Everett J. Moding

Strategies for optimizing the response of cancer and normal tissues to radiation

Approximately 50% of all patients with cancer receive radiation therapy at some point during the course of their treatment, and the majority of these patients are treated with curative intent. Despite recent advances in the planning of radiation treatment and the delivery of image-guided radiation therapy, acute toxicity and potential long-term side effects often limit the ability to deliver a sufficient dose of radiation to control tumours locally. In the past two decades, a better understanding of the hallmarks of cancer and the discovery of specific signalling pathways by which cells respond to radiation have provided new opportunities to design molecularly targeted therapies to increase the therapeutic window of radiation therapy. Here, we review efforts to develop approaches that could improve outcomes with radiation therapy by increasing the probability of tumour cure or by decreasing normal tissue toxicity.

A Plasmonic Gold Nanostar Theranostic Probe for In Vivo Tumor Imaging and Photothermal Therapy.

Nanomedicine has attracted increasing attention in recent years, because it offers great promise to provide personalized diagnostics and therapy with improved treatment efficacy and specificity. In this study, we developed a gold nanostar (GNS) probe for multi-modality theranostics including surface-enhanced Raman scattering (SERS) detection, x-ray computed tomography (CT), two-photon luminescence (TPL) imaging, and photothermal therapy (PTT). We performed radiolabeling, as well as CT and optical imaging, to investigate the GNS probes biodistribution and intratumoral uptake at both macroscopic and microscopic scales. We also characterized the performance of the GNS nanoprobe for in vitro photothermal heating and in vivo photothermal ablation of primary sarcomas in mice. The results showed that 30-nm GNS have higher tumor uptake, as well as deeper penetration into tumor interstitial space compared to 60-nm GNS. In addition, we found that a higher injection dose of GNS can increase the percentage of tumor uptake. We also demonstrated the GNS probes superior photothermal conversion efficiency with a highly concentrated heating effect due to a tip-enhanced plasmonic effect. In vivo photothermal therapy with a near-infrared (NIR) laser under the maximum permissible exposure (MPE) led to ablation of aggressive tumors containing GNS, but had no effect in the absence of GNS. This multifunctional GNS probe has the potential to be used for in vivo biosensing, preoperative CT imaging, intraoperative detection with optical methods (SERS and TPL), as well as image-guided photothermal therapy.

A next-generation dual-recombinase system for time and host specific targeting of pancreatic cancer

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP–based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell–autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.

Dual-Energy Micro-CT Functional Imaging of Primary Lung Cancer in Mice Using Gold and Iodine Nanoparticle Contrast Agents: A Validation Study

Purpose To provide additional functional information for tumor characterization, we investigated the use of dual-energy computed tomography for imaging murine lung tumors. Tumor blood volume and vascular permeability were quantified using gold and iodine nanoparticles. This approach was compared with a single contrast agent/single-energy CT method. Ex vivo validation studies were performed to demonstrate the accuracy of in vivo contrast agent quantification by CT. Methods Primary lung tumors were generated in LSL-KrasG12D; p53FL/FL mice. Gold nanoparticles were injected, followed by iodine nanoparticles two days later. The gold accumulated in tumors, while the iodine provided intravascular contrast. Three dual-energy CT scans were performed–two for the single contrast agent method and one for the dual contrast agent method. Gold and iodine concentrations in each scan were calculated using a dual-energy decomposition. For each method, the tumor fractional blood volume was calculated based on iodine concentration, and tumor vascular permeability was estimated based on accumulated gold concentration. For validation, the CT-derived measurements were compared with histology and inductively-coupled plasma optical emission spectroscopy measurements of gold concentrations in tissues. Results Dual-energy CT enabled in vivo separation of gold and iodine contrast agents and showed uptake of gold nanoparticles in the spleen, liver, and tumors. The tumor fractional blood volume measurements determined from the two imaging methods were in agreement, and a high correlation (R2 = 0.81) was found between measured fractional blood volume and histology-derived microvascular density. Vascular permeability measurements obtained from the two imaging methods agreed well with ex vivo measurements. Conclusions Dual-energy CT using two types of nanoparticles is equivalent to the single nanoparticle method, but allows for measurement of fractional blood volume and permeability with a single scan. As confirmed by ex vivo methods, CT-derived nanoparticle concentrations are accurate. This method could play an important role in lung tumor characterization by CT.

Dual-Energy Micro-Computed Tomography Imaging of Radiation-Induced Vascular Changes in Primary Mouse Sarcomas

PURPOSE To evaluate the effects of radiation therapy on primary tumor vasculature using dual-energy (DE) micro-computed tomography (micro-CT). METHODS AND MATERIALS Primary sarcomas were generated with mutant Kras and p53. Unirradiated tumors were compared with tumors irradiated with 20 Gy. A liposomal-iodinated contrast agent was administered 1 day after treatment, and mice were imaged immediately after injection (day 1) and 3 days later (day 4) with DE micro-CT. CT-derived tumor sizes were used to assess tumor growth. After DE decomposition, iodine maps were used to assess tumor fractional blood volume (FBV) at day 1 and tumor vascular permeability at day 4. For comparison, tumor vascularity and vascular permeability were also evaluated histologically by use of CD31 immunofluorescence and fluorescently-labeled dextrans. RESULTS Radiation treatment significantly decreased tumor growth from day 1 to day 4 (P<.05). There was a positive correlation between CT measurement of tumor FBV on day 1 and extravasated iodine on day 4 with microvascular density (MVD) on day 4 (R(2)=0.53) and dextran accumulation (R(2)=0.63) on day 4, respectively. Despite no change in MVD measured by histology, tumor FBV significantly increased after irradiation as measured by DE micro-CT (0.070 vs 0.091, P<.05). Both dextran and liposomal-iodine accumulation in tumors increased significantly after irradiation, with dextran fractional area increasing 5.2-fold and liposomal-iodine concentration increasing 4.0-fold. CONCLUSIONS DE micro-CT is an effective tool for noninvasive assessment of vascular changes in primary tumors. Tumor blood volume and vascular permeability increased after a single therapeutic dose of radiation treatment.

p53 Functions in Endothelial Cells to Prevent Radiation-Induced Myocardial Injury in Mice

Without the transcription factor p53 in cardiac endothelial cells, the heart is more susceptible to radiation-induced damage. Keeping the Heart Safe from Radiation The cells that make up the muscular part of the heart, or the myocardium, are generally not actively dividing. Therefore, one would not expect radiation therapy aimed at eliminating rapidly dividing cancer cells to cause heart disease. However, radiation-related heart disease is a clinically important long-term side effect of radiation therapy. The transcription factor p53 is activated in response to radiation and other DNA-damaging stresses; whether it promotes or attenuates radiation-related heart disease is unclear. Lee et al. generated mice lacking p53 in endothelial cells, the cells that line blood vessels, and whole-heart irradiation in these mice resulted in damage to the vasculature of the heart, triggering ischemia (disrupted blood flow) in the myocardium and eventual heart failure. p53 inhibitors that protect normal cells from the effects of radiation therapy have been proposed as an approach to improve the therapeutic ratio of radiation therapy, but these data suggest that combining radiation therapy with p53 inhibitors may actually increase the risk of cardiac injury. Radiation therapy, which is used for the treatment of some cancers, can cause delayed heart damage. In the heart, p53 influences myocardial injury that occurs after multiple types of stress. Here, we demonstrated that p53 functioned in endothelial cells to protect mice from myocardial injury after whole-heart irradiation. Mice with an endothelial cell–specific deletion of p53 succumbed to heart failure after whole-heart irradiation as a result of myocardial necrosis, systolic dysfunction, and cardiac hypertrophy. Moreover, the onset of cardiac dysfunction was preceded by alterations in myocardial vascular permeability and density, which resulted in cardiac ischemia and myocardial hypoxia. Mechanistic studies with primary cardiac endothelial cells irradiated in vitro indicated that p53 signaling caused mitotic arrest and protected cardiac endothelial cells from cell death resulting from abnormal mitosis or mitotic catastrophe. Furthermore, mice lacking the cyclin-dependent kinase inhibitor p21, which is a transcriptional target of p53, were also sensitized to myocardial injury after whole-heart irradiation. Together, our results demonstrate that the p53-p21 axis functions to prevent radiation-induced myocardial injury in mice.

Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium

Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy

Tumor cells, rather than endothelial cells, are critical targets that regulate primary sarcoma eradication by radiation therapy. Not all cells are eradicated equally Personalized cancer therapies dominate the news. But radiation therapy continues to be an essential part of the treatment regimens of nearly half of all cancer patients—sometimes achieving complete tumor regression through the safe delivery of high doses of radiation. Previous research with transplanted tumor models in mice has suggest that radiation targets, not only the tumor cells themselves, but also components of the surrounding milieu, which comprises blood vessels and various cell types that influence tumor growth. Now Moding et al. challenge the earlier findings in studies conducted with primary sarcomas in mice that carried, in either tumor or endothelial cells, genetic mutations that modulate radiation sensitivity. The authors found that it was the tumor, rather than endothelial, cells that mediate primary sarcoma shrinkage by radiation therapy and that selective small-molecule inhibition of a DNA-damage response enzyme can enhance radiosensitization of some tumors. Cancer clinics currently use high-dose stereotactic body radiation therapy as a curative treatment for several kinds of cancers. However, the contribution of vascular endothelial cells to tumor response to radiation remains controversial. Using dual recombinase technology, we generated primary sarcomas in mice with targeted genetic mutations specifically in tumor cells or endothelial cells. We selectively mutated the proapoptotic gene Bax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. Bax deletion from endothelial cells did not affect radiation-induced cell death in tumor endothelial cells or sarcoma response to radiation therapy. Although Atm deletion increased endothelial cell death after radiation therapy, deletion of Atm from endothelial cells failed to enhance sarcoma eradication. In contrast, deletion of Atm from tumor cells increased sarcoma eradication by radiation therapy. These results demonstrate that tumor cells, rather than endothelial cells, are critical targets that regulate sarcoma eradication by radiation therapy. Treatment with BEZ235, a small-molecule protein kinase inhibitor, radiosensitized primary sarcomas more than the heart. These results suggest that inhibiting ATM kinase during radiation therapy is a viable strategy for radiosensitization of some tumors.

Generation of primary tumors with Flp recombinase in FRT-flanked p53 mice

SUMMARY The site-specific recombinases Cre and Flp can mutate genes in a spatially and temporally restricted manner in mice. Conditional recombination of the tumor suppressor gene p53 using the Cre-loxP system has led to the development of multiple genetically engineered mouse models of human cancer. However, the use of Cre recombinase to initiate tumors in mouse models limits the utilization of Cre to genetically modify other genes in tumor stromal cells in these models. To overcome this limitation, we inserted FRT (flippase recognition target) sites flanking exons 2–6 of the endogenous p53 gene in mice to generate a p53FRT allele that can be deleted by Flp recombinase. We show that FlpO-mediated deletion of p53 in mouse embryonic fibroblasts impairs the p53-dependent response to genotoxic stress in vitro. In addition, using FSF-KrasG12D/+; p53FRT/FRT mice, we demonstrate that an adenovirus expressing FlpO recombinase can initiate primary lung cancers and sarcomas in mice. p53FRT mice will enable dual recombinase technology to study cancer biology because Cre is available to modify genes specifically in stromal cells to investigate their role in tumor development, progression and response to therapy.

A comparison of radial keyhole strategies for high spatial and temporal resolution 4D contrast-enhanced MRI in small animal tumor models.

PURPOSE Dynamic contrast-enhanced (DCE) MRI has been widely used as a quantitative imaging method for monitoring tumor response to therapy. The simultaneous challenges of increasing temporal and spatial resolution in a setting where the signal from the much smaller voxel is weaker have made this MR technique difficult to implement in small-animal imaging. Existing protocols employed in preclinical DCE-MRI acquire a limited number of slices resulting in potentially lost information in the third dimension. This study describes and compares a family of four-dimensional (3D spatial + time), projection acquisition, radial keyhole-sampling strategies that support high spatial and temporal resolution. METHODS The 4D method is based on a RF-spoiled, steady-state, gradient-recalled sequence with minimal echo time. An interleaved 3D radial trajectory with a quasi-uniform distribution of points in k-space was used for sampling temporally resolved datasets. These volumes were reconstructed with three different k-space filters encompassing a range of possible radial keyhole strategies. The effect of k-space filtering on spatial and temporal resolution was studied in a 5 mM CuSO(4) phantom consisting of a meshgrid with 350-μm spacing and in 12 tumors from three cell lines (HT-29, LoVo, MX-1) and a primary mouse sarcoma model (three tumors∕group). The time-to-peak signal intensity was used to assess the effect of the reconstruction filters on temporal resolution. As a measure of heterogeneity in the third dimension, the authors analyzed the spatial distribution of the rate of transport (K(trans)) of the contrast agent across the endothelium barrier for several different types of tumors. RESULTS Four-dimensional radial keyhole imaging does not degrade the system spatial resolution. Phantom studies indicate there is a maximum 40% decrease in signal-to-noise ratio as compared to a fully sampled dataset. T1 measurements obtained with the interleaved radial technique do not differ significantly from those made with a conventional Cartesian spin-echo sequence. A bin-by-bin comparison of the distribution of the time-to-peak parameter shows that 4D radial keyhole reconstruction does not cause significant temporal blurring when a temporal resolution of 9.9 s is used for the subsamples of the keyhole data. In vivo studies reveal substantial tumor heterogeneity in the third spatial dimension that may be missed with lower resolution imaging protocols. CONCLUSIONS Volumetric keyhole imaging with projection acquisition provides a means to increase spatiotemporal resolution and coverage over that provided by existing 2D Cartesian protocols. Furthermore, there is no difference in temporal resolution between the higher spatial resolution keyhole reconstruction and the undersampled projection data. The technique allows one to measure complex heterogeneity of kinetic parameters with isotropic, microscopic spatial resolution.