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>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;">632200</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>
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			<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>
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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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		<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>
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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>
			<td></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 ...

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

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

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 tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

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 zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

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 target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

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 stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture 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 standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

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

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...