging, the animals were frozen at -20 degrees Celsius and sliced at 3 mm using a standard meat slicer available in the correlative pathology laboratory. Selected slices from the brain, lungs, abdomen, and pelvis were further sliced to 4 microns and processed for histological analysis with standard hematoxylin and eosin staining. The histology provided a cellular-based atlas corresponding to the high-resolution images. Following image reconstruction, the MR and CT images were imported into a radiation therapy treatment planning system. Registration and fusion was performed between the MR and CT for each image set and delineation of the normal tissues was performed. The histological stains were rigidly registered to the corresponding anatomical slice in the MR image using an in-house algorithm developed for correlative pathology. Relative 2D deformation between the MR slice and the histology was corrected using a 2D pointbased thin-plate spline deformable registration. Results: Rigid registration between the animal body MR and CT scans was performed with no detectable residual error as the gel immobilization provided a consistent anatomical positioning between both imaging sessions. Boney anatomy was automatically segmented from the CT image using a thresholding technique on all four specimens (male and female mouse and rat). Soft tissue, not well visualized on the CT image, was easily identified on the MR images. All tissues, including brain, lungs, heart, liver, stomach, bowels, and male and female pelvic anatomy was segmented in each MR image. Segmentation and validation of the histology slices is ongoing. In addition to providing a reference dataset for education purposes, a feasibility study investigating how such a reference dataset may aid in liver animal irradiation experiments was performed. Reference contoured MR images of the female mouse were registering to a kV CBCT image obtained on a liver mouse using the preclinical irradiation system to provide enhanced anatomical image guidance and normal tissue delineations capabilities for a conformal liver radiation therapy plan. Conclusions: Multi-modality high resolution reference anatomical datasets were generated for a female and male mouse and rat for purposes of education and anatomical reference. 315 TUMOR MICROENVIRONMENTAL REACTIONS INFLUENCING RESPONSE TO RADIOTHERAPY C. Ruegg University of Fribourg Purpose: About half of cancer patients receive radiotherapy during the course of their disease, either as curative-intent or palliative treatments. While in some cases disease control is satisfactory, in others is
S168 not and cancers further progress. Others and we have observed that experimental tumor growing in preirradiated stroma are locally more invasive and metastatic pointing to a critical role of the irradiated tumor microenvironment in promoting invasion and metastasis. The purpose of our studies was to identify stomal events promoting the increased aggressiveness of tumors relapsing after radiotherapy and to unravel the underlying mechanisms. Material and Methods: To address the question, we used an in vivo model of local irradiation in mice and tumor implantation, in vitro cell biology experiments, and pharmacological of genetic interventions. Results: We have observed that irradiation suppresses de novo angiogenesis and favors the appearance of tumor-associated hypoxia. This suppression is due, at least in part, to a TGFbeta dependent mechanism. Tumors growing within a previously irradiated and angiogenesis-deficient stroma have reduced growth while they display increased hypoxia, necrosis, local invasion and lung metastasis formation. Initial studies revealed that hypoxia selects for tumor variant expressing high levels of the matricellular protein CYR61. CYR61 promotes survival under hypoxia and also increase motility invasiveness and metastasis. Subsequently we have observed that CD11b+Gr1+ inflammatory cells are increasingly recruited at the periphery of tumors growing in preirradiated beds. Analysis of these cells revealed that they are mobilized from the bone marrow by tumor hypoxia in a HIFdependent manner. Mobilized CD11b+Gr1+ cells also home to premetastatic lungs. Cell transfer experiments revealed that CD11b+ circulating in the blood of tumorbearing preirradiated mice promote lung metastasis when transferred to tumor-bearign control (i.e. non preirradiated) mice. At last we have identified a factor induced by tumor hypoxia and its cognate receptor expressed on CD11b+ cells that mediated mobilization of metastasis-promoting CD11b+ cells. This ligandreceptor axis has not been previously associated with metastatic tumor spreading. Neutralization of this factor or its cognate receptor significantly suppressed tumor metastasis without impinging on primary tumor growth. Conclusions: Our studies demonstrating that irradiated tumor bed promotes tumor invasion and etastasis by at least two mechanisms: selection of CYR61 expressing cells and attraction of metastasis-promoting bone marrow-derived cells. These results indicate a therapeutic approach for preventing escape, which can be promptly tested in the clinic. 316 SRXRF ANALYSIS ON THE ACCUMULATION OF DACHPT-LOADED POLYMERIC MICELLES IN TUMOR BEFORE AND AFTER IRRADIATION K. Mizuno, H. Fujisawa, H. Cabral, K. Kataoka University of Tokio, Japan Platinum anticancer drug-containing polymeric micelles have long circulation in the bloodstream and high accumulation at some solid tumors reducing the side effects and improving the efficacy of incorporated drugs. Based on such extraordinary properties, these micelles have progressed into clinical trials in Europe and Asia (NC-6004; NC-4016; Nanocarrier, Japan). These micelles are also regarded as effective radio-
ICTR-PHE 2012 sensitizers when used in combination with radiation. To achieve the optimal therapeutic effect, the pharmacokinetics of the drugs should be fully understood before and after irradiation. However, the micro-distribution of these drugs inside tissues has not been well studied due to the unfeasibility of visual identification of low molecular weight platinum compounds. Moreover, the modification with fluorescent dyes might change their pharmacokinetics. In this research, therefore, the micro-distribution of platinum was directly observed by Synchrotron Radiation X-ray Fluorescence Spectrometry (SR-XRF) in which the characteristic X-rays induced by irradiation with high-energy photons were measured. Materials and Methods: Human pancreatic BxPC3 tumors were subcutaneously implanted to 9-week-old Balb/c nu/nu mice. BxPC3 tumor is hypovascular and known to have cancer cell nests. After 3 weeks, the tumors were irradiated with X-ray at a dose of 10Gy produced by an X-ray tube (Pantak H350) with a voltage of 200kV and a current of 20mA. A lead plate with a hole about 1cm in diameter was used for shielding the mice body from radiation exposure. After 1 hour, 1 day and 5 days, the polymeric micelles containing 1,2-diaminocyclohexane platinum (∂) (DACHPt) with a diameter of 30nm was injected intravenously at a dose of 20mg/kg. The polymeric micelles release the contained DACHPt in the presence of chlorine ion. Tumors were taken 24 hours later, then frozen with O.C.T. compound and sliced into 20μm thickness. SRXRF imaging was conducted at SPring-8, Hyogo, Japan, using the beam line BL37XU (15keV, 1012 1013photons/s). The area of 500μmx500μm was scanned with a special resolution of 10μm to obtain the microelemental distribution. The tumor section next to the section used for SR-XRF analysis was used for hematoxylin-enosin (HE) staining. Results and Discussions: Platinum was accumulated mainly in the regions of fibrotic tissue (50-100μM), and its concentration was gradually decreased into the cancer cell nests (<25μM). In the tumor section of a mouse injected with DACHPt loaded micelles 5 days after irradiation, fibrotic tissue regions were increased. In contrast, the cancer cell nests were shrank, and their borders to the fibrotic tissue became fold-shaped, which were smooth and round-shaped before irradiation. The ratio of the averaged platinum concentration in the nest to that in the stroma was 1.5 times increased after irradiation. This might be caused by the structural changes of the tumor. Especially due to the increase of the surface area of the cancer cell nests, more micelles could pass through the collagen membrane and accumulated inside the nests. Conclusions: The micro-distribution of platinum was directly observed by SR-XRF. The micelles permeated the tumor blood vessel, and were retained inside the tumor. Exposure to ionizing radiation could change the accumulation of the micelles. Further study on the effect of radiation to the micro-structure and environment of tumors is necessary to achieve optimum chemoradiotherapy using nano-sized drugs.
ICTR-PHE 2012 field of view (FOV) of 100 x 60 x 32 mm, with 2 averages, resulting in a scan time of 23 hours and 46 minutes. The rat MR imaging was performed using a BGA20S gradient coil and a 15.5 cm I.D. quadrature cylindrical RF resonator. A 3D spin-echo acquisition sequence was used with an echo time of 10 ms and a repetition time of 625 ms. The MR images were acquired with a 350 micron isotropic resolution, a FOV of 170 x 134.4 x 62.2 mm, with 2 averages, resulting in a scan time of 22 hours and 56 minutes. Following imaging, the animals were frozen at -20 degrees Celsius and sliced at 3 mm using a standard meat slicer available in the correlative pathology laboratory. Selected slices from the brain, lungs, abdomen, and pelvis were further sliced to 4 microns and processed for histological analysis with standard hematoxylin and eosin staining. The histology provided a cellular-based atlas corresponding to the high-resolution images. Following image reconstruction, the MR and CT images were imported into a radiation therapy treatment planning system. Registration and fusion was performed between the MR and CT for each image set and delineation of the normal tissues was performed. The histological stains were rigidly registered to the corresponding anatomical slice in the MR image using an in-house algorithm developed for correlative pathology. Relative 2D deformation between the MR slice and the histology was corrected using a 2D pointbased thin-plate spline deformable registration. Results: Rigid registration between the animal body MR and CT scans was performed with no detectable residual error as the gel immobilization provided a consistent anatomical positioning between both imaging sessions. Boney anatomy was automatically segmented from the CT image using a thresholding technique on all four specimens (male and female mouse and rat). Soft tissue, not well visualized on the CT image, was easily identified on the MR images. All tissues, including brain, lungs, heart, liver, stomach, bowels, and male and female pelvic anatomy was segmented in each MR image. Segmentation and validation of the histology slices is ongoing. In addition to providing a reference dataset for education purposes, a feasibility study investigating how such a reference dataset may aid in liver animal irradiation experiments was performed. Reference contoured MR images of the female mouse were registering to a kV CBCT image obtained on a liver mouse using the preclinical irradiation system to provide enhanced anatomical image guidance and normal tissue delineations capabilities for a conformal liver radiation therapy plan. Conclusions: Multi-modality high resolution reference anatomical datasets were generated for a female and male mouse and rat for purposes of education and anatomical reference. 315 TUMOR MICROENVIRONMENTAL REACTIONS INFLUENCING RESPONSE TO RADIOTHERAPY C. Ruegg University of Fribourg Purpose: About half of cancer patients receive radiotherapy during the course of their disease, either as curative-intent or palliative treatments. While in some cases disease control is satisfactory, in others is
S168 not and cancers further progress. Others and we have observed that experimental tumor growing in preirradiated stroma are locally more invasive and metastatic pointing to a critical role of the irradiated tumor microenvironment in promoting invasion and metastasis. The purpose of our studies was to identify stomal events promoting the increased aggressiveness of tumors relapsing after radiotherapy and to unravel the underlying mechanisms. Material and Methods: To address the question, we used an in vivo model of local irradiation in mice and tumor implantation, in vitro cell biology experiments, and pharmacological of genetic interventions. Results: We have observed that irradiation suppresses de novo angiogenesis and favors the appearance of tumor-associated hypoxia. This suppression is due, at least in part, to a TGFbeta dependent mechanism. Tumors growing within a previously irradiated and angiogenesis-deficient stroma have reduced growth while they display increased hypoxia, necrosis, local invasion and lung metastasis formation. Initial studies revealed that hypoxia selects for tumor variant expressing high levels of the matricellular protein CYR61. CYR61 promotes survival under hypoxia and also increase motility invasiveness and metastasis. Subsequently we have observed that CD11b+Gr1+ inflammatory cells are increasingly recruited at the periphery of tumors growing in preirradiated beds. Analysis of these cells revealed that they are mobilized from the bone marrow by tumor hypoxia in a HIFdependent manner. Mobilized CD11b+Gr1+ cells also home to premetastatic lungs. Cell transfer experiments revealed that CD11b+ circulating in the blood of tumorbearing preirradiated mice promote lung metastasis when transferred to tumor-bearign control (i.e. non preirradiated) mice. At last we have identified a factor induced by tumor hypoxia and its cognate receptor expressed on CD11b+ cells that mediated mobilization of metastasis-promoting CD11b+ cells. This ligandreceptor axis has not been previously associated with metastatic tumor spreading. Neutralization of this factor or its cognate receptor significantly suppressed tumor metastasis without impinging on primary tumor growth. Conclusions: Our studies demonstrating that irradiated tumor bed promotes tumor invasion and etastasis by at least two mechanisms: selection of CYR61 expressing cells and attraction of metastasis-promoting bone marrow-derived cells. These results indicate a therapeutic approach for preventing escape, which can be promptly tested in the clinic. 316 SRXRF ANALYSIS ON THE ACCUMULATION OF DACHPT-LOADED POLYMERIC MICELLES IN TUMOR BEFORE AND AFTER IRRADIATION K. Mizuno, H. Fujisawa, H. Cabral, K. Kataoka University of Tokio, Japan Platinum anticancer drug-containing polymeric micelles have long circulation in the bloodstream and high accumulation at some solid tumors reducing the side effects and improving the efficacy of incorporated drugs. Based on such extraordinary properties, these micelles have progressed into clinical trials in Europe and Asia (NC-6004; NC-4016; Nanocarrier, Japan). These micelles are also regarded as effective radio-
ICTR-PHE 2012 sensitizers when used in combination with radiation. To achieve the optimal therapeutic effect, the pharmacokinetics of the drugs should be fully understood before and after irradiation. However, the micro-distribution of these drugs inside tissues has not been well studied due to the unfeasibility of visual identification of low molecular weight platinum compounds. Moreover, the modification with fluorescent dyes might change their pharmacokinetics. In this research, therefore, the micro-distribution of platinum was directly observed by Synchrotron Radiation X-ray Fluorescence Spectrometry (SR-XRF) in which the characteristic X-rays induced by irradiation with high-energy photons were measured. Materials and Methods: Human pancreatic BxPC3 tumors were subcutaneously implanted to 9-week-old Balb/c nu/nu mice. BxPC3 tumor is hypovascular and known to have cancer cell nests. After 3 weeks, the tumors were irradiated with X-ray at a dose of 10Gy produced by an X-ray tube (Pantak H350) with a voltage of 200kV and a current of 20mA. A lead plate with a hole about 1cm in diameter was used for shielding the mice body from radiation exposure. After 1 hour, 1 day and 5 days, the polymeric micelles containing 1,2-diaminocyclohexane platinum (∂) (DACHPt) with a diameter of 30nm was injected intravenously at a dose of 20mg/kg. The polymeric micelles release the contained DACHPt in the presence of chlorine ion. Tumors were taken 24 hours later, then frozen with O.C.T. compound and sliced into 20μm thickness. SRXRF imaging was conducted at SPring-8, Hyogo, Japan, using the beam line BL37XU (15keV, 1012 1013photons/s). The area of 500μmx500μm was scanned with a special resolution of 10μm to obtain the microelemental distribution. The tumor section next to the section used for SR-XRF analysis was used for hematoxylin-enosin (HE) staining. Results and Discussions: Platinum was accumulated mainly in the regions of fibrotic tissue (50-100μM), and its concentration was gradually decreased into the cancer cell nests (<25μM). In the tumor section of a mouse injected with DACHPt loaded micelles 5 days after irradiation, fibrotic tissue regions were increased. In contrast, the cancer cell nests were shrank, and their borders to the fibrotic tissue became fold-shaped, which were smooth and round-shaped before irradiation. The ratio of the averaged platinum concentration in the nest to that in the stroma was 1.5 times increased after irradiation. This might be caused by the structural changes of the tumor. Especially due to the increase of the surface area of the cancer cell nests, more micelles could pass through the collagen membrane and accumulated inside the nests. Conclusions: The micro-distribution of platinum was directly observed by SR-XRF. The micelles permeated the tumor blood vessel, and were retained inside the tumor. Exposure to ionizing radiation could change the accumulation of the micelles. Further study on the effect of radiation to the micro-structure and environment of tumors is necessary to achieve optimum chemoradiotherapy using nano-sized drugs.