We would like to thank the Stanford Wu Tsai Neuroscience Microscopy Service, the Stanford Center for Innovations in In Vivo Imaging (SCi3) - small animal imaging center, NIH S10 Shared Instrumentation Grant (S10RR026917-01, PI Michael Moseley, Ph.D.), and Stanford Preclinical Imaging Facility at Porter Drive for providing the equipment and infrastructure for this project. This work was supported by a grant from the National Institute for Child Health and Human Development, grant number R01HD103638. We would like to thank the Schnitzer Group, Stanford University; the Zuo lab, University of Santa Cruz; and the Neurovascular Imaging Laboratory, Boston Photonic Center, University of Boston, for educational discussions on two-photon imaging and cranial window models.

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

<ol>
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The authors declare that they have no conflict of interest.

We present a method for real-time in vivo NP tracking using 2P-IVM through a cranial window to evaluate the tumor delivery of fluorescent-labeled iron oxide NPs. The surgical technique for this procedure requires a steady hand and advanced experimental surgical skills. It is advisable to practice using carcasses or phantoms before moving forward to live animal experience. As an alternative, Hoeferlin et al. implemented a robotic drill to reduce thermal damage, minimize surgical technique variability, and standar...

Two-photon intravital microscopy (2P-IVM) is a fluorescence imaging technique that allows the visualization of living tissue1.
First developed in the 1990s, 2P-IVM has been used for in vivo analysis of the retina2, kidney3, small intestine4, cochlea5, heart6, trachea7, and the brain in various preclinical models8,9. In the field of neuroscience, 2P-IVM has gained importance as a technique for real-time imaging of the healthy brain in awake animals10, as well as studying diseases of the nervous system such as Alzheimer&#39;s11, Parkinson&#39;s12 and glioblastoma (GBM)13,14,15,16.
2P-IVM offers an elegant solution for studying the tumor microenvironment during the development of GBM. While some previous studies focused on in vitro17 and ex vivo models18, others implemented orthotopic19 and xenotropic20&#160;in vivo models for examining GBM. Madden et al. performed native imaging of CNS-1 rat glioma cell line in a mouse model13. Using an orthotopic GL261-DsRed murine model, Ricard et al. performed an intravenous administration of a fluorophore to enhance the blood vessels in the tumor region in 2P-IVM14.
Here, we apply 2P-IVM for tracking the tumor delivery of fluorescent-labeled iron oxide nanoparticles (NP) in an orthotopic mouse model of GBM. Using a cranial window, this method allows us to study the real-time spatiotemporal distribution of NPs in the brain in detail.

<table><tbody><tr><td>0.9% sodium chloride infusion solution</td><td>Baxter Corp</td><td>533-JB1301P</td><td></td></tr><tr><td>&lt;br/&gt; Dulbecco&#039;s Modified Eagle Medium</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>1 mL syringes</td><td>BD</td><td>Luer-Lok syringe, REF309628</td><td></td></tr><tr><td>10%&amp;nbsp;FBS</td><td>Thermo fisher</td><td>Cytiva SH30910.03HI</td><td></td></tr><tr><td>10% DMSO</td><td>Sigma-Aldrich</td><td>D8418-50ML</td><td></td></tr><tr><td>2-photon microscope</td><td>Prairie Technologies, Bruker</td><td>Prairie Ultima IV</td><td></td></tr><tr><td>Alcohol applicators, 70%</td><td>Medline Industries, LP</td><td>MDS093810</td><td></td></tr><tr><td>Alcohol, spray bottle</td><td>Decon Labs Inc</td><td>Decon SaniHol, 04-355-122</td><td></td></tr><tr><td>Aluminum foil</td><td>Reynold Brands</td><td>Reynold Wrap non-stick aluminum foil</td><td></td></tr><tr><td>Anesthesia machine</td><td>Patterson Scientific</td><td>SAS3</td><td></td></tr><tr><td>Anesthesia monitoring&amp;nbsp;</td><td>Kent Scientific&amp;nbsp;</td><td>MouseSTAT Jr. Rodent Pulsoxymeter</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x), liquid</td><td>Invitrogen</td><td>15240-096</td><td></td></tr><tr><td>Betadine applicators</td><td>Professional Disposables International, Inc</td><td>S41125</td><td></td></tr><tr><td>Biopsy punch, 5 mm&amp;nbsp;</td><td>Miltex</td><td>Size 5</td><td></td></tr><tr><td>Buprenorphine sustained release</td><td>Zoo Pharm</td><td>Bup SR Lab, 1.0 mg/mL</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>C6 rat glioma cell line</td><td>ATCC (American Tissue Culture Collection)</td><td>CCL-107</td><td></td></tr><tr><td>Cannulas</td><td>BD</td><td>16 G, 1.1/2&amp;rdquo;, 30 G, 1&amp;rdquo;</td><td></td></tr><tr><td>Carprofen</td><td>Pfizer&amp;nbsp;</td><td>Rimadyl, 50 mg/ml</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>Cefazoline</td><td>Sagent Pharmaceuticals</td><td>25021-101-10, 1 g/vial</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>Cell strainer, 40 &amp;micro;m</td><td>Fisher Scientific</td><td>87711</td><td></td></tr><tr><td>Cotton tip applicators, 6&amp;rdquo;&amp;nbsp;</td><td>Dyad Medical Sourcing, LLC</td><td>HCS1005</td><td></td></tr><tr><td>Dental cement</td><td>Stoelting Co</td><td>51459</td><td>Dental cement kit, clear, 2 components</td></tr><tr><td>Dexamethasone</td><td>Bimeda</td><td>138RX, 2 mg/mL</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>DietGel</td><td>ClearH2O</td><td>Recovery, 72-06-502</td><td></td></tr><tr><td>Drape&amp;nbsp;</td><td>Cardinal Health</td><td>Bio Shield Wrap</td><td></td></tr><tr><td>Drill</td><td>Saeyang Microtech</td><td>Escort Pro, B08350</td><td></td></tr><tr><td>Drill tips&amp;nbsp;</td><td>Hager &amp;amp; Meissinger GmbH</td><td>REF310104001001005</td><td>Size 005, US 1/4</td></tr><tr><td>FIJI imaging analysis software</td><td>National Institute of Health</td><td>https://imagej.net/software/fiji/</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>13-812-41</td><td></td></tr><tr><td>Gauze</td><td>Fisher HealthCare</td><td>Sterile Cotton Gauze Pad, 4 x 4&amp;rdquo;, 22-415-469</td><td></td></tr><tr><td>Gelfoam&amp;nbsp;</td><td>Ethicon Inc.&amp;nbsp;</td><td>Surgifoam absorbable gelatin sponge, Ref. 1972</td><td></td></tr><tr><td>Germinator&amp;nbsp;</td><td>Cellpoint Scientific&amp;nbsp;</td><td>Germinator 500, No. 11688</td><td></td></tr><tr><td>Glass coverslips, 5 mm diameter</td><td>Fisher Scientific</td><td>Menzel Cover glass&amp;nbsp;</td><td></td></tr><tr><td>Gloves, non-sterile</td><td>Fisher Scientific</td><td>Nitrile powder-free medical examination gloves</td><td></td></tr><tr><td>Gloves, sterile</td><td>Medline Industries, LP</td><td>MDS104070</td><td></td></tr><tr><td>Hair removal cream</td><td>Church &amp;amp; Dwight</td><td>Nair Hair remover lotion&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton Company Inc</td><td>Gastight #1701, 10 &amp;micro;L</td><td></td></tr><tr><td>HBSS without Ca, Mg</td><td>Fisher Scientific</td><td>PI88284</td><td></td></tr><tr><td>Head bar</td><td>Hongway</td><td>5 mm inner diameter O-rings</td><td></td></tr><tr><td>Heating pad</td><td>Stoelting Co.&amp;nbsp;</td><td>Rodent warmer X2</td><td></td></tr><tr><td>Insulin syringes</td><td>Exel International Medical Products&amp;nbsp;</td><td>29G x 1/2&amp;Prime;</td><td></td></tr><tr><td>Iron oxide nanoparticles</td><td>Covis Pharma GmbH</td><td>Feraheme ferumoxytol injection, 510 mg/17 mL, 59338077501</td><td></td></tr><tr><td>Isoflurane</td><td>Dechra</td><td>26675-46-7</td><td></td></tr><tr><td>Mice</td><td>Jackson Laboratories</td><td>NSG, Strain 005557</td><td></td></tr><tr><td>Microscope (surgery)</td><td>Seiler Medical</td><td>Seiler IQ Q-100-220</td><td></td></tr><tr><td>Nanoparticles</td><td>Custom</td><td></td><td>Iron oxide nanoparticles (Ferumoxytol) labeled with fluorescein isothiocyanate</td></tr><tr><td>Ophtalmic ointment</td><td>Major pharmaceuticals</td><td>Lubrifresh P.M. nighttime ointment, 203964</td><td></td></tr><tr><td>Oxygen</td><td>Linde Gas &amp;amp; Equipment Inc.&amp;nbsp;</td><td>High Pressure Steel K Style Cylinder, 249CF, 2000PSIG, CGA 540</td><td></td></tr><tr><td>Plastic cups</td><td>Georgia-Pacirif Consumer Products</td><td>Dixie Portion Cup, 2 oz., Plastic, Clear, PK2400</td><td></td></tr><tr><td>Polyethylene tubing</td><td>Braintree Scientific</td><td>50-195-5494</td><td></td></tr><tr><td>Scale</td><td>Ohaus Corp</td><td>CR2200</td><td></td></tr><tr><td>Scalpel</td><td>Integra Life Sciences Production Corp</td><td>Integra Miltex Stainless steel disposable scalpel</td><td></td></tr><tr><td>Scissors</td><td>Fisher Scientific</td><td>13-804-18</td><td></td></tr><tr><td>Sealant</td><td>Henkel Corp</td><td>Loctite 4014</td><td></td></tr><tr><td>Single use lab gown</td><td>High Tech Conversions</td><td>17-444-081</td><td></td></tr><tr><td>Stereotactic frame</td><td>Stoelting Co.&amp;nbsp;</td><td>Stoelting New Standard TM</td><td></td></tr><tr><td>Sterile Vacuum Bottle Top Filtration Systems</td><td>Fisher Scientific</td><td>S2GPU05RE, MilliporeSigma NO.:S2GPU05RE</td><td></td></tr><tr><td>Styrofoam box</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Surgical gloves</td><td>Cardinal Health</td><td>19-163-108</td><td></td></tr><tr><td>Surgical glue, 3M Vetbond tissues adhesive</td><td>3M Animal Care Prodcuts</td><td>1469SB</td><td></td></tr><tr><td>Tail vein cathether</td><td>Custom</td><td></td><td>Consists of two 30 G cannulas connected with sillicone tubing&amp;nbsp;</td></tr><tr><td>TrypLE Express (1x), no phenol red</td><td>Invitrogen</td><td>12604-039</td><td></td></tr><tr><td>Ultraviolett torch</td><td>Spring sunshine technology</td><td>Consciot</td><td></td></tr></tbody></table>

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.

Here, we performed cranial window surgery and engrafted C6 cells in an NSG mouse model of GBM (n = 5). A proper seal between all components involved in the creation of the window (Figure 1A) will ensure the windows&#39; durability for long-term imaging and, additionally, reduce morbidity. Using the stage adapted for in vivo 2P-IVM (Figure 2), we could image animals under anesthesia for up to 2 h without any major motion artifacts. Approximately 10 min a...

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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.

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 ...

two-photon-intravital-microscopy-of-glioblastoma-in-a-murine-model

Research

JoVE Journal

Cancer Research

1.9K Views.  Stanford University School of Medicine. Glioblastoom is een van de meest voorkomende primaire hersenkanker en wordt in verband gebracht met snelle groei en een slechte levensverwachting. Het ontwikkelen van effectieve therapieën voor glioblastoom blijft een grote uitdaging, aangezien de toediening van therapieën wordt beperkt door de bloed-hersenbarrière. Een methode om de accumulatie en distributie van macromoleculen in de hersenen direct in beeld te brengen, zou ons vermogen om de afgifte van geneesmiddelen te begrijpen en te ...

Video: Auteur in de schijnwerpers: multimodale beeldvormingsstrategieën voor het optimaliseren van medicijnafgifte en vroege detectie bij de behandeling van glioblastoom

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 ...

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

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.

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.

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 ...

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

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 ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

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 ...

Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

Source: Alieva, M. and Rios, A. C. <a href="https://www.jove.com/v/59278/longitudinal-intravital-imaging-brain-tumor-cell-behavior-response-to">Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.</a>&#160;J. Vis. Exp.&#160;(2019)
In this video, we perform intravital imaging of pre-injected cancer cells in a mouse model via an implanted cranial window. This&#160;high-resolution time-lapse imaging technique enables the study of dynamic behavior of cancer cells in the live mouse.

Source: Alieva, M. and Rios, A. C. Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vivo studies

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 ...

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

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