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.12</li>
	<li>Culture approximately 2.0 x 106 cells from a human glioma cell line with DMEM plus 10% fetal bovine serum (FBS) in a 15-cm dish; or culture 2.0 x 106 human glioblastoma patient-derived (GBM-PDX) spheres with DMEM/F12 1:1 medium with B27 supplement plus human recombinant epidermal growth factor (EGF; 20 ng/mL) and FGF (10 ng/mL) in a T75 flask.</li>
	<li>Maintain all cells at 37 &#176;C, 5% CO2, and 100% relative humidity.</li>
	<li>Dissociate the cells.
	<ol>
		<li>For the glioma cells: remove the supernatant from the plates and adding 3-5 mL of trypsin 1X to the glioma cells.</li>
		<li>For the GBM spheres: collect the cells by pipetting the cells into a 15-mL tube.
		<ol>
			<li>Spin down the GBM spheres at 200 x g for 5 min using a tabletop centrifuge at room temperature.</li>
			<li>Remove the supernatant and add 3-5 mL of cell detachment solution.</li>
		</ol>
		</li>
		<li>Incubate all cells at 37 &#176;C for 10-15 min.</li>
	</ol>
	</li>
	<li>Carefully dissociate the cells by pipetting up and down several times (ensure that spheres and glioma cells are completed dissociated) and spin down at 200 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the cells with 5 mL of medium by pipetting up and down several times. Determine the cell viability by trypan blue exclusion.</li>
	<li>Plate 1.0 x 106 of target cells in a 10-cm dish with 5 mL of medium by pipetting.</li>
	<li>Transduce cells with lentivirus expressing fluorescent proteins at multiplicity of infection (MOI) 5 as is described by Tiscornia et al.12 and incubate at 37 &#176;C.</li>
	<li>After 24 h, remove the medium from the transduced cells, add 5 mL of fresh medium, and incubate for additional 48 h at 37 &#176;C.</li>
	<li>Dissociate the infected cells into a single-cell suspension as described in steps 1.4-1.6. Spin down the cells at 200 x g for 5 min and resuspend in 500 &#181;L of sorting solution (phosphate-buffered saline (PBS) with 1% FBS).</li>
	<li>Pipette the cell suspension into FACS tubes. Co-stain the cells with 1 &#181;L/mL of 4&#39;,6-diamidino-2-phenylindole (DAPI) dihydrochloride to exclude dead cells. Negative controls are required to set up the flow cytometer gates (non-transduced cells and non-transduced cells/ DAPI stained).</li>
	<li>Sort the fluorescent-positive/DAPI-negative cells into sorting solution, as described by Basu et al.13, and collect the sorted cells into a 15-mL conical tube.</li>
	<li>Spin down the sorted cells at 200 x g for 5 min, remove the supernatant, and resuspend in 5 mL of culture medium. Seed the sorted cells in a 10-cm dish by pipetting. Incubate at 37 &#176;C for at least 48-72 h.</li>
	<li>Expand the sorted cells by dissociating the cells following steps 1.4-1.6 and plating into multiple dishes for in vivo experiments. 
	NOTE: For more details about cell sorting preparation and purification, see Basu et al.13</li>
</ol>
2. Intracranial Injection of iRFP-tagged Glioblastoma Cells into Immunodeficient Mice
NOTE: Before starting the surgery, make sure that the surgical room and tools are clean for the procedure. Use immunodeficient athymic nude (Foxn1nu) male or female mice, between 4-5 weeks old and 17-19 g for intracranial injections. Animals should be housed for at least 3 days after arrival and before surgery.
<ol>
	<li>Cell preparation
	<ol>
		<li>Harvest and dissociate fluorescent positive-glioma cells into a single-cell suspension (steps 1.4-1.6) and resuspend 0.5 or 1.0 x 106 cells in 2-5 &#181;L of PBS per injection per animal. Pipette the cell suspension into a 1.5-mL microcentrifuge tube and place on ice.</li>
	</ol>
	</li>
	<li>Anesthesia preparation and administration
	<ol>
		<li>Prepare 200 &#181;L of saline solution containing ketamine (100 mg/kg) and xylazine (10 mg/kg) per 20 grams of mouse body weight. Weigh the animals and calculate the appropriate dose of anesthesia.</li>
		<li>Provide an intraperitoneal injection of anesthesia and apply ophthalmic ointment to the eyes to prevent dehydration. For more details about this step see Kathleen et al.14 Place the animal on a pre-warmed water thermal pad at 37 &#176;C.</li>
		<li>Check that the animals are anesthetized by monitoring the toe pinch response (pedal reflex), usually within 5-10 min.</li>
	</ol>
	</li>
	<li>Intracranial injection
	<ol>
		<li>Load a 5 &#181;L Hamilton syringe (blunt tip needle) with cell suspension and mount in the probe holder.</li>
		<li>Place the mouse in a small animal stereotactic frame and fix the head using the ear bars and the incisor adapter following the described details by Cetin et al.15</li>
		<li>Disinfect the head with 70% ethanol and betadine solution. Make sure that the surgical site is complete sterile. Perform a middle incision with a small scalpel and separate the skin and connective tissues.</li>
		<li>Place the Hamilton syringe on the bregma point using the micromanipulator.</li>
		<li>Move the probe holder 1.0 mm anteroposterior and 2.0 mm lateral from the bregma point. Mark this position with a pencil.</li>
		<li>At this location, carefully make a hole in the skull with a micromotor hand-held drill by applying slight pressure downward until the blood vessels become visible. Do not disrupt the arachnoid mater and brain parenchyma.</li>
		<li>Introduce the needle into the burr hole and advance 3.0 mm below the pial surface.</li>
		<li>Set up the volume (2-5 &#181;L) and flow rate (1 &#181;L/min) of injection using a motorized stereotaxic injector or manually perform the injection.</li>
		<li>After the injection, remove the needle gradually by ascending 1.0 mm every 1 min and clean the injection site with 70% ethanol.</li>
		<li>Close the skin wound with surgical staples or by making surgical sutures (often referred to as stitches) and see Kathleen et al.14 for more details.</li>
	</ol>
	</li>
	<li>Animal recovery
	<ol>
		<li>Transfer the animal to a water thermal pad (37 &#176;C) and monitor the body temperature, respiration rate, and heart rate, until full recovery. Administer analgesia per standard protocols. 
		NOTE: For more information about the stereotactic procedure, surgery, and coordinates, see other reports5,14,15.</li>
	</ol>
	</li>
</ol>
3. FMT Imaging
NOTE: According to the experimental aim, the iRFP-tagged glioblastoma cells can be monitored in vivo before, during, or after treatment. For imaging purpose, anesthetize animals using an isoflurane induction chamber, maintain in an imaging cassette during the scanning, and image in the docking station of the FMT imager.
<ol>
	<li>Remove the animal from the cage and place in the isoflurane induction chamber. Close the chamber and turn on oxygen flow and vaporizer to 3-5%.</li>
	<li>Monitor the animal until it is completely anesthetized, usually within 1-2 min. Unconscious animals should not respond to external stimuli.</li>
	<li>Remove the anesthetized animal from the chamber and put the animal in the imaging cassette by placing the head adaptor first, followed by placing the animal facing down. Make sure that the head is located in the center of the cassette.</li>
	<li>Place the top plate of the cassette on top and tighten the adjustment knobs until 17 mm.</li>
	<li>Once that the animal is secure in the imaging cassette, proceed to imaging by inserting the cassette into the internal docking station of the FMT imager, where the isoflurane is pumped to keep the animal anesthetized.</li>
	<li>Run the imager and analyzer software by double-clicking the software icon.</li>
	<li>In the &#34;Experimental tab&#34; window, select the &#34;Database&#34; and &#34;Study group&#34;.</li>
	<li>Go to the &#34;Scan tab&#34; window and select the subject to image by clicking &#34;select subject&#34;. Also, select the laser channel in the &#34;Laser channel&#34; panel. For the FP635-labeled cells, select &#34;laser channel 635 nm&#34;, and for FP720-labeled cells, select &#34;laser channel 680 nm&#34;.</li>
	<li>Acquire an image by clicking the &#34;Capture&#34; option in the &#34;Scan tab&#34; window. Then, adjust the scan field by clicking and dragging the scan field in the captured image to a determined number of source locations, usually within 20-25 sources for the ipsilateral side.</li>
	<li>Check the &#34;Add to reconstruction queue&#34; option and hit &#34;Scan&#34; in the &#34;Scan tab&#34; window.</li>
	<li>Wait until the scanning is completed and then remove the imaging cassette from the docking station.</li>
	<li>Remove the top plate of the cassette and place the animal back into the cage.</li>
	<li>Repeat the steps 3.1-3.12 for the rest of the animals.</li>
</ol>
4. Image Analysis
<ol>
	<li>From the imager and analyzer software, select the &#34;Analysis tab&#34; window.</li>
	<li>Load the dataset and the subject to analyze by clicking the &#34;+&#34; button in the &#34;Dataset selection&#34; panel of the &#34;Analysis tab&#34; window.</li>
	<li>Once the image has been loaded in the &#34;Analysis tab&#34; window, perform the region of interest (ROI) analysis by selecting the ellipsoid icon, located in the upper left corner of the &#34;Analysis tab&#34; window.</li>
	<li>Adjust the threshold to zero in the &#34;statistic data&#34; panel by right clicking the &#34;threshold&#34; column in the &#34;Analysis tab&#34; window.</li>
	<li>The total value in the &#34;Statistic data&#34; panel represents the signal from the fluorescent-tagged glioblastoma cells implanted in the mouse brain.</li>
	<li>Repeat steps 4.2-4.4 for the rest of the animals. Load or remove a new subject by clicking the &#34;+&#34; or &#34;&#8722;&#34; button, respectively, in the &#34;Dataset selection&#34; panel of the &#34;Analysis tab&#34; window. 
	NOTE: For more information about the FMT imaging and software operation, see20.</li>
</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><tbody><tr><td>DMEM/High Glucose&amp;nbsp;</td><td>HyClone/GE</td><td>SH30022.1</td><td></td></tr><tr><td>DMEM/F12 1:1&amp;nbsp;</td><td>Gibco</td><td>11320-082</td><td></td></tr><tr><td>FBS</td><td>HyClone/GE</td><td>SH30071.03</td><td></td></tr><tr><td>Accutase</td><td>Innovative cell technologies</td><td>AT-104</td><td></td></tr><tr><td>Trypsin</td><td>HyClone/GE</td><td>SH30236.01</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>human recombinant EGF&amp;nbsp;</td><td>Stemcell Technologies</td><td>2633</td><td></td></tr><tr><td>human recombinant FGF</td><td>Stemcell Technologies</td><td>2634</td><td></td></tr><tr><td>DPBS</td><td>Corning</td><td>21-031-00</td><td></td></tr><tr><td>FACS tubes</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>62248</td><td></td></tr><tr><td>Blasticidin</td><td>ThermoFisher Scientific</td><td>A1113903</td><td></td></tr><tr><td>p24 ELISA&amp;nbsp;</td><td>Clontech</td><td>632200</td><td></td></tr><tr><td>Xylazine</td><td>Akorn</td><td>NDC 59399-110-20</td><td></td></tr><tr><td>Ketamine</td><td>Zoetis</td><td>NADA 043-403</td><td>Controlled substance</td></tr><tr><td>Ointment</td><td>Dechron</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Absorbable suture</td><td>CpMedical</td><td>VQ392</td><td></td></tr><tr><td>5 ul syringe</td><td>Hamilton</td><td>26200-U</td><td>Catalog number as sold by Sigma-Aldrich</td></tr><tr><td>Cell Sorter</td><td>Sony</td><td>SH8007</td><td></td></tr><tr><td>Mouse stereotaxic frame&amp;nbsp;</td><td>Stoelting</td><td>51730</td><td></td></tr><tr><td>Motorized stereotaxic injector</td><td>Stoelting</td><td>53311</td><td></td></tr><tr><td>Micromotor hand-held drill</td><td>Foredom</td><td>K1070</td><td></td></tr><tr><td>Mouse warming pad&amp;nbsp;</td><td>Ken Scientific Corporation</td><td>TP-22G</td><td></td></tr><tr><td>Fluorescence Tomography System&amp;nbsp;</td><td>PerkinElmer</td><td>FMT 2500 XL</td><td></td></tr><tr><td>TrueQuant Imaging Software&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</td><td>7005319</td><td></td></tr><tr><td>Ultra-centrifuge Optima L-80 XP</td><td>Beckman Coulter</td><td>392049</td><td></td></tr><tr><td>Tissue Culture 100mm Dishes</td><td>Olympus Plastics</td><td>25-202</td><td></td></tr><tr><td>Tissue Culture 150mm Dishes</td><td>Olympus Plastics</td><td>25-203</td><td></td></tr><tr><td>Tissue Culture Flasks T75</td><td>Corning</td><td>430720U</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Olympus Plastics</td><td>28-101</td><td></td></tr><tr><td>Centrifuge Avanti J-20</td><td>Beckman Coulter</td><td>J320XP-IM-5</td><td></td></tr><tr><td>Tube, Polypropylene, Thinwall, 5.0 mL</td><td>Beckman Coulter</td><td>326819</td><td></td></tr><tr><td>Tube, Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Athymic nude mice</td><td>Charles River Laboratories</td><td>Strain Code&amp;nbsp; 490 (Homozygous)</td><td>Prior approval by the Institutional Animal Care Program and by the Institutional Biosafety Committee required.&amp;nbsp;&amp;nbsp;&amp;nbsp;</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 ...

fluorescence-molecular-tomography-for-vivo-imaging-glioblastoma

Research

JoVE Journal

Cancer Research

8.9K Views.  Ludwig Institute for Cancer Research. Orthotopic intracraniële injectie van tumorcellen wordt in kankeronderzoek te bestuderen hersenen tumor biologie, progressie en evolutie therapeutische respons uitgegaan. Hier presenteren we fluorescentie moleculaire tomografie van tumor xenografts, die biedt real-time intravital imaging en kwantificering van een tumor massa in preklinische glioblastoma modellen.

Video: Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Neuroscience

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Medicine

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

Biology

In Vivo Fluorescence Imaging to Localize Antibodies in a Mouse Tumor Xenograft Model

Source: Shivange, G. et al., <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.</a>&#160;J. Vis. Exp. (2020)
The video demonstrates a fluorescence imaging method for in vivo antibody localization in a mouse tumor xenograft model. It involves the injection of tumor cells in a mouse to generate solid xenografts beneath the skin. Subsequent injection of fluorescent antibodies confirms specific localization through enhanced fluorescence signals within the tumor xenograft.

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Fluorescence Molecular Tomography: An Imaging Technique for In Vivo Imaging of Fluorescent Protein-tagged Glioblastoma Xenografts in Mouse Model

Source: Benitez, J. A. et al., <a href="https://www.jove.com/video/57448" target="_blank">Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts.</a> J. Vis. Exp. (2018)
In this video, we describe fluorescence molecular tomography or FMT for in vivo imaging of fluorescent protein-tagged glioblastoma xenografts in mouse models. The technique is useful for elucidating the effects of anti-tumor therapeutics at the molecular level.

Source: Benitez, J. A. et al., Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts. J. Vis. Exp. (2018)
In this video, we ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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