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Detecting Epidermal Growth Factor Receptor Tumor Activity In Vivo During Cetuximab Therapy of Murine Gliomas

Posted Dec 31, 2009 Updated Apr 7, 2023
By Summer L. Gibbs-Strauss PhD , Kimberley S. Samkoe PhD , Julia A. O'Hara PhD , Scott C. Davis PhD , P. Jack Hoopes DVM PhD , Tayyaba Hasan PhD , Brian W. Pogue PhD
9 min read

Rationale and Objectives

Noninvasive molecular imaging of glioma tumor receptor activity was assessed with diagnostic in vivo fluorescence monitoring during targeted therapy. The study goals were to assess the range of use for treatment monitoring and stratification of tumor types using epidermal growth factor (EGF) receptor (EGFR) status with administration of fluorescently labeled EGF and determine its utility for tumor detection compared to magnetic resonance imaging (MRI).

Materials and Methods

EGFR+ and EGFR− glioma tumor lines (human glioma [U251-GFP] and rat gliosarcoma [9L-GFP], respectively) were used to assess these goals, having a 20-fold difference between their EGF uptakes.

Results

Treatment with cetuximab in the EGFR+ tumor-bearing animals led to decreased EGF tumor uptake, whereas for the EGFR− tumors, no change in fluorescence signal followed treatment. This diagnostic difference in EGFR expression could be used to stratify the tumor-bearing animals into groups of potential responders and nonresponders, and receiver-operating characteristic curve analysis revealed an area under the curve (AUC) of 0.92 in separating these tumors. The nonlocalized growth pattern of U251-GFP tumors resulted in detection difficulty on standard MRI, but high EGFR expression made them detectable by fluorescence imaging (AUC = 1.0). The EGFR+ U251-GFP tumor–bearing animals could be noninvasively stratified into treated and untreated groups on the basis of fluorescence intensity difference ( P = .035, AUC = 0.90).

Conclusions

EGFR expression was tracked in vivo with fluorescence and determined to be of use for the stratification of EGFR+ and EGFR− tumors, the detection of EGFR+ tumors, and monitoring of molecular therapy.

Although malignant gliomas account for a small percentage of cancers diagnosed, they have proved to be some of the least responsive to aggressive therapies, including surgical resection, radiation therapy, and temozolomide chemotherapy . As a result, average patient survival from the date of diagnosis is in the range of 9 to 12 months , and morbidity rates approach 100%. Given the poor performance of conventional therapies, new treatment approaches are necessary. Glioblastoma multiforme (GBM) has a defined set of “molecular lesions” as well as identified disruptions in signaling pathways and therefore may respond more readily to newly developed targeted therapies . One such target is epidermal growth factor (EGF) receptor (EGFR), which is overexpressed in 40% to 50% of all GBMs . Overexpression of EGFR appears to be correlated with glioma grade, and an unfavorable prognostic relationship has been shown between EGFR amplification and overall survival of patients with GBM . Part of the difficulty in the development and testing of targeted therapies is the need to track biomarkers and surrogate measures of response to therapy in individual subjects. In particular, molecular expression is known to increase or decrease significantly in response to therapy, and the ability to track expression during courses of therapy may become critically important. In this study, a novel optical method was considered to monitor targeted therapy diagnostically in vivo.

Overexpression of EGFR in GBM as well as many other types of cancer has been shown to promote the development and progression of malignancy given its association with cellular proliferation, angiogenesis, metastasis, and apoptosis inhibition . Anti-EGFR monoclonal antibodies target the extracellular domain of the receptor and have been shown to successfully block its activation . The IMC-C225, or cetuximab, antibody has been commercially developed by ImClone Systems Inc., (New York, NY) and is in clinical trials for several cancer types. Clinical trials for GBMs are still in their preliminary stages, although preclinical data suggested that systemic cetuximab treatment decreased proliferation and increased apoptosis in xenograft models of GBM grown both subcutaneously and orthotopically, when the cell line overexpressed EGFR . The ability to noninvasively track monoclonal antibody binding, tumor response, and EGFR expression in situ would have considerable value if it could be performed reliably with high sensitivity. Tumor tissue changes at the molecular level occur prior to any detectable tumor size changes, so the ability to dynamically and quantitatively assess and track the molecular profile in vivo could provide substantial patient benefit. Additionally, there is evidence to support the fact that signaling pathways change in response to receptor blocking therapy , so having the ability to track dynamic changes could significantly improve the potential of developing multireceptor targeting approaches.

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Materials and methods

Cell Culture and Brain Tumor Model

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In Vitro Fluorescence Monitoring of Cetuximab

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Multichannel Transmission Fluorescence Spectroscopy

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Figure 1, (a) Eight of the 16 available channels from the multichannel transmission spectroscopic system were used to encircle the head of the mouse in a coronal plane during measurement collection. The in vivo experimental timeline is shown (b) , in which the experiment began with tumor implantation on day 0. Each mouse in the study underwent magnetic resonance imaging (M), administration of endothelial growth factor–conjugated IRDye 800CW (LI-COR Biosciences, Lincoln, NE) (D), and fluorescence spectroscopic monitoring (S) on the days shown. Only mice in the treated groups received cetuximab therapy (C) on the days shown.

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Spectral Data Postprocessing

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Magnetic Resonance Imaging (MRI) of Murine Brain Tumors

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In Vivo Fluorescence Monitoring of Cetuximab

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Ex Vivo Fluorescence Analysis

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Statistical Analysis

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Results

In Vitro Fluorescence Monitoring of Cetuximab Therapy

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Figure 2, The endothelial growth factor (EGF) uptake of the (a) human glioma (U251-GFP) cells and (b) rat gliosarcoma (9L-GFP) cells as measured by EGF-bound Alexa Fluor 647 (EGF-AF647; Invitrogen Corporation, Carlsbad, CA) fluorescence, following varied incubation times with difference concentrations of cetuximab (Erbitux; ImClone Systems, New York, NY). “Ctrl” (control) indicates fluorescence for cells that were not incubated with EGF-AF647 to show the background signal at the excitation and emission wavelength used to measure the EGF-AF647 fluorescence. “Untreated” indicates the EGF-AF647 fluorescence signal from cells that were not treated with cetuximab therapy. Each bar represents the average of three samples, and the error bars show the standard deviation of the mean.

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Detection of EGFR+ and EGFR− Glioma with MRI and In Vivo Spectroscopy

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Table 1

ROC Curve Analysis for MRI, EGF-IRDye In Vivo Spectroscopy, and Ex Vivo Measurements

9L-GFP U251-GFP Treated vs Control Untreated vs Control Treated vs Untreated Treated vs Control Untreated vs Control Treated vs Untreated 9L-GFP Untreated vs U251-GFP Untreated T1 + Gd 1.00 1.00 0.57 0.50 0.54 0.50 0.96 T2 FLAIR 1.00 1.00 0.75 0.50 0.58 0.53 1.00 T1 FFE 1.00 0.94 0.69 0.75 0.88 0.70 0.75 T1 IR 1.00 1.00 1.00 0.85 0.83 0.60 1.00 T1 difference 1.00 1.00 0.50 0.60 0.50 0.57 1.00 Week 1, 24 h 0.50 0.50 0.50 0.78 0.88 0.51 0.92 Week 2, 24 h 0.85 0.72 0.67 0.88 1.00 0.90 0.80 Ex vivo 1.00 1.00 0.67 0.58 1.00 0.79 0.73

EGF-IRDye, endothelial growth factor–conjugated IRDye 800CW (LI-COR Biosciences, Lincoln, NE); FFE, fast-field echo; FLAIR, fluid-attenuated inversion recovery; Gd, gadolinium; IR, inversion recovery; 9L-GFP, rat gliosarcoma; ROC, receiver-operating characteristic curve; U251-GFP, human glioma.

Values represent area under the ROC curves.

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Noninvasive In Vivo Fluorescence Stratification of EGFR+ and EGFR− Glioma

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Figure 3, ( a ) Endothelial growth factor (EGF)–conjugated IRDye 800CW (LI-COR Biosciences, Lincoln, NE) fluorescence on day 14 of the experiment for the treated and untreated human glioma (U251-GFP) and rat gliosarcoma (9L-GFP) groups as well as the control (Ctrl) group. (b) Untreated EGF receptor–positive U251-GFP and EGF receptor–negative 9L-GFP tumors could be stratified using receiver-operating characteristic analysis (area under the curve = 0.92). FPF, false-positive fraction; TPF, true-positive fraction; Tx, treatment.

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Noninvasive Cetuximab Treatment Monitoring Using Fluorescently Labeled EGF

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Figure 4, Endothelial growth factor (EGF)–conjugated IRDye 800CW (EGF-IRDye; LI-COR Biosciences, Lincoln, NE) fluorescence during the second week of the experiment, 24 hours after administration (day 22) for the treated and untreated (a) human glioma (U251-GFP) and (b) rat gliosarcoma (9L-GFP) tumor-bearing animals. The mice were sacrificed on day 24 of the experiment and their brains analyzed by (c) green fluorescent protein (GFP) fluorescence for tumor size, shown as a percentage of the brain and (d) EGF-IRDye fluorescence. The EGF-IRDye tumor (T)–to–brain tissue (N) fluorescence was normalized to tumor size as determined by GFP fluorescence. Ctrl, control; Tx, treatment.

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Ex Vivo EGF-IRDye Fluorescence Verification and Tumor Size Comparison

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Figure 5, Ex vivo coronal images of representative examples from the human glioma (U251-GFP) tumor-bearing group. Two example mice each from the U251-GFP cetuximab-treated group (a,b) and the U251-GFP cetuximab-untreated group (c,d) are shown. Endothelial growth factor (EGF)–conjugated IRDye 800CW (EGF-IRDye; LI-COR Biosciences, Lincoln, NE) fluorescence (row 1) was compared to green fluorescent protein (GFP) fluorescence (row 2) and hematoxylin and eosin (H&E)–stained images (row 3) and demonstrated decreased fluorescence in the treated group compared to the untreated group. Representative examples of in vivo magnetic resonance images showed that the U251-GFP tumors were difficult to detect using T1 turbo spin-echo (TSE) contrast-enhanced (CE) magnetic resonance imaging (row 4) or T2 fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (row 5).

Figure 6, Ex vivo coronal images of representative examples from the rat gliosarcoma (9L-GFP) tumor-bearing group. Two example mice each from the 9L-GFP cetuximab-treated group (a,b) and the 9L-GFP cetuximab-untreated group (c,d) . Endothelial growth factor (EGF)–conjugated IRDye 800CW (EGF-IRDye; LI-COR Biosciences, Lincoln, NE) fluorescence (row 1) was compared to green fluorescent protein (GFP) fluorescence (row 2) and hematoxylin and eosin (H&E)–stained images (row 3) and demonstrated heterogenous fluorescence within the tumor tissue that was similar for the treated and untreated tumors. Representative examples of in vivo magnetic resonance images showed visible tumor masses by T1 turbo spin-echo (TSE) contrast-enhanced (CE) magnetic resonance imaging (row 4), T2 TSE and T2 fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (row 5).

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Discussion

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Conclusions

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Academic Radiology, Volume 17, Issue 1
Journals General Radiology
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