Name: fluorescence molecular tomography for in vivo imaging of glioblastoma xenografts
Uploaded: 2018-04-26T14:00:00
Description: Watch this Scientific Journal Video about Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts at JoVE.com

We thank Dr. Frederick Lang, MD Anderson Cancer Center for GBM-PDX neurospheres. This work was supported by the Defeat GBM Research Collaborative, a subsidiary of National Brain Tumor Society (Frank Furnari), R01-NS080939 (Frank Furnari), the James S. McDonnell Foundation (Frank Furnari); Jorge Benitez was supported by an award from the American Brain Tumor Association (ABTA); Ciro Zanca was partially supported by an American-Italian Cancer Foundation postdoctoral research fellowship. Frank Furnari receives salary and additional support from the Ludwig Institute for Cancer Research.

The use of experimental research animals and infectious agents, such as lentivirus to transduce the cancer cells, require prior approval by the institutional animal care program and by the institutional biosafety committee. This protocol follows the animal care guidelines of the University of California San Diego (UCSD).
1. Labeling of Glioblastoma Cells with FP635 or FP720 Construct
<ol>
	<li>Produce and purify lentivirus according to the protocol described by Tiscornia et al.<sup ...

<ol>
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Tumor xenografts have been extensively used in cancer research and a number of well-established imaging techniques has been developed: BLI; magnetic resonance imaging (MRI); positron emission tomography (PET), computed tomography (CT); FMT. Each of these approaches comes with pros and cons, but ultimately complement each other with the type of information provided. One of the most commonly used in vivo imaging technology is BLI5,6,<sup class="...

Cancer is one of the leading causes of illness-related deaths in humans in the industrialized world. With an extremely high death toll, new treatments are urgently required. Glioblastoma multiforme (GBM) is an extremely lethal type of brain cancer, composed of heterogeneous populations of brain tumor, stromal, and immune cells. According to the Central Brain tumor registry of the USA, the incidence of primary malignant and non-malignant brain tumors is approximately 22 cases per 100,000. Approximately 11,000 new cases are expected to be diagnosed in the USA in 20171.
Preclinical studies investigate the likelihood of a drug, procedure, or treatment to be effective prior to testing in humans. One of the earliest laboratory steps in preclinical studies is identifying potential molecular targets for drug treatment by using cancer cells implanted in a host organism, defined as human xenograft models. Within this context, intracranial brain tumor xenograft models using patient-derived xenografts (PDXs) have been widely used to study brain tumor biology, progression, evolution, and therapeutic response, and more recently for biomarkers development, drug screening, and personalized medicine2,3,4.
One of the most affordable and non-invasive in vivo imaging methods to monitor intracranial xenografts is bioluminescence imaging (BLI)5,6,7,8. However, some BLI limitations include substrate administration and availability, enzyme stability, and light quenching and scattering during imaging acquisition9. Here we report the infrared FMT as an alternative imaging method to monitor preclinical glioblastoma models. In this method, signal acquisition and quantification of intracranially implanted PDXs, expressing a near-infrared fluorescent protein iRFP72010,11 (henceforth termed as FP720) or turboFP635 (henceforth termed as FP635), is performed with a FMT imaging system. Using the FMT technology, the orthotopic tumors can be monitored in vivo before, during, or after treatment, in a non-invasive, substrate-free, and quantitative manner for preclinical observations.

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			<td height="20" style="height:21px;width:343px;">DMEM/High Glucose&#160;</td>
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			<td height="20" style="height:21px;width:343px;">DMEM/F12 1:1&#160;</td>
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			<td>11320-082</td>
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			<td>SH30071.03</td>
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			<td height="20" style="height:21px;width:343px;">Accutase</td>
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			<td>17504044</td>
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			<td height="20" style="height:21px;">human recombinant EGF&#160;</td>
			<td>Stemcell Technologies</td>
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			<td style="width:213px;">NDC 59399-110-20</td>
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			<td height="20" style="height:21px;width:343px;">Ketamine</td>
			<td style="width:191px;">Zoetis</td>
			<td style="width:213px;">NADA 043-403</td>
			<td>Controlled substance</td>
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			<td style="width:213px;">NDC 17033-211-38</td>
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			<td style="width:191px;">CpMedical</td>
			<td style="width:213px;">VQ392</td>
			<td></td>
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			<td height="20" style="height:21px;width:343px;">5 ul syringe</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:213px;">26200-U</td>
			<td>Catalog number as sold by Sigma-Aldrich</td>
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			<td height="20" style="height:21px;width:343px;">Cell Sorter</td>
			<td style="width:191px;">Sony</td>
			<td style="width:213px;">SH8007</td>
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			<td height="20" style="height:21px;width:343px;">Mouse stereotaxic frame&#160;</td>
			<td style="width:191px;">Stoelting</td>
			<td style="width:213px;">51730</td>
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			<td height="20" style="height:21px;width:343px;">Motorized stereotaxic injector</td>
			<td style="width:191px;">Stoelting</td>
			<td style="width:213px;">53311</td>
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			<td height="20" style="height:21px;width:343px;">Micromotor hand-held drill</td>
			<td style="width:191px;">Foredom</td>
			<td style="width:213px;">K1070</td>
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			<td height="20" style="height:21px;width:343px;">Mouse warming pad&#160;</td>
			<td style="width:191px;">Ken Scientific Corporation</td>
			<td style="width:213px;">TP-22G</td>
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			<td height="20" style="height:21px;">Fluorescence Tomography System&#160;</td>
			<td>PerkinElmer</td>
			<td style="width:213px;">FMT 2500 XL</td>
			<td></td>
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			<td height="20" style="height:21px;">TrueQuant Imaging Software&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td style="width:213px;">7005319</td>
			<td></td>
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			<td height="20" style="height:21px;width:343px;">Ultra-centrifuge Optima L-80 XP</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">392049</td>
			<td></td>
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			<td height="20" style="height:21px;width:343px;">Tissue Culture 100mm Dishes</td>
			<td style="width:191px;">Olympus Plastics</td>
			<td style="width:213px;">25-202</td>
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			<td height="20" style="height:21px;width:343px;">Tissue Culture 150mm Dishes</td>
			<td style="width:191px;">Olympus Plastics</td>
			<td style="width:213px;">25-203</td>
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			<td height="20" style="height:21px;width:343px;">Tissue Culture Flasks T75</td>
			<td style="width:191px;">Corning</td>
			<td style="width:213px;">430720U</td>
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			<td height="20" style="height:21px;width:343px;">50 mL conical tubes</td>
			<td style="width:191px;">Corning</td>
			<td style="width:213px;">430290</td>
			<td></td>
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			<td height="20" style="height:21px;width:343px;">15 mL conical tubes</td>
			<td style="width:191px;">Olympus Plastics</td>
			<td style="width:213px;">28-101</td>
			<td></td>
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			<td height="20" style="height:21px;width:343px;">Centrifuge Avanti J-20</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">J320XP-IM-5</td>
			<td></td>
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			<td height="20" style="height:21px;width:343px;">Tube, Polypropylene, Thinwall, 5.0 mL</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">326819</td>
			<td></td>
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			<td height="20" style="height:21px;width:343px;">Tube, Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">326823</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Athymic nude mice</td>
			<td style="width:191px;">Charles River Laboratories</td>
			<td style="width:213px;">Strain Code&#160; 490 (Homozygous)</td>
			<td>Prior approval by the Institutional Animal Care Program and by the Institutional Biosafety Committee required.&#160;&#160;&#160;</td>
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fluorescence molecular tomography for in vivo imaging of glioblastoma xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Glioblastoma cells U87EGFRvIII (U87 cells over-expressing the EGF receptor variant III) were cultured according to the step 1.2. Lentivirus was produced and purified according to step 1.1. The viral concentration was determined by p24 ELISA analysis. Cells were transduced with lentivirus carrying infrared fluorescent proteins according to step 1.8. The plasmid encoding FP72010,11 was kindly provided by Dr. V.V. Verkhusha and the F...

Watch this Scientific Journal Video about Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts at JoVE.com

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

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Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...