In vivo visualization of GL261-luc2 mouse glioma cells by use of Alexa Fluor–labeled TRP-2 antibodies

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Object

For patients with glioblastoma multiforme, median survival time is approximately 14 months. Longer progression-free and overall survival times correlate with gross-total resection of tumor. The ability to identify tumor cells intraoperatively could result in an increased percentage of tumor resected and thus increased patient survival times. Available labeling methods rely on metabolic activity of tumor cells; thus, they are more robust in high-grade tumors, and their utility in low-grade tumors and metastatic tumors is not clear. The authors demonstrate intraoperative identification of tumor cells by using labeled tumor-specific antibodies.

Methods

GL261 mouse glioma cells exhibit high expression of a membrane-bound protein called second tyrosinase-related protein (TRP-2). The authors used these cells to establish an intracranial, immunocompetent model of malignant glioma. Antibodies to TRP-2 were labeled by using Alexa Fluor 488 fluorescent dye and injected into the tail vein of albino C57BL/6 mice. After 24 hours, a craniotomy was performed and the tissue was examined in vivo by using an Optiscan 5.1 handheld portable confocal fiber-optic microscope. Tissue was examined ex vivo by using a Pascal 5 scanning confocal microscope.

Results

Labeled tumor cells were visible in vivo and ex vivo under the respective microscopes.

Conclusions

Fluorescently labeled tumor-specific antibodies are capable of binding and identifying tumor cells in vivo, accurately and specifically. The development of labeled markers for the identification of brain tumors will facilitate the use of intraoperative fluorescence microscopy as a tool for increasing the extent of resection of a broad variety of intracranial tumors.

Abbreviations used in this paper:5-ALA = 5-aminolevulinic acid; DMEM = Dulbecco's modified Eagle medium; PBS = phosphate-buffered saline; SAIVI = Small Animal In Vivo Imaging; TRP-2 = second tyrosinase-related protein.

Abstract

Object

For patients with glioblastoma multiforme, median survival time is approximately 14 months. Longer progression-free and overall survival times correlate with gross-total resection of tumor. The ability to identify tumor cells intraoperatively could result in an increased percentage of tumor resected and thus increased patient survival times. Available labeling methods rely on metabolic activity of tumor cells; thus, they are more robust in high-grade tumors, and their utility in low-grade tumors and metastatic tumors is not clear. The authors demonstrate intraoperative identification of tumor cells by using labeled tumor-specific antibodies.

Methods

GL261 mouse glioma cells exhibit high expression of a membrane-bound protein called second tyrosinase-related protein (TRP-2). The authors used these cells to establish an intracranial, immunocompetent model of malignant glioma. Antibodies to TRP-2 were labeled by using Alexa Fluor 488 fluorescent dye and injected into the tail vein of albino C57BL/6 mice. After 24 hours, a craniotomy was performed and the tissue was examined in vivo by using an Optiscan 5.1 handheld portable confocal fiber-optic microscope. Tissue was examined ex vivo by using a Pascal 5 scanning confocal microscope.

Results

Labeled tumor cells were visible in vivo and ex vivo under the respective microscopes.

Conclusions

Fluorescently labeled tumor-specific antibodies are capable of binding and identifying tumor cells in vivo, accurately and specifically. The development of labeled markers for the identification of brain tumors will facilitate the use of intraoperative fluorescence microscopy as a tool for increasing the extent of resection of a broad variety of intracranial tumors.

Glioblastoma multiforme is an aggressive form of brain tumor that typically recurs; the overall patient median survival time is approximately 14 months.19 For these patients, progression-free and overall survival times correlate with the percentage of tumor remaining after resection.13,19,22,26 The ability to obtain a gross-total resection is complicated by tumor characteristics such as their highly proliferative and diffuse infiltrative nature and difficulty identifying tumor cells by sight in the intraoperative bed. Magnetic resonance imaging is used to determine the ideal surgical approach, and intraoperative navigation with the use of a surgical microscope and/or intraoperative MRI47 helps define the location and extent of disease. However, the ability to label tumor cells in vivo could result in increased sensitivity and specificity, leading to an increased percentage of tumor resected and thus increased patient survival time.

One of the most recent intraoperative imaging aids is 5-aminolevulinic acid (5-ALA), which is metabolized within the cell, producing protoporphyrin IX, a fluorescent compound that can be seen intraoperatively by using fluorescence microscopes equipped with the appropriate wavelength of light.3,7,8,23,29,30,32,38,39,41 Discovery and subsequent development of 5-ALA for use as a tumor-specific imaging dye has facilitated resection beyond the tumor bulk into the diffusely infiltrative margins because it enhances the surgeon's ability to visualize tumor cells. Indeed, in a number of trials in which patients with malignant gliomas were given 5-ALA before tumor resection, 5-ALA led to an increased extent of tumor resection.7,26,29,32,37–39,42,43 In addition, the extent of resection improves when 5-ALA and intraoperative confocal microscopy are used in combination with neuronavigation, PET, and other imaging modalities.10,11,14,26,27,30,37 Despite these improvements, however, questions remain as to the sensitivity and specificity of this approach, particularly (but not exclusively) for low-grade tumors.4,10,14,27,39–41 Studies designed to identify mechanisms of false-positive and false-negative results have shown effects from differences in the levels of tumor cell metabolism, intraoperative microscope sensitivity, 5-ALA uptake, 5-ALA metabolism (resulting from intrinsic molecular characteristics of the tumor and/or interactions with other therapies), diffusion of protoporphyrin IX, and other factors.4,8,16,23,27,29,36,40–42 Despite these caveats, the improved patient outcomes reported after use of 5-ALA underscore the utility of tumor markers that can be used intraoperatively to improve tumor cell identification and facilitate more complete resections.

Use of an intraoperative tumor marker without the limitations described above would help facilitate the use of intraoperative imaging for the resection of brain tumors. We describe proof-of-concept for the identification of tumor cells in vivo by exploiting a membrane-bound protein marker in the GL261 mouse model of malignant glioma. The identification and exploitation of unique protein expression in tumors can enhance resection and ensure accurate tumor cell detection regardless of metabolic activity. We have identified one such protein in the GL261 mouse glioma cell line: second tyrosinase-related protein (TRP-2). This single-pass membrane-bound surface protein has been shown by real-time reverse transcription polymerase chain reaction to contain mRNA transcript in excess of 100,000-fold over that in healthy brain.18 We chose this protein as a marker for GL261 cells in vivo because of its overexpression and cellular location. Alexa Fluor 488– or Alexa Fluor 750– (Molecular Probes, Life Technologies Corp.) labeled antibodies bound to tumor cells in vivo and were detectable by handheld portable fiber-optic confocal endomicroscopes: a 488-nm visible-wavelength system (Optiscan 5.1, Optiscan Pty. Ltd., and Carl Zeiss Surgical, GmbH) and a 780-nm near-infrared laser system (Optiscan Pty. Ltd and Carl Zeiss Surgical, GmbH). This proof-of-concept work suggests that the future utility of intraoperative portable confocal endomicroscopy for the resection of gliomas may lie in the design and use of tumor-specific labeled antibodies.

Methods

Cell Culture

GL261 mouse glioma cells were obtained from the Division of Cancer Treatment and Diagnosis Tumor Repository (National Cancer Institute), and B16-F1 mouse melanoma cells were obtained from the American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle medium (DMEM, Invitrogen Corp.) supplemented with 10% fetal calf serum at 37°C with 5% CO2. To facilitate quantitative measurement of tumor size, GL261 cells were stably transfected with the gene encoding luc2 by using the pGL4.51[luc2/CMV/Neo] vector (Promega Corp.) and FuGENE6 Transfection Reagent (Roche Applied Science), according to conditions specified by the manufacturer. These cells are designated GL261-luc2 and are maintained in DMEM with 10% fetal calf serum and 100 μg/ml Geneticin (G418, Invitrogen Corp.). Just before implantation, GL261-luc2 cells were harvested by trypsinization, washed, and resuspended at a concentration of 1–2 × 107 cells/ml in DMEM without fetal calf serum.1

Antibody Labeling

TRP-2 antibodies were labeled with either Alexa Fluor 488 or Alexa Fluor 750 (Molecular Probes, Invitrogen Corp.) by using the Small Animal In Vivo Imaging (SAIVI) kits (Invitrogen Corp.) and following the manufacturer's instructions for Alexa Fluor 750, and using the following modifications for labeling with Alexa Fluor 488: 5 μl of sodium bicarbonate (2×), 5 μl of Alexa Fluor 488 (0.3 mg), 2 μl of regulator solution (component B), and 100 μl of IgG (TRP-2 antibody, 1 mg/ml) were combined. The solution was incubated for 60 minutes in the dark, put through the purification column, and 12 fractions of the labeled antibody were collected (900 μl/fraction) and analyzed by using a NanoDrop ND–1000 spectrophotometer (Thermo Scientific). The excitation/emission wavelength of the Alexa Fluor 488 fluorescent tag is within the spectral range of the Optiscan 5.1 system, and the Alexa Fluor 750 is detectable by the Optiscan near-infrared system. Alexa Fluor 750 was used for in vivo imaging with the IVIS Spectrum in vivo imaging system (Life Sciences, a PerkinElmer Co.) because it emits light at 750 nm and thus minimizes interference from autofluorescence, which typically occurs at lower wavelengths of light.

Animal Implantation

C57BL/6-cBrd/cBrd/Cr (albino C57BL/6) mice (National Cancer Institute at Frederick Animal Production Program) were anesthetized by an intraperitoneal injection of ketamine (10 mg/kg) and xylazine (80 mg/kg). GL261-luc2 cells were implanted by following the procedure for intracranial implantation as previously described.2,31 To verify that the implanted GL261-luc2 cells developed tumors in the mice, we gave each mouse a subcutaneous injection of luciferin (15 mg/ml in phosphate-buffered saline [PBS], 150 μg luciferin/kg body weight) 20 minutes before imaging with the IVIS Spectrum in vivo imaging system to determine photon count within the brain. Two cohorts, consisting of 10 mice each, were imaged on Days 23 (Cohort 1) and 9 (Cohort 2) after implantation. Mice were housed in the animal care facility of St. Joseph's Hospital and Medical Center according to the guidelines outlined in the National Institutes of Health Guide for Care and Use of Laboratory Animals and with approval of the St. Joseph's Institutional Animal Care and Use Committee.

In Vitro Cell Imaging

GL261-luc2 cells were plated in a Lab-Tek II 4-well chamber slide with lid (Nalge Nunc International) and maintained at 37°C with 5% CO2. The Alexa Fluor 488–labeled TRP-2 antibody was diluted (4:1) in 5× DMEM plus 50% fetal calf serum. The cells were incubated with labeled TRP-2 antibodies for 48 hours at 37°C and 5% CO2, cells were washed twice with 0.5 ml PBS to remove any unbound antibody, and PBS was added to each well before imaging with the Optiscan 5.1 system. After imaging, cells were washed with PBS and incubated for 1–2 hours in 0.5 ml DMEM plus 10% fetal calf serum. Cells were fixed in 4% paraformaldehyde, rinsed twice with PBS, and a cover slip was placed with VECTASHIELD Mounting Medium with DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories Inc.) before imaging with the Zeiss Pascal 5 confocal microscope (Carl Zeiss MicroImaging, LLC).

In Vivo Cell Imaging

One day before intracranial imaging with the Optiscan 5.1 system, the tail vein of the mice was injected with 100 μl of Alexa Fluor 488–labeled antibody from fraction 9. Mice were anesthetized by an intraperitoneal injection of ketamine (10 mg/kg) and xylazine (80 mg/kg) before undergoing craniotomy. The Optiscan probe was affixed to a stereotactic frame and positioned over the exposed brain. With use of the dorsal/ventral adjustment dials, the Optiscan probe was placed on top of the tumor, ensuring that the probe was in contact with the tissue. Fluorescent tumor cells were imaged in the brain, and the contralateral non-tumor–containing brain was used as a control.

Ex Vivo Immunofluorescence and Histologic Analysis

After in vivo imaging, the brain was removed from the mice and covered in baby powder before being flash frozen in liquid nitrogen and stored at −80°C. The frozen brain was embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek U.S.A. Inc.), cryosectioned at 10 μm, and fixed in 95% ethanol. Standard hematoxylin and eosin staining was performed to verify the presence of tumor in the imaged area. For immunofluorescent detection of TRP-2, 4-μm sections of tissue were fixed in cold 95% ethanol and stored at −20°C until use. Sections were removed from storage at −20°C, incubated in Image IT (Invitrogen Corp.) for 30 minutes, and slides were rinsed in PBS and incubated with TRP-2 antibody diluted 1:2500 (ab74073; Abcam). The slides were then incubated at 4°C overnight, rinsed in PBS, and incubated with Alexa Fluor 488 donkey anti–rabbit IgG (Molecular Probes) for 2 hours at room temperature in the dark and then rinsed as previously described. Tissue sections were counterstained with DAPI in antifade (Vector Laboratories) and imaged on a Zeiss Pascal 5 confocal microscope.

Results

Antibody Concentrations and Degree of Labeling

Antibody concentration was determined at 280 nm, and fluorescence was quantified at 495 nm for Alexa Fluor 488 (Fig. 1 upper) and at 770 nm for Alexa Fluor 750. Degree of labeling was measured by using the formula specified in the SAIVI labeling kit (Fig. 1 lower); however, the correction factor is specific to Alexa Fluor 488 (correction factor = 0.11) and was obtained from Molecular Probes. The formulas used were as follows: [A280 − (A495 × 0.11)]/203,000 = [protein concentration (M)] and A495/71,000 × [protein] = [DOL(moles of dye/moles of protein)].

Fig. 1.
Fig. 1.

TRP-2 labeling. Upper: Antibody concentration of each column fraction as measured by NanoDrop fluorescence at 280 nm. Lower: Degree of labeling of each column fraction.

Fractions 5 and 6 were used for experiments with Alexa Fluor 750–nm labeled TRP-2. Fraction 9 was used for TRP-2 labeled with Alexa Fluor 488 nm because it contained the highest concentration of antibodies and the highest degree of labeling per antibody molecule (1.28 mg/ml, degree of labeling = 89.3).

In Vitro Detection of TRP-2 Expression

To demonstrate that the TRP-2 antibodies retained the ability to specifically bind to the TRP-2 antigens without nonspecific binding after adding a fluorescent tag, we performed immunohistochemical examination by using the Alexa Fluor 488–labeled antibodies on normal mouse brain tissue as a negative control (Fig. 2A). To demonstrate binding of the labeled antibodies, we performed standard immunocytochemical examination on GL261 (Fig. 2B) and B16 mouse melanoma cells (Fig. 2C, positive control) by using the Alexa Fluor 488–labeled TRP-2. Imaging performed by using a Zeiss Pascal 5 laser scanning confocal microscope demonstrated extracellular membrane-bound TRP-2 on the cells (Fig. 2B and C), but no binding was seen in the normal brain tissue. To determine whether the Optiscan system could detect the fluorescently labeled TRP-2 antibodies, it was used to image GL261 mouse glioma cells in vitro. Figure 2D shows that fluorescently labeled TRP-2 antibodies bound to the cell surface are detectable with this system.

Fig. 2.
Fig. 2.

In vitro analysis of TRP-2 expression. Alexa Fluor 488–labeled TRP-2 antibody expression in normal brain (A); GL261 glioma cells in vitro (B and D); B16 melanoma cells in vitro (C) as seen by using the Zeiss Pascal 5 confocal microscope (A–C) and the Optiscan 5.1 system (D).

In Vivo Mouse Malignant Glioma Model System

To determine whether the size of the tumor would affect the binding of the labeled antibodies, perhaps resulting from differences in the extent of the breakdown of the blood-brain barrier, we used 2 cohorts of animals at 2 times after implantation with GL261-luc2 cells. The size of the tumors was determined by bioluminescence after a subcutaneous injection of luciferin. Figure 3 shows the tumor sizes, expressed as photon counts, for the 2 cohorts of animals used in these experiments.

Fig. 3.
Fig. 3.

Tumor size as measured by bioluminescence. Photon counts of Cohort 1 and Cohort 2 on Day 23 and Day 9 after implantation. Data are shown as mean photon counts ± 95% confidence intervals.

Standard histologic examination was performed to demonstrate the pathologic features of the GL261 tumor. Figure 4A shows a tumor section stained with hematoxylin and eosin, demonstrating the cellularity of the tumor and the appearance of the invading front. Immunofluorescence was used to demonstrate the TRP-2 antibody affinity for GL261 tumor cells. The specificity of TRP-2 antibodies can be visualized by both the bulk tumor area in green and by the individual tumor cells, which are advancing away from the tumor border (Fig. 4B).

Fig. 4.
Fig. 4.

A: H & E–stained border between tumor and normal brain. B: Immunofluorescence demonstrating the specificity of TRP-2 antibodies. Arrows indicate individual migrating tumor cells.

In Vivo Imaging of TRP-2–Positive Tumor Cells

The optimum time for imaging with the Optiscan system after antibody infusion was determined by labeling the TRP-2 antibody with Alexa Fluor 750 and using the IVIS in vivo imaging system to image over a 72-hour period (Fig. 5). The need for serial in vivo imaging precluded the use of a craniotomy, so Alexa Fluor 750 was used instead of Alexa Fluor 488 to circumvent the confounding effects of autofluorescence seen at 488 nm in the absence of a craniotomy. The background consisting of free dye and unbound antibodies decreased over the first 24 hours, and then the signal remained relatively stable. We therefore performed the Optiscan imaging 24 hours after infusion.

Fig. 5.
Fig. 5.

Distribution of the Alexa Fluor 750–labeled TRP-2 antibody over a 72-hour period. Mice were identified with an “M” and a number. Each mouse was imaged at 2 time points. Two control mice did not receive labeled TRP-2, and each was imaged 1 time. Fluorescence images of mice injected with Alexa Fluor–750 labeled antibodies were obtained at 8, 24, 32, 48, and 72 hours after injection.

Labeled antibodies were injected into the tail vein of the mice, and 24 hours later the mice underwent a craniotomy and tumor cells were visualized through the Optiscan endomicroscope. Fluorescence was localized to the cellular membrane, without staining of nuclei and extracellular space. Nuclei appeared as dark shadows contrasted against the fluorescent membrane. Images of tumor areas revealed atypia, mitosis, and pleomorphism. However, the tumor mass was not stained homogeneously, precluding visualization of the interface between the tumor and normal brain (Fig. 6A). Within normal brain, TRP-2 provided extremely weak fluorescence, and the architectural characteristics of normal brain could not be identified (Fig. 6B). A mouse that had not received an implantation of tumor cells was used as a negative control to demonstrate that normal cells were not labeled with the TRP-2 antibodies (Fig. 6C).

Fig. 6.
Fig. 6.

In vivo visualization of tumor cells by using the Optiscan 5.1 endomicroscope. Mice received an intravenous injection of labeled anti–TRP-2 antibodies 24 hours before imaging. A: Central area of tumor. B: Normal brain adjacent to tumor. C: Brain of mouse that did not have an implanted tumor.

Discussion

Intraoperative imaging of brain tumors has greatly enhanced the accuracy and extent of their surgical resection. However, despite advances in imaging technology, the identification of isolated invasive tumor cells is still difficult, particularly in lower grade tumors. The use of 5-ALA before surgery as a fluorescent label for tumor cells is gaining popularity, and it seems to be helpful for the resection of high-grade gliomas; however, there are some caveats to its use. Work by Valdés et al.,41 using ex vivo analyses of tissue labeled in vivo, demonstrated that the 5-ALA metabolite protoporphyrin IX identified regions of increasing malignancy in low-grade and high-grade gliomas; however, approximately 40% of biopsy sites that were positive for tumor according to standard microscopy were not visibly fluorescent under the operating microscope. This work suggests the need for increased sensitivity for intraoperative detection of protoporphyrin IX fluorescence, but increased sensitivity of fluorescence detection might also increase the false-positive detection of tumor cells. It has been shown for both metastatic and recurrent brain tumors that 5-ALA can promote the resection of normal nontumor brain tissue because of inaccurate fluorescent labeling with protoporphyrin IX.39,40 These authors suggested that the increased resection of normal brain might be caused by the degradation of the blood-brain barrier, which might allow the metabolized form of 5-ALA, protoporphyrin IX, to leak out of the tumor cells into nontumor edematous areas of the brain. Although the extent to which this may or may not occur is somewhat controversial, there is a need for the development of instruments with increased sensitivity for the detection of intraoperative fluorescence and a need for methods to increase specificity.21,28 Additional studies are also needed to identify therapeutic agents and intrinsic molecular characteristics that affect the metabolism of 5-ALA, such as has been seen with phenytoin,16 cadherin 13 expression,34 aquaporin,33 and ferrochelatase expression.36

Other dyes, such as methylene blue, are being studied for their ability to enhance the morphologic image;46 however, the utility of this approach relies on the surgeon's ability to recognize sometimes subtle morphological characteristics. Eschbacher et al.9 showed that a variety of brain tumors (including meningiomas, schwannomas, gliomas, and a hemangioblastoma) could be visualized by using confocal microscopy. This method was compared with use of routine neuropathological techniques to examine corresponding biopsy samples. The intraoperative confocal endomicroscopic imaging correlated well with standard histology, and 26 (92.9%) of 28 lesions were diagnosed correctly. These studies demonstrate a use for intraoperative confocal endomicroscopy; however, the full use of this technology will be realized when tumor-specific markers that take advantage of the strengths of this technology are available.

The work presented here is proof-of-concept that fluorescently labeled tumor-specific antigens are capable of binding and identifying tumor cells in vivo accurately and specifically in a mouse model of malignant glioma. We searched the literature to find an appropriate surface protein for these studies, and we found the cellular protein TRP-2, a membrane-bound protein that is highly expressed in the mouse malignant glioma cell line, GL261. We exploited this overexpression to identify GL261 mouse glioma cells in vitro, in vivo, and ex vivo. We efficiently labeled TRP-2 with Alexa Fluor 488 and Alexa Fluor 750 and visualized the bound protein within the mouse brain. Alexa Fluor 488 was used in conjunction with the Optiscan 5.1 system. Although the use of this dye is not optimal for most imaging modalities because of interference from tissue autofluorescence at this wavelength, it imitates similar procedures that are being investigated in a clinical environment. We found that binding of these fluorescent antibodies was specific and efficient enough to identify the tumor boundary in vivo without attenuation of the dye or interference from autofluorescence. By using this method, we were able to correlate regions of the tumor in real time by using the Zeiss Optiscan 5.1 handheld confocal microscope and ex vivo by using a standard Zeiss Pascal 5 scanning confocal microscope. This technique enabled accurate identification of tumor cells in vivo, while bound antigen was absent on the surface of the normal non-tumor–containing brain tissue.

Our work supports the concept that identification of individual tumor cells can be done intraoperatively, regardless of cell type or metabolism, in a mouse model system. The development and use of antigens specific to unique tumor cell types would obviate the need for tumor cells to metabolize the compound to produce a fluorescent product, thus allowing for a more dependable and reliable cell marker during surgery. Moving this model to the clinic will first require the identification of antigens unique to brain tumor cells and the discovery of nontoxic labeling compounds that can be attached to these antigens without reducing their ability to cross the human blood-brain barrier and bind to tumor cells. Support for the feasibility of this approach in the future comes from the fact that surface molecules unique to glioma cells such as EGFRvIII have been identified.17,20,35,44,45 Furthermore, the explosion of genomic information available as a result of initiatives such as The Cancer Genome Atlas is likely to facilitate the identification of a number of additional targets. The use of therapeutic antibodies for the treatment of malignant glioma is not a new idea,5,6,12,24,25,48 suggesting that the use of antibodies for labeling tumor cells should be possible.15 The development of labeled markers for the identification of a variety of brain tumors can facilitate the use of intraoperative fluorescence microscopy as a tool to increase the extent of resection of brain tumors.

Conclusions

Fluorescently labeled tumor-specific antibodies are capable of binding and identifying tumor cells in vivo, accurately and specifically. The development of labeled markers for the identification of brain tumors will facilitate the use of intraoperative fluorescence microscopy as a tool for increasing the extent of resection of a broad variety of intracranial tumors.

Acknowledgments

We thank Caliper Life Sciences, a PerkinElmer company (Hopkinton, MA) for assistance with the in vivo time course imaging and Carl Zeiss Meditec AG (Oberkochen, Germany) for providing the Optiscan 5.1 instrument. We also thank Eric C. Woolf for technical assistance.

Disclosure

This work was supported by Students Supporting Brain Tumor Research (to A.C.S.), the Barrow Neurological Foundation, and the Newsome Endowed Chair in Neurosurgery Research held by Dr. Preul.

Author contributions to the study and manuscript preparation include the following. Conception and design: Scheck, Preul. Acquisition of data: Fenton, Martirosyan, Abdelwahab. Analysis and interpretation of data: Scheck, Fenton, Martirosyan, Abdelwahab, Coons. Drafting the article: Fenton, Martirosyan, Abdelwahab. Critically revising the article: Scheck. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Scheck. Study supervision: Scheck, Preul.

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  • 36

    Teng LNakada MZhao SGEndo YFuruyama NNambu E: Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy. Br J Cancer 104:7988072011

  • 37

    Tsugu AIshizaka HMizokami YOsada TBaba TYoshiyama M: Impact of the combination of 5-aminolevulinic acid-induced fluorescence with intraoperative magnetic resonance imaging-guided surgery for glioma. World Neurosurg 76:1201272011

  • 38

    Tykocki TMichalik RBonicki WNauman P: Fluorescence-guided resection of primary and recurrent malignant gliomas with 5-aminolevulinic acid. Preliminary results. Neurol Neurochir Pol 46:47512012

  • 39

    Utsuki SMiyoshi NOka HMiyajima YShimizu SSuzuki S: Fluorescence-guided resection of metastatic brain tumors using a 5-aminolevulinic acid-induced protoporphyrin IX: pathological study. Brain Tumor Pathol 24:53552007

  • 40

    Utsuki SOka HSato SShimizu SSuzuki STanizaki Y: Histological examination of false positive tissue resection using 5-aminolevulinic acid-induced fluorescence guidance. Neurol Med Chir (Tokyo) 47:2102142007

  • 41

    Valdés PAKim ABrantsch MNiu CMoses ZBTosteson TD: δ-aminolevulinic acid-induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy. Neuro Oncol 13:8468562011

  • 42

    Valdés PALeblond FKim AHarris BTWilson BCFan X: Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker. Clinical article. J Neurosurg 115:11172011

  • 43

    von Campe GMoschopulos MHefti M: 5-Aminolevulinic acid-induced protoporphyrin IX fluorescence as immediate intraoperative indicator to improve the safety of malignant or high-grade brain tumor diagnosis in frameless stereotactic biopsies. Acta Neurochir (Wien) 154:5855882012

  • 44

    Wikstrand CJMcLendon REFriedman AHBigner DD: Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII. Cancer Res 57:413041401997

  • 45

    Wikstrand CJReist CJArcher GEZalutsky MRBigner DD: The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target. J Neurovirol 4:1481581998

  • 46

    Wirth DSnuderl MSheth SKwon CSFrosch MPCurry W: Identifying brain neoplasms using dye-enhanced multimodal confocal imaging. J Biomed Opt 17:0260122012

  • 47

    Wirtz CRTronnier VMBonsanto MMKnauth MStaubert AAlbert FK: Image-guided neurosurgery with intraoperative MRI: update of frameless stereotaxy and radicality control. Stereotact Funct Neurosurg 68:39431997

  • 48

    Wu AHXiao JAnker LHall WAGregerson DSCavenee WK: Identification of EGFRvIII-derived CTL epitopes restricted by HLA A0201 for dendritic cell based immunotherapy of gliomas. J Neurooncol 76:23302006

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Article Information

Address correspondence to: Adrienne C. Scheck, Ph.D., Neuro-Oncology and Neurosurgery Research, Barrow Neurological Institute of SJHMC, 350 W. Thomas Rd., Phoenix, AZ 85013. email: adrienne.scheck@dignityhealth.org.

Please include this information when citing this paper: DOI: 10.3171/2013.12.FOCUS13488.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    TRP-2 labeling. Upper: Antibody concentration of each column fraction as measured by NanoDrop fluorescence at 280 nm. Lower: Degree of labeling of each column fraction.

  • View in gallery

    In vitro analysis of TRP-2 expression. Alexa Fluor 488–labeled TRP-2 antibody expression in normal brain (A); GL261 glioma cells in vitro (B and D); B16 melanoma cells in vitro (C) as seen by using the Zeiss Pascal 5 confocal microscope (A–C) and the Optiscan 5.1 system (D).

  • View in gallery

    Tumor size as measured by bioluminescence. Photon counts of Cohort 1 and Cohort 2 on Day 23 and Day 9 after implantation. Data are shown as mean photon counts ± 95% confidence intervals.

  • View in gallery

    A: H & E–stained border between tumor and normal brain. B: Immunofluorescence demonstrating the specificity of TRP-2 antibodies. Arrows indicate individual migrating tumor cells.

  • View in gallery

    Distribution of the Alexa Fluor 750–labeled TRP-2 antibody over a 72-hour period. Mice were identified with an “M” and a number. Each mouse was imaged at 2 time points. Two control mice did not receive labeled TRP-2, and each was imaged 1 time. Fluorescence images of mice injected with Alexa Fluor–750 labeled antibodies were obtained at 8, 24, 32, 48, and 72 hours after injection.

  • View in gallery

    In vivo visualization of tumor cells by using the Optiscan 5.1 endomicroscope. Mice received an intravenous injection of labeled anti–TRP-2 antibodies 24 hours before imaging. A: Central area of tumor. B: Normal brain adjacent to tumor. C: Brain of mouse that did not have an implanted tumor.

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Tabori URienstein SDromi YLeider-Trejo LConstantini SBurstein Y: Epidermal growth factor receptor gene amplification and expression in disseminated pediatric low-grade gliomas. J Neurosurg 103:4 SupplApplication of Fluorescent Technology in Neurosurgery3573612005

36

Teng LNakada MZhao SGEndo YFuruyama NNambu E: Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy. Br J Cancer 104:7988072011

37

Tsugu AIshizaka HMizokami YOsada TBaba TYoshiyama M: Impact of the combination of 5-aminolevulinic acid-induced fluorescence with intraoperative magnetic resonance imaging-guided surgery for glioma. World Neurosurg 76:1201272011

38

Tykocki TMichalik RBonicki WNauman P: Fluorescence-guided resection of primary and recurrent malignant gliomas with 5-aminolevulinic acid. Preliminary results. Neurol Neurochir Pol 46:47512012

39

Utsuki SMiyoshi NOka HMiyajima YShimizu SSuzuki S: Fluorescence-guided resection of metastatic brain tumors using a 5-aminolevulinic acid-induced protoporphyrin IX: pathological study. Brain Tumor Pathol 24:53552007

40

Utsuki SOka HSato SShimizu SSuzuki STanizaki Y: Histological examination of false positive tissue resection using 5-aminolevulinic acid-induced fluorescence guidance. Neurol Med Chir (Tokyo) 47:2102142007

41

Valdés PAKim ABrantsch MNiu CMoses ZBTosteson TD: δ-aminolevulinic acid-induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy. Neuro Oncol 13:8468562011

42

Valdés PALeblond FKim AHarris BTWilson BCFan X: Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker. Clinical article. J Neurosurg 115:11172011

43

von Campe GMoschopulos MHefti M: 5-Aminolevulinic acid-induced protoporphyrin IX fluorescence as immediate intraoperative indicator to improve the safety of malignant or high-grade brain tumor diagnosis in frameless stereotactic biopsies. Acta Neurochir (Wien) 154:5855882012

44

Wikstrand CJMcLendon REFriedman AHBigner DD: Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII. Cancer Res 57:413041401997

45

Wikstrand CJReist CJArcher GEZalutsky MRBigner DD: The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target. J Neurovirol 4:1481581998

46

Wirth DSnuderl MSheth SKwon CSFrosch MPCurry W: Identifying brain neoplasms using dye-enhanced multimodal confocal imaging. J Biomed Opt 17:0260122012

47

Wirtz CRTronnier VMBonsanto MMKnauth MStaubert AAlbert FK: Image-guided neurosurgery with intraoperative MRI: update of frameless stereotaxy and radicality control. Stereotact Funct Neurosurg 68:39431997

48

Wu AHXiao JAnker LHall WAGregerson DSCavenee WK: Identification of EGFRvIII-derived CTL epitopes restricted by HLA A0201 for dendritic cell based immunotherapy of gliomas. J Neurooncol 76:23302006

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