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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The authors declare no conflicts of interests.

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.

<table border="0" cellpadding="0" cellspacing="0" width="1489">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DMEM/High Glucose&#160;</td>
			<td style="width:191px;">HyClone/GE</td>
			<td style="width:213px;">SH30022.1</td>
			<td style="width:744px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DMEM/F12 1:1&#160;</td>
			<td style="width:191px;">Gibco</td>
			<td>11320-082</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">FBS</td>
			<td style="width:191px;">HyClone/GE</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Accutase</td>
			<td style="width:191px;">Innovative cell technologies</td>
			<td style="width:213px;">AT-104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Trypsin</td>
			<td style="width:191px;">HyClone/GE</td>
			<td style="width:213px;">SH30236.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">B27 supplement</td>
			<td style="width:191px;">Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">human recombinant EGF&#160;</td>
			<td>Stemcell Technologies</td>
			<td style="width:213px;">2633</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">human recombinant FGF</td>
			<td>Stemcell Technologies</td>
			<td style="width:213px;">2634</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DPBS</td>
			<td style="width:191px;">Corning</td>
			<td style="width:213px;">21-031-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">FACS tubes</td>
			<td style="width:191px;">Falcon</td>
			<td style="width:213px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DAPI</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:213px;">62248</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Blasticidin</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:213px;">A1113903</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">p24 ELISA&#160;</td>
			<td style="width:191px;">Clontech</td>
			<td style="width:213px;">632200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Xylazine</td>
			<td style="width:191px;">Akorn</td>
			<td style="width:213px;">NDC 59399-110-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Ointment</td>
			<td style="width:191px;">Dechron</td>
			<td style="width:213px;">NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Absorbable suture</td>
			<td style="width:191px;">CpMedical</td>
			<td style="width:213px;">VQ392</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Cell Sorter</td>
			<td style="width:191px;">Sony</td>
			<td style="width:213px;">SH8007</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Motorized stereotaxic injector</td>
			<td style="width:191px;">Stoelting</td>
			<td style="width:213px;">53311</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Fluorescence Tomography System&#160;</td>
			<td>PerkinElmer</td>
			<td style="width:213px;">FMT 2500 XL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">TrueQuant Imaging Software&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td style="width:213px;">7005319</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
	</tbody>
</table>

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 ...

The overall goal of this experimental procedure is to establish a preclinical brain tumor imaging model using fluorescence molecular tomography or FMT. This method can help answer key questions in the fields of brain tumor biology, cancer research, and drug discovery about tumor aggressiveness, heterogeneity, and treatment responsiveness. The main advantage of this technique is that the tumors can be monitored in vivo before, during, or after treatment in a non-invasive, suture free, and quantitative manner.Another advantage of this technique is the possibility to apply to other types of xenograft models implanted in different organs of the animal. Begin by seeding one times 10 to the sixth glioblastoma cells in five milliliters of medium into a 10 centimeter dish for 24 hour incubation in a cell culture incubator. The next morning, transduce the cells with lentivirus, expressing an appropriate fluorescent protein of interest at a multiplicity of infection of five and return the cells to the incubator.After 24 hours, replace the supernatant with five milliliters of fresh medium and incubate the culture for another 48 hours. At the end of the incubation, replace the medium with three to five milliliters of tripson for 10 to 15 minutes at 37 degrees Celsius followed by careful pipetting to fully dissociate the detached cells. Collect the cells by centrifugation and re-suspend the pellet in 500 microliters of sorting solution.Split the cells into the appropriate number of fax tubes and co-stain the cells with DAPI for dead cell exclusion. Then load the tubes onto the flow cytometer and sort the live cells according to their fluorescent construct expression into a 15 milliliter conical tube containing fresh sorting solution. After centrifugation, re-suspend the construct positive, DAPI negative cells in five milliliters of culture medium and seed them onto a new 10 centimeter dish for their culture at 37 degrees Celsius for 48 to 72 hours.At the end of the incubation, split the cells for seeding into multiple dishes for subsequent in vivo experiments. On the day of the injection, dissociate the fluorescent positive gliomacells into a single cell suspension and re-suspend them at a 0.5 or one times 10 to the sixth cells per two to five microliters of PBS per injection per animal concentration in a 1.5 milliliter microcentrifuge tube on ice. After confirming a lack of response to toe pinch, apply ointment to the eyes of the immuno-deficient Athymic Nude recipient mouse and load a five microliter Hamilton syringe equipped with a blunt tipped needle with the cells for the injection.Mount the syringe into a probe holder and place the mouse in a small, animal sterotactic frame. Fix the head using the ear bars and the incisor adaptor and disinfect the head with 70%ethanol and batadine solution. Using a small scalpel, make a middle incision and separate the skin and connective tissues.Using the micromanipulator, place the Hamilton syringe on the bregma point and move the probe holder one millimeter anteroposterior and two millimeters lateral from the bregma point. After marking the position with a pencil, use a micromotor handheld drill to carefully make a hole in the skull, applying a slight downward pressure until the blood vessels become visible. Introduce the needle into the burr hole three millimeters below the pial surface and inject the cells at a one microliter per minute flow right.When the full experimental cell volume has been injected, gradually remove the needle at a one millimeter per minute ascension rate and clean the injection site with 70%ethanol. Then close the skin wound according to standard protocols and monitor the animal on a warming pad until it has fully recovered. To image the injected cells, place the anesthetized recipient animal in the imaging cassette of a fluorescence molecular tomography imager, head adapter first in the prone position, with the head in the center of the cassette.With the cassette closed, tighten the adjustment knobs to 17 millimeters. When the animal is secure, insert the cassette into the internal docking station and open the imager and analyzer software. In the experimental tab window, select the appropriate database and study group and open the scan tab window.Click select subject to select the subject to image and select the laser channel in the laser channel panel. Click capture to acquire an image and click and drag that scan field in the captured image to identify the source locations. Check the add to reconstruction que option and click scan in the scan tab window.When the scanning is complete, remove the imaging cassette from the docking station and return the animal to its cage with monitoring until full recovery. To analyze the images, in the imager and analyzer software open the analysis tab window and click the plus sign button in the data set selection panel to load the data set and subject for analysis. Then use the ellipsoid icon to select the region of interest and right click the threshold column to adjust the threshold to zero in the statistic data panel.Glioblastoma cells labeled with near-infrared fluorescent proteins, as just demonstrated, exhibit distinct fluorescent profiles that can be distinguished by fluorescence molecular tomography, even up to five days after injection. Further, the lack of background signal between the constructs facilitates dual in vivo imaging of cancer cell populations of interest. In this experiment, fluorescent construct labeled glioblastoma spheres co-transduced with lentivirus encoding SHRNA targeting death-domain associated protein were injected into recipient immunodeficient Athymic Nude mice, as just demonstrated.Reverse transcription quantitative preliminaries chain reaction confirmed the efficacy of the SHRNA targeting in both cancer cell lines and fluorescence molecular tomography revealed a decrease in fluorescence signal in the mice engrafted with SHRNA glioblastoma spheres compared to animals implanted with SH control transduced cells. After watching this video, you should have a good understanding of how to generate preclinical data for brain cancer research using a fluorescence molecular tomography system.

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Research

JoVE Journal

Cancer Research

8.8K 조회수.  Ludwig Institute for Cancer Research. The overall goal of this experimental procedure is to establish a preclinical brain tumor imaging model using fluorescence molecular tomography or FMT. This method can help answer key questions in the fields of brain tumor biology, cancer research, and drug discovery about tumor aggressiveness, heterogeneity, and treatment responsiveness. The main advantage of this technique is that the tumors can be monitored in vivo before, during, or after treatment in a non-invasive, suture free, and quan...