The Krebs tricarboxylic acid cycle (TCA) is central to metabolic energy production and is known to be altered in many disease states. Real time molecular imaging of TCA cycle in vivo will be important in understanding the metabolic basis of several diseases. Positron emission tomography (PET) using FDG-glucose (2-[18F]fluoro-2-deoxy-D-glucose) is already being used as a metabolic imaging agent in clinics. However, FDG-glucose does not reveal anything past glucose uptake and phosphorylation. We have developed a new metabolic imaging agent, hyperpolarized diethyl 1-13C 2,3-d2 succinate, that allows for real time in vivo imaging and spectroscopy of the TCA cycle. Diethyl succinate can be hyperpolarized using parahydrogen induced polarization (PHIP) in an aqueous solution with signal enhancement of 5000 compared to Boltzmann polarization. 13C magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) were achieved in vivo seconds after injection of 10 to 20 μmol of hyperpolarized diethyl succinate into normal mice. The downstream metabolites of hyperpolarized diethyl succinate were identified in vivo as malate, succinate, fumarate and aspartate. The metabolism of diethyl succinate was altered after exposing the animal to 3-nitropropionate, a known irreversible inhibitor of succinate dehydrogenase. Based on our results, hyperpolarized diethyl succinate allows for in real time in vivo MRI and MRS with a high signal to noise ratio and with visualization of multiple steps of the TCA cycle. Hyperpolarization of diethyl succinate and its in vivo applications may reveal an entirely new regime wherein the local status of TCA cycle metabolism is interrogated on the time scale of seconds to minutes with unprecedented chemical specificity and MR sensitivity.
parahydrogen; hyperpolarization; 13C MRI; TCA cycle; cancer imaging; PHIP
The prognosis for patients with high grade gliomas is poor, with a median survival of 1 year. Treatment efficacy assessment is typically unavailable until 5-6 months post diagnosis. Investigators hypothesize that quantitative magnetic resonance imaging can assess treatment efficacy 3 weeks after therapy starts, thereby allowing salvage treatments to begin earlier. The purpose of this work is to build a predictive model of treatment efficacy by using quantitative magnetic resonance imaging data and to assess its performance. The outcome is 1-year survival status. We propose a joint, two-stage Bayesian model. In stage I, we smooth the image data with a multivariate spatiotemporal pairwise difference prior. We propose four summary statistics that are functionals of posterior parameters from the first-stage model. In stage II, these statistics enter a generalized non-linear model as predictors of survival status. We use the probit link and a multivariate adaptive regression spline basis. Gibbs sampling and reversible jump Markov chain Monte Carlo methods are applied iteratively between the two stages to estimate the posterior distribution. Through both simulation studies and model performance comparisons we find that we can achieve higher overall correct classification rates by accounting for the spatiotemporal correlation in the images and by allowing for a more complex and flexible decision boundary provided by the generalized non-linear model.
Bayesian analysis; Image analysis; Multivariate adaptive regression splines; Multivariate pairwise difference prior; Quantitative magnetic resonance imaging; Spatiotemporal model
The dual modality of TGFβ, both as a potent tumor suppressor and a stimulator of tumor progression, invasion, and metastasis, make it a critical target for therapeutic intervention in human cancers. The ability to perform real-time, noninvasive imaging of TGFβ-activated Smad signaling in live cells and animal models would significantly improve our understanding of the regulation of this unique signaling cascade. To advance these efforts, we developed a highly sensitive molecular imaging tool that repetitively, non-invasively and dynamically reports on TGFBR1 kinase activity.
The bioluminescent TGFβR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation wherein inhibition of TGFβ receptor1 kinase activity leads to an increase in reporter signaling. The reporter was stably transfected into mammalian cells and used to image in vivo and in vitro bioluminescent activity as a surrogate for monitoring TGFBR1 kinase activity.
The reporter was successfully used to monitor direct and indirect inhibitors of TGFβ-induced Smad2 and SMAD3 phosphorylation in live-cells and tumor xenografts and adapted for high throughput screening to identify a role for receptor tyrosine kinase-inhibitors as modulators of TGFβ signaling.
The reporter is a dynamic, non-invasive imaging modality for monitoring TGFβ-induced Smad2 signaling in live cells and tumor xenografts. It has immense potential for identifying novel effectors of R-Smad phosphorylation; for validating drug-target interaction; and for studying TGFβ signaling in different metastasis models.
bioluminescence; kinase activity; non-invasive molecular imaging; receptor-regulated Smads; TGFβ
Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.
To present the use of a quality control ice-water phantom for DW-MRI. DW-MRI has emerged as an important cancer imaging biomarker candidate for diagnosis and early treatment response assessment. Validating imaging biomarkers through multi-center trials requires calibration and performance testing across sites.
Materials and Methods
The phantom consisted of a center tube filled with distilled water surrounded by ice-water. Following preparation of the phantom approximately 30 minutes was allowed to reach thermal equilibrium. DW-MRI data was collected at 7 institutions, 20 MRI scanners from three vendors and 2 field strengths (1.5 and 3T). The phantom was also scanned on a single system on 16 different days over a 25 day period. All data was transferred to a central processing site at the University of Michigan for analysis.
Results revealed that the variation of measured ADC values between all systems tested was ±5% indicating excellent agreement between systems. Reproducibility of a single system over a 25 day period was also found to be within ±5% ADC values. Overall, the use of an ice water phantom for assessment of ADC was found to be a reasonable candidate for use in multi-center trials.
The ice water phantom described here is a practical and universal approach to validate the accuracy of ADC measurements with ever changing MRI sequence and hardware design and can be readily implemented in multicenter clinical trial designs.
diffusion; MRI; phantom; ice water; quality control
FADD (Fas-associated protein with death domain) is a cytosolic adapter protein essential for mediating death receptor-induced apoptosis. It has also been implicated in a number of non-apoptotic activities including embryogenesis, cell-cycle progression, cell proliferation, and tumorigenesis. Our recent studies have demonstrated that high levels of phosphorylated FADD in tumor cells correlates with increased activation of the anti-apoptotic transcription factor NF-κB and is a biomarker for aggressive disease and poor clinical outcome. These findings suggest that inhibition of FADD phosphorylation is a viable target for cancer therapy. A high throughput screen using a cell-based assay for monitoring FADD-kinase activity identified NSC 47147 as a small molecule inhibitor of FADD phosphorylation. The compound was evaluated in live cells and mouse tumors for its efficacy as an inhibitor of FADD-kinase activity through the inhibition of CK1α. NSC 47147 was shown to decrease levels of phosphorylated FADD and NF-κB activity such that combination therapy lead to greater induction of apoptosis and enhanced tumor control as compared to either agent alone. The studies described here demonstrate the utility of bioluminescent cell based assays for the identification of active compounds and the validation of drug target interaction in a living subject. In addition, the presented results provide proof of principle studies as to the validity of targeting FADD-kinase activity as a novel cancer therapy strategy.
FADD; phosphorylation; non-invasive molecular imaging; NF-κB; chemotherapy
Epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades including cell proliferation, differentiation and migration. To investigate the EGFR signaling events in real time and in living cells and animals, we here describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC-1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thus providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with EGF. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.
EGFR; EPS-15; Molecular Imaging; Split Luciferase; Reporter
Magnetic resonance (MR) techniques using hyperpolarized 13C have successfully produced examples of angiography and intermediary metabolic imaging, but to date no receptor imaging has been attempted. The goal of this study is to synthesize and evaluate a novel hyperpolarizable molecule, tetrafluoropropyl 1-13C-propionate-d3 (TFPP), for detecting atheromatous plaque in vivo. TFPP binds to lipid bilayers and its use in hyperpolarized MR could prove to be a major step towards receptor imaging.
The precursor, Tetrafluoropropyl 1-13C-acrylate (TFPA) binds to dimyristoylphosphatidylcholine (DMPC) lipid bilayers with a 1.6 ppm chemical shift in the 19F MR spectrum. This molecule was designed to be hyperpolarized through addition of parahydrogen to 13C acrylate moiety by Parahydrogen Induced Polarization (PHIP). 13C TFPA was hyperpolarized to Tetrafluoropropyl 1-13C-propionate (TFPP) to a similar extent to that of hydroxyethylacrylate (HEA) to hydroxyethylpropionate (HEP); 17% +/− 4 % for TFPP vs 20% for HEP; T1 relaxation times (45s ± 2 vs 55s ± 2) were comparable and the hyperpolarized properties of TFPP were characterized. HEA, like TFPA has a chemical structure with an acrylate moiety but do not have the lipid binding Tetrafluoropropyl functional group. Hyperpolarized 13C TFPP binds to lipid bilayer appearing as a second, chemically shifted 13C hyperpolarized MR resonance with further reduction in longitudinal relaxation time (T1 = 21s ± 1). In aortas harvested from Low Density Lipoprotein Receptor (LDLR) knock-out mice fed with a high fat diet for nine months, and in which atheroma is deposited in aorta and heart, 13C TFPP showed greater binding to lipid on the intimal surface than in normal diet control mice. When 13C TFPP was hyperpolarized and administered in vivo to atheromatous mice in a pilot study, increased binding was observed on the endocardial surface of the intact heart compared to normal fed controls.
Hyperpolarized 13C TFPP has bio-sensing specificity for lipid, coupled with 42,000 fold sensitivity gain in MR signal at 4.7 Tesla. Binding of TFPP with lipids results in the formation of a characteristic second peak in MR spectroscopy. TFPP therefore has the potential to act as an in vivo molecular probe for atheromatous plaque imaging and may serve as a model of receptor targeted bioimaging with enhanced MR sensitivity.
13C; heart; receptor imaging; atheroma; hyperpolarization; TFPP; PHIP; MR
We demonstrate that rapamycin can induce regression of adenomatous polyposis coli (Apc) mutation-dependent colonic adenomas in genetically engineered mice (CPC;Apc). An endoscope was used to visualize adenomas in CPC;Apc mice weekly for 10 weeks. The lesion surface areas were measured using a distance gauge and digitally generated grid. Coronal scans were performed on magnetic resonance imaging (MRI) to localize adenomas, and tumor volumes were measured from regions of interest drawn on consecutive axial scans. Rapamycin (5 mg/kg) was administered intraperitoneally daily for 5 weeks. Endoscopy and MRI were performed weekly to monitor adenoma regression. Caliper measurements and immunohistochemistry (IHC) were performed on adenomas postmortem. Dimensions from n = 30 adenomas in n = 7 animals were measured. Adenoma surface areas on endoscopy correlated with volumes on MRI and with postmortem caliper measurements, R2 = 0.84 and R2 = 0.81, respectively. The mean adenoma doubling times on endoscopy and MRI were 0.95 ± 0.14 and 1.21 ± 0.16 weeks, respectively. The minimum detectable adenoma surface area and volume on endoscopy and MRI was 0.69 mm2 and 1.76 mm3, respectively. On histology, the rapamycin-treated adenomas showed limited regions of dysplasia. Rapamycin therapy resulted in much lower mammalian target of rapamycin signaling and cell proliferation. Lower expression of phospho-S6 and reduced numbers of Ki67-positive cells were seen on IHC compared to vehicle-treated lesions. Endoscopy can be validated by MRI as a robust methodology for quantitative monitoring of therapy, representing a promising approach for future preclinical efforts to assess utility of novel colorectal cancer prevention strategies.
Redundant receptor tyrosine kinase (RTK) signaling is a mechanism for therapeutic resistance to EGFR inhibition. A strategy to reduce parallel signaling by co-expressed RTKs is inhibition of N-linked glycosylation (NLG), an endoplasmic reticulum (ER) co-translational protein modification required for receptor maturation and cell surface expression. We therefore investigated the feasibility of blocking NLG in vivo to reduce over-expression of RTKs.
We developed a model system to dynamically monitor NLG in vitro and in vivo using bioluminescent imaging techniques. Functional imaging of NLG is accomplished with a luciferase reporter (ER-LucT) modified for ER-translation and glycosylation. After in vitro validation, this reporter was integrated with D54 glioma xenografts to perform non-invasive imaging of tumors, and inhibition of NLG was correlated with RTK protein levels and tumor growth.
The ER-LucT reporter demonstrates the ability to sensitively and specifically detect NLG inhibition. Using this molecular imaging approach we performed serial imaging studies to determine safe and efficacious in vivo dosing of the GlcNAc-1-phosphotransferase inhibitor, tunicamycin, which blocks N-glycan precursor biosynthesis. Molecular analyses of tunicamycin treated tumors showed reduced levels of EGFR and Met, two RTKs over-expressed in gliomas. Furthermore, D54 and U87MG glioma xenograft tumor experiments demonstrated significant reductions in tumor growth following NLG inhibition and radiation therapy, consistent with an enhancement in tumor radiosensitivity.
This study suggests NLG inhibition is a novel therapeutic strategy for targeting EGFR and RTK signaling in both gliomas and other malignant tumors.
Glycosylation; Radiation; EGFR; Met
Currently, radiologic response of brain tumors is assessed according to the Macdonald criteria 10 weeks from the start of therapy. There exists a critical need to identify non-responding patients early in the course of their therapy for consideration of alternative treatment strategies. Our study assessed the effectiveness of the Parametric Response Map (PRM) imaging biomarker to provide for an earlier measure of patient survival prediction.
Forty-five high grade glioma patients received concurrent chemoradiation. Quantitative MRI including apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) maps were acquired pre-treatment and 3 weeks mid-treatment on a prospective institutional-approved study. PRM, a voxel-by-voxel image analysis method, was evaluated as an early prognostic biomarker of overall survival. Clinical and conventional MR parameters were also evaluated.
Multivariate analysis showed that PRMADC+ in combination with PRMrCBV- obtained at week 3 had a stronger correlation to one-year and overall survival rates than any baseline clinical or treatment response imaging metric. The composite biomarker identified three distinct patient groups, non-responders (median survival (MS) of 5.5 months CI: 4.4-6.6) months, partial responders (MS of 16 months CI: 8.6-23.4) and responders (MS has not yet been reached.)
Inclusion of PRMADC+ and PRMrCBV- into a single imaging biomarker metric provided early identification of patients resistant to standard chemoradiation. In comparison to the current standard of assessment of response at 10 weeks (MacDonald Criteria) the composite PRM biomarker potentially provides a useful opportunity for clinicians to identify patients who may benefit from alternative treatment strategies.
DW-MRI; DSC-MRI; glioma; prospective trial; treatment response
The receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF) modulate signaling cascades implicated in cellular proliferation, survival, migration, invasion, and angiogenesis. Therefore, dysregulation of HGF/c-Met signaling can compromise the cellular capacity to moderate these activities, and lead to tumorigenesis, metastasis, and therapeutic resistance in various human malignancies. To facilitate studies investigating HGF/cMet receptor coupling or c-Met signaling events in real time and in living cells and animals, we here describe a genetically engineered reporter wherein bioluminescence can be used as a surrogate for c-Met tyrosine kinase activity. C-Met kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to the activation or inhibition of the HGF/c-Met signaling pathway. Treatment of tumor bearing animals with a c-Met inhibitor and the HGF neutralizing antibody stimulated the reporter’s bioluminescence activity in a dose dependent manner and led to a regression of U-87 MG tumor xenografts. Results obtained from these studies provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate c-Met activity and validate c-Met as a target for human glioblastoma therapy.
c-Met; non-invasive molecular imaging; bioluminescence; kinase activity
Here we describe the Parametric Response Map (PRM), a voxel-wise approach for image analysis and quantification of hemodynamic alterations during treatment for 44 patients with high-grade glioma. Relative cerebral blood volume (rCBV) and flow (rCBF) maps were acquired before treatment and after 1 and 3 weeks of therapy. We compared the standard approach using region-of-interest analysis for change in rCBV or rCBF to the change in perfusion parameters on the basis of PRM (PRMrCBV and PRMrCBF) for their accuracy in predicting overall survival. Neither the percentage change of rCBV or rCBF predicted survival, whereas the regional response evaluations based upon PRM were highly predictive of survival. Even when accounting for baseline rCBV, which is prognostic, PRMrCBV proved more predictive of overall survival.
The past decade has seen momentous development in brain cancer research in terms of novel imaging-assisted surgeries, molecularly targeted drug-based treatment regimens or adjuvant therapies and in our understanding of molecular footprints of initiation and progression of malignancy. However, mortality due to brain cancer has essentially remained unchanged in the last three decades. Thus, paradigm-changing diagnostic and therapeutic reagents are urgently needed. Nanotheranostic platforms are powerful tools for imaging and treatment of cancer. Multifunctionality of these nanovehicles offers a number of advantages over conventional agents. These include targeting to a diseased site thereby minimizing systemic toxicity, the ability to solubilize hydrophobic or labile drugs leading to improved pharmacokinetics and their potential to image, treat and predict therapeutic response. In this article, we will discuss the application of newer theranostic nanoparticles in targeted brain cancer imaging and treatment.
Nanoparticle; MRI; Photodynamic therapy; F3 and RGD peptides; toxicity; cancer therapy
The aim of this study was to empirically test the effect of chemotherapy-induced tissue changes in a glioma model as measured by several diffusion indices calculated from non-monoexponential formalisms over a wide range of b-values. We also compared these results to the conventional two-point apparent diffusion coefficient (ADC) calculation using nominal b-values.
Diffusion weighted imaging was performed over an extended range of b-values (120–4000s/mm2) on intracerebral rat 9L gliomas prior to and following a single dose of 1,3-bis(2-chloroethyl)-1-nitrosourea. Diffusion indices from three formalisms of DW signal decay ((a) two-point analytical calculation using either low or high b-values, (b) a stretched exponential formalism and (c) a biexponential fit) were tested for responsiveness to therapy-induced differences between control and treated groups.
Diffusion indices sensitive to “fast diffusion” produced the largest response to treatment, which resulted in significant differences between groups. These trends were not observed for “slow diffusion” indices. Although the highest rate of response was observed from the biexponential formalism, this was not found to be significantly different from the conventional monoexponential ADC method. In conclusion, parameters from the more complicated non-monoexponential formalisms did not provide additional sensitivity to treatment response in this glioma model beyond that observed from the two-point conventional monoexponential ADC method.
Glycogen synthase kinase-3 (GSK3 β) and casein kinase-1 alpha (CK-1α) are multifunctional kinases that play critical role in the regulation of a number of cellular processes. In spite of their importance, molecular imaging tools for non-invasive and real-time monitoring of their kinase activities have not been devised. Here, we report development of bioluminescent GSK3β and CK-1α reporter (BGCR) based on firefly luciferase complementation. Treatment of SW620 cells stably expressing the reporter with inhibitors of GSK3β (SB415286 and LiCl) or CK1α (CKI-7) resulted in dose and time dependent increase in BGCR activity which were validated using western blotting. No increase in bioluminescence was observed in case of S37A mutant (GSK3β inhibitors) or with S45A mutant (CKI-7) demonstrating the specificity of the reporter. Imaging of mice tumor xenograft generated with BGCR expressing SW620 cells following treatment with LiCl showed unique oscillations in GSK3β activity which were corroborated by phospho-GSK3β immunoblotting. Taken together, BGCR is novel molecular imaging tool that reveals unique insight into GSK3β and CK1α kinase activities and may provide powerful tool in experimental therapeutics for rapid optimization of dose and schedule of targeted therapies and for monitoring therapeutic response.
Molecular imaging; GSK3 β; CK1α; split luciferase; reporter
Protein kinases are important regulators of signal-transduction pathways. Dysregulated kinase activity is observed in a variety of human diseases such as cancer, making them targets for the development of molecular therapies. Identification of new drugs is greatly aided by molecular imaging tools which enable real time, non-invasive, dynamic and quantitative imaging of kinase activity in vivo. We have recently described a new reporter platform based on conformation dependent complementation of firefly luciferase to monitor serine/threonine kinase (Akt) activity. The reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase activity in living subjects and also provide a platform for cell based high-throughput drug screening for modulators of kinase activity.
protein kinase; non-invasive imaging; kinase activity
The ultimate goal of any cancer therapy is to target the elimination of neoplastic cells. Although newer therapeutic strategies are in constant development, therapeutic assessment has been hampered by the inability to assess, rapidly and quantitatively, efficacy in vivo. Diffusion imaging and, more recently, sodium MRI have demonstrated their distinct abilities to detect therapy-induced alterations in tumor cellularity, which has been demonstrated to be indicative of therapeutic efficacy. More importantly, both imaging modalities detect tumor response much earlier than traditional methodologies that rely on macroscopic volumetric changes. In this study, the correlation between tumor sodium and diffusion was further tested to demonstrate the sensitivity of sodium imaging to gauge tumor response to therapy by using a 9L rat gliosarcoma treated with varying doses of BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea]. This orthotopic model has been demonstrated to display variability in response to BCNU therapy where initial insult has been shown to lead to drug-resistance. In brief, a single 26.6 mg/kg BCNU dose yielded dramatic responses in both diffusion and sodium MRI. However, a second equivalent BCNU dose yielded a much smaller change in diffusion and sodium, suggesting a drop in tumor sensitivity to BCNU. The MRI responses of animals treated with 13.3 mg/kg BCNU were much lower and similar responses were observed after the initial and secondary applications of BCNU. Furthermore, these results were further validated using volumetric measurements of the tumor and also ex vivo determination of tumor sensitivity to BCNU. Overall, these experiments demonstrate the sensitivity and applicability of sodium and diffusion MRI as tools for dynamic assessment of tumor response to therapy.
tumor; diffusion; sodium; magnetic resonance imaging; 1,3-bis(2-chloroethyl)-1-nitrosourea; BCNU; chemotherapy resistance
An imaging biomarker that would provide for an early quantitative metric of clinical treatment response in cancer patients would provide for a paradigm shift in cancer care. Currently, non-image based clinical outcome metrics include morphology, clinical and laboratory parameters however, these are obtained relatively late following treatment. Diffusion-weighted MRI (DW-MRI) holds promise for use as a cancer treatment response biomarker as it is sensitive to macromolecular and microstructural changes which can occur at the cellular level earlier than anatomical changes during therapy. Studies have shown that successful treatment of a many tumor types can be detected using DW-MRI as an early increase in the apparent diffusion coefficient (ADC) values. Additionally, low pre-treatment ADC values of various tumors are often predictive of better outcome. These capabilities, once validated, could provide for an important opportunity to individualize therapy thereby minimizing unnecessary systemic toxicity associated with ineffective therapies with the additional advantage of improving overall patient health care and associated costs. In this report, we provide a brief technical overview of DW-MRI acquisition protocols, quantitative image analysis approaches and review studies which have implemented DW-MRI for the purpose of early prediction of cancer treatment response.
diffusion-weighted MRI; oncology; treatment monitoring; biomarker
Apoptosis is an essential process for the maintenance of normal physiology. The ability to non-invasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta galactosidase gene flanked by two ER regulatory domains and intervening DEVD sequences was constructed to serve as a tool for invivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable LACZ activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of LACZ activity. Enzymatic activation of the reporter during an apoptotic event enabled non-invasive measurement of LACZ activity in living cells, which correlated with traditional measures of apoptosis in a dose and time dependent manner. Furthermore, using a near-infrared fluorescent substrate of beta galactosidase (DDAOG), non-invasive invivo imaging of apoptosis was achieved using a xenograft tumor model in response to pro-apoptotic therapy. Lastly, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals especially in context of skin biology.
Molecular imaging; proteaseiImaging; caspase 3; beta galactosidase
The ability to quantitate early effects of tumor therapeutic response using noninvasive imaging would have a major impact in clinical oncology. One area of active research interest is the ability to use MR techniques to detect subtle changes in tumor cellular density. In this study, sodium and proton diffusion MRI were compared for their ability to detect early cellular changes in tumors treated with a cytotoxic chemotherapy. Subcutaneous 9L gliosarcomas were treated with a single dose of 1,3-bis(2- chloroethyl) -1- nitrosourea. Both sodium and diffusion imaging modalities were able to detect changes in tumor cellularity as early as 2 days after treatment, which continued to evolve as increased signal intensities reached a maximum ~8 days posttreatment. Early changes in tumor sodium and apparent diffusion coefficient values were predictive of subsequent tumor shrinkage, which occurred ~10 days later.
Overall, therapeutical induced changes in sodium and diffusion values were found to have similar dynamic and spatial changes. These findings suggest that these imaging modalities detected similar early cellular changes after treatment. The results of this study support the continued clinical testing of diffusion MRI for evaluation of early tumor treatment response and demonstrate the complementary insights of sodium MRI for oncology applications.
Oncology; Proton diffusion; Sodium; Magnetic resonance; Tumor; Chemotherapy
This study investigates the comparative changes in the sodium MRI signal and proton diffusion following treatment using a 9L rat glioma model to develop markers of earliest response to cancer therapy. Sodium MRI and proton diffusion mapping were performed on untreated (n = 5) and chemotherapy 1,3-bis(2-chloroethyl)-1-nitrosourea-treated rats (n = 5). Animals were scanned serially at 2- to 3-day intervals for up to 30 days following therapy. The time course of Na concentration in a tumor showed a dramatic increase in the treated brain tumor compared to the untreated tumor, which correlates in time with an increase in tumor water diffusion. The largest posttreatment increase in sodium signal occurred 7–9 days following treatment and correlated to the period of the greatest chemotherapy-induced cellular necrosis based on diffusion and histopathology. Both Na MRI and proton ADC mapping revealed early changes in tumor sodium content and cellularity. This study demonstrates the possibility of Na MRI to function as a biomarker for monitoring early tumor treatment and validates the use of monitoring changes in diffusion MRI values for assessing tumor cellularity.
sodium; MRI; chemotherapy; diffusion; glioma
Fas-Associated protein with Death Domain (FADD) was originally reported as a pro-apoptotic adaptor molecule that mediates receptor induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Over-expression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, we developed a cell based reporter for FADD kinase activity. The reporter assay, optimized for a high throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.
FADD; phosphorylation; non-invasive molecular imaging; bioluminescence; kinase activity
Development of noninvasive, real-time molecular imaging tools to assess responsiveness of a given therapy may be a critical component of the success of individualized therapy approach for patients. Toward this, we have previously developed and validated molecular sensors for Akt and caspase-3 activity, and in this report, we have explored the utility of these reporters in assessing the responsiveness of tumors to a combination of gemcitabine (Gem) and cetuximab (Cet) delivered in two opposite schedules. We found that human head and neck cancer (UMSCC1) xenografts responded significantly better in a schedule where cetuximab was administered after gemcitabine when compared with the schedule of cetuximab followed by gemcitabine. Wilcoxon two-sample tests suggested that the difference in tumor volumes in two schedules became significant on day 7 (P > .05 on day 4, and P < .05 on days 7 and 10), and the difference in activity of Akt in two schedules became significant on day 4 (P < .05 on days 4, 6, and 10). Using Akt reporter activity and cubic spline interpolation, the distinction between the two schedules could be detected 2 days before using the tumor volume, suggesting that molecular imaging of Akt may allow early prediction of therapy responsiveness. We did not observe a significant difference between the two schedules in the caspase-3 activity. In summary, this proof-of-concept study provides a basis for using molecular imaging of Akt as an early indicator of therapeutic efficacy.
The goal of this work was to develop a fast 3D chemical shift imaging technique for the non-invasive measurement of hyperpolarized 13C-labeled substrates and metabolic products at low concentration.
MATERIALS AND METHODS
Multiple echo 3D balanced steady state MR imaging (ME-3DbSSFP) was performed in vitro on a syringe containing hyperpolarized [1,3,3-2H3; 1-13C]2-hydroxyethylpropionate (HEP) adjacent to a 13C-enriched acetate phantom, and in vivo on a rat before and after IV injection of hyperpolarized HEP at 1.5 T. Chemical shift images of the hyperpolarized HEP were derived from the multiple echo data by Fourier transformation along the echoes on a voxel by voxel basis for each slice of the 3D data set.
ME-3DbSSFP imaging was able to provide chemical shift images of hyperpolarized HEP in vivo, and in a rat with isotropic 7 mm spatial resolution, 93 Hz spectral resolution and 16 second temporal resolution for a period greater than 45 seconds.
Multiple echo 3D bSSFP imaging can provide chemical shift images of hyperpolarized 13C-labeled compounds in vivo with relatively high spatial resolution and moderate spectral resolution. The increased signal-to-noise ratio (SNR) of this 3D technique will enable the detection of hyperpolarized 13C-labeled metabolites at lower concentrations as compared to a 2D technique.
hyperpolarized 13C; chemical shift imaging; balanced steady state free precession imaging; spectroscopic imaging