RATIONALE: Treatment of glioblastoma (GBM) remains challenging due in part to its histologic intratumoral heterogeneity that contributes to its overall poor treatment response. Our goal was to evaluate a voxel-based biomarker, the functional diffusion map (fDM), as an imaging biomarker to detect heterogeneity of tumor response in a radiation dose escalation protocol using a genetically engineered murine GBM model. EXPERIMENTAL DESIGN: Twenty-four genetically engineered murine GBM models [Ink4a-Arf-/-/Ptenloxp/loxp/Ntv-a RCAS/PDGF(+)/Cre(+)] were randomized in four treatment groups (n = 6 per group) consisting of daily doses of 0, 1, 2, and 4 Gy delivered for 5 days. Contrast-enhanced T1-weighted and diffusion-weighted magnetic resonance imaging (MRI) scans were acquired for tumor delineation and quantification of apparent diffusion coefficient (ADC) maps, respectively. MRI experiments were performed daily for a week and every 2 days thereafter. For each animal, the area under the curve (AUC) of the percentage change of the ADC (AUCADC) and that of the increase in fDM values (AUCfDM+) were determined within the first 5 days following therapy initiation. RESULTS: Animal survival increased with increasing radiation dose. Treatment induced a dose-dependent increase in tumor ADC values. The strongest correlation between survival and ADC measurements was observed using the AUCfDM+ metric (R2 = 0.88). CONCLUSION: This study showed that the efficacy of a voxel-based imaging biomarker (fDM) was able to detect spatially varying changes in tumors, which were determined to be a more sensitive predictor of overall response versus whole-volume tumor measurements (AUCADC). Finally, fDM provided for visualization of treatment-associated spatial heterogeneity within the tumor.
Induction of apoptosis plays a crucial role in the response of tumors to treatment. Thus, we investigated the pharmacodynamics and tumor saturation kinetics of a death receptor 5 antibody (anti-DR5) when combined with chemotherapeutics. For our investigations, we applied an imaging method that allows monitoring of apoptosis noninvasively in living mice. A stably transfected apoptosis reporter based on split luciferase technology facilitates to screen various chemotherapeutics and anti-DR5 on their ability to induce apoptosis in glioblastoma cells in vitro as well as in vivo. We found that doxorubicin (DOX) treatment in vitro led to significant apoptosis induction within 48 hours and to a 2.3-fold increased anti-DR5 binding to the cell surface. In contrast, cisplatin and 5-fluorouracil (5-FU) treatment altered anti-DR5 binding only marginally. Induction of apoptosis by treatment with anti-DR5 was dose- and time-dependent (both in vitro and in vivo). Simultaneous visualization of fluorescence-labeled anti-DR5 in tumor tissue and apoptosis revealed maximal apoptosis induction immediately after the compound had reached tumor site. Regarding combination therapy of anti-DR5 and DOX, we found that the sequential application of DOX before anti-DR5 resulted in synergistically enhanced apoptosis reporter activity. In striking contrast, anti-DR5 given before DOX did not lead to increased apoptosis induction. We suggest that DOX-induced recruitment of DR5 to the cell surface impacts the enhanced apoptotic effect that can be longitudinally monitored by apoptosis imaging. This study demonstrates that the combination of apoptosis and fluorescence imaging is an excellent method for optimizing dosing and treatment schedules in preclinical cancer models.
Loss of bone mass due to disease, such as osteoporosis and metastatic cancer to the bone, is a leading cause of orthopedic complications and hospitalization. Onset of bone loss resulting from disease increases the risk of incurring fractures and subsequent pain, increasing medical expenses while reducing quality of life. Although current standard CT-based protocols provide adequate prognostic information for assessing bone loss, many of the techniques for evaluating CT scans rely on measures based on whole-bone summary statistics. This reduces the sensitivity at identifying local regions of bone resorption, as well as formation. In this study, we evaluate the effectiveness of a voxel-based image post-processing technique, called the Parametric Response Map (PRM), for identifying local changes in bone mass in weight-bearing bones on CT scans using an established animal model of osteoporosis. Serial CT scans were evaluated weekly using PRM subsequent to ovariectomy or sham surgeries over the period of one month. For comparison, bone volume fraction and mineral density measurements were acquired and found to significantly differ between groups starting 3 weeks post-surgery. High resolution ex vivo measurements acquired four weeks post-surgery validated the extent of bone loss in the surgical groups. In contrast to standard methodologies for assessing bone loss, PRM results were capable of identifying local decreases in bone mineral by week 2, which were found to be significant between groups. This study concludes that PRM is able to detect changes in bone mineral with higher sensitivity and spatial differentiation than conventional techniques for evaluating CT scans, which may aid in clinical decision making for patients suffering from bone loss.
ovariectomy; osteoporosis; imaging; CT; response; biomarker
Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However, tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor, tunicamycin, we show that expression levels of several RTKS (EGFR, ErbB2, ErbB3, and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines, two cell lines resistant to epidermal growth factor receptor–targeted therapies, we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison, experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity, suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies.
In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50–100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.
The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of trans-membrane proteins, including receptor tyrosine kinases (RTKs), which are vital for the growth and survival of malignancies. To identify compounds which disrupt the function of the ER and thus could potentially impair cancer cell survival signaling, we adapted a set of glycosylation-sensitive luciferase reporters for the development and optimization of a cell-based high-throughput screen (HTS). Secondary screens for false-positive luciferase activation and tertiary lectin-based and biochemical analyses were also devised for compound triage. Through a pilot screen of 2802 compounds from the National Cancer Institute (NCI) chemical libraries, we identified aclacinomycin (Acm) as a compound that preferentially affects ER function. We report that Acm reduces plasma membrane expression of glycoproteins including epidermal growth factor receptor (EGFR) and Met but does not inhibit N-linked glycosylation or generalized protein translation. Fluorescence microscopy co-localization experiments were also performed and demonstrated Acm accumulation in the ER in further support of the overall HTS design. The consequences of Acm treatment on cell survival were analyzed through clonogenic survival analysis. Consistent with the reduction of EGFR levels, pretreatment with Acm sensitizes the EGFR-mutant non-small cell lung cancer (NSCLC) cell lines HCC827 and HCC2935 to ionizing radiation and did not affect the sensitivity of the RTK-independent and KRAS-mutant A549 NSCLC cell line. Thus, Acm and similar compounds targeting the ER may represent a novel approach for radiosensitizing tumor cells dependent on RTK function.
There is an urgent need for the development of novel therapies to treat pancreatic cancer, which is among the most lethal of all cancers. KRAS activating mutations, which are found in >90% of pancreatic adenocarcinomas, drive tumor dependency on the Ras/MAPK and Akt signaling pathways. Radiation is currently being explored as a component of the standard treatment regimen for pancreatic cancer. This study’s purpose was to test the hypothesis that MEK inhibitors will offer clear therapeutic benefit when integrated into radiotherapy treatment regimens for treatment of this disease. We explored the activation of the MAPK and Akt pathways in response to radiation in multiple pancreatic tumor cell lines. Small molecule inhibitors of MEK (PD0325901) and Akt (API-2) were subsequently evaluated for their radiosensitizing potential alone and in combination. In vivo efficacy was tested in subcutaneous MIA-PaCa2 xenografts. Phosphorylated levels of ERK-1/2 and Akt were found to increase in response to radiation treatment in our pancreatic tumor cell line panel. MEK inhibitor-induced radiosensitization was observed in vitro and in vivo. The further addition of an Akt inhibitor to the MEK inhibitor/radiation regimen resulted in enhanced therapeutic gain as determined by increased radiosensitization and tumor cell death. In conclusion, MEK inhibition results in growth arrest, apoptosis, and radiosensitization of multiple preclinical pancreatic tumor models, and the effects can be enhanced by combination with an Akt inhibitor. These results provide rationale for further testing of a treatment regimen in pancreatic cancer that combines MEK inhibition with radiation, optimally in conjunction with Akt inhibition.
MEK-1/2; Akt; PI3-kinase; Radiation; Pancreatic Cancer
Chronic obstructive pulmonary disease (COPD) is increasingly being recognized as a highly heterogeneous disorder, composed of varying pathobiology. Accurate detection of COPD subtypes by image biomarkers are urgently needed to enable individualized treatment thus improving patient outcome. We adapted the Parametric Response Map (PRM), a voxel-wise image analysis technique, for assessing COPD phenotype. We analyzed whole lung CT scans of 194 COPD individuals acquired at inspiration and expiration from the COPDGene Study. PRM identified the extent of functional small airways disease (fSAD) and emphysema as well as provided CT-based evidence that supports the concept that fSAD precedes emphysema with increasing COPD severity. PRM is a versatile imaging biomarker capable of diagnosing disease extent and phenotype, while providing detailed spatial information of disease distribution and location. PRMs ability to differentiate between specific COPD phenotypes will allow for more accurate diagnosis of individual patients complementing standard clinical techniques.
PURPOSE: The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2)-mediated increased DNA repair capability, which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate diffusion-weighted magnetic resonance imaging (DW-MRI) as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. EXPERIMENTAL DESIGN: We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. DW-MRI and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. RESULTS: AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53 wild-type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. CONCLUSION: DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA damage-induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM.
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
Genetically engineered mouse (GEM) models of ovarian cancer that closely recapitulate their human tumor counterparts may be invaluable tools for preclinical testing of novel therapeutics. We studied murine ovarian endometrioid adenocarcinomas (OEAs) arising from conditional dysregulation of canonical WNT and PI3K/AKT/mTOR pathway signaling to investigate their response to conventional chemotherapeutic drugs and mTOR or AKT inhibitors.
OEAs were induced by injection of adenovirus expressing Cre recombinase (AdCre) into the ovarian bursae of Apcflox/flox;Ptenflox/flox mice. Tumor-bearing mice or murine OEA-derived cell lines were treated with cisplatin and paclitaxel, mTOR inhibitor rapamycin, or AKT inhibitors API-2 or perifosine. Treatment effects were monitored in vivo by tumor volume and bioluminescence imaging, in vitro by WST-1 proliferation assays, and in OEA tissues and cells by immunoblotting and immunostaining for levels and phosphorylation status of PI3K/AKT/mTOR signaling pathway components.
Murine OEAs developed within 3 weeks of AdCre injection and were not preceded by endometriosis. OEAs responded to cisplatin + paclitaxel, rapamycin, and AKT inhibitors in vivo. In vitro studies showed that response to mTOR and AKT inhibitors, but not conventional cytotoxic drugs, was dependent on the status of PI3K/AKT/mTOR signaling. AKT inhibition in APC−/PTEN− tumor cells resulted in compensatory up-regulation of ERK signaling.
The studies demonstrate the utility of this GEM model of ovarian cancer for pre-clinical testing of novel PI3K/AKT/mTOR signaling inhibitors and provide evidence for compensatory signaling, suggesting that multiple rather than single agent targeted therapy will be more efficacious for treating ovarian cancers with activated PI3K/AKT/mTOR signaling.
ovarian carcinoma; endometriosis; PI3K/AKT/mTOR signaling; mouse model; bioluminescence imaging
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
Improvements in technology and resources are helping to advance our understanding of cancer-initiating events as well as factors involved with tumor progression, adaptation, and evasion of therapy. Tumors are well known to contain diverse cell populations and intratumor heterogeneity affords neoplasms with a diverse set of biologic characteristics that can be used to evolve and adapt. Intratumor heterogeneity has emerged as a major hindrance to improving cancer patient care. Polygenic cancer drug resistance necessitates reconsidering drug designs to include polypharmacology in pursuit of novel combinatorial agents having multitarget activity to overcome the diverse and compensatory signaling pathways in which cancer cells use to survive and evade therapy. Advances will require integration of different biomarkers such as genomics and imaging to provide for more adequate elucidation of the spatially varying location, type, and extent of diverse intratumor signaling molecules to provide for a rationale-based personalized cancer medicine strategy.
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
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
The epidermal growth factor receptor (EGFR) has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90) is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT) WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA) and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.
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
The effectiveness of the radiosensitizer gemcitabine (GEM) was evaluated in a mouse glioma along with the imaging biomarker diffusion-weighted magnetic resonance imaging (DW-MRI) for early detection of treatment effects. A genetically engineered murine GBM model [Ink4a-Arf−/− PtenloxP/loxP/Ntv-a RCAS/PDGF(+)/Cre(+)] was treated with gemcitabine (GEM), temozolomide (TMZ) +/− ionizing radiation (IR). Therapeutic efficacy was quantified by contrast-enhanced MRI and DW-MRI for growth rate and tumor cellularity, respectively. Mice treated with GEM, TMZ and radiation showed a significant reduction in growth rates as early as three days post-treatment initiation. Both combination treatments (GEM/IR and TMZ/IR) resulted in improved survival over single therapies. Tumor diffusion values increased prior to detectable changes in tumor volume growth rates following administration of therapies. Concomitant GEM/IR and TMZ/IR was active and well tolerated in this GBM model and similarly prolonged median survival of tumor bearing mice. DW-MRI provided early changes to radiosensitization treatment warranting evaluation of this imaging biomarker in clinical trials.
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