The authors warmly thank Dr. KK Fenrich, Dr. M-C. Amoureux, P. Weber and A. Jaouen for helpful discussions; M. Hocine, C. Meunier, M. Metwaly, S. Bensemmane, J. Bonnardel, the staff of the animal facility at IBDML and the staff of the PicSIL imaging platform at IBDML for technical support. This work was supported by grants from Institut National du Cancer (INCA-DGOS-INSERM6038) to GR, Agence Nationale de la Recherche (ANR JCJC PathoVisu3Dyn), F&#233;d&#233;ration pour la Recherche sur le Cerveau (FRC) to FD, by fellowships from the F&#233;d&#233;ration de la Recherche M&#233;dicale and Canc&#233;ropole PACA to CR.

All experimental procedures were performed in accordance with the French legislation and in compliance with the European Community Council Directive of November 24, 1986 (86/609/EEC) for the care and use of laboratory animals. The research on animals was authorized by the Direction D&#233;partementale des Services V&#233;t&#233;rinaires des Bouches-du-Rh&#244;ne (license D-13-055-21) and approved by the ethical committee of Provence Cote d&#39;Azur n&#176;14 (Project 87-04122012).
1. Spheroids P...

<ol>
	<li>DeAngelis, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumors.">Brain tumors.</a> N Engl J Med. 344, 114-123 (2001).</li><li>Ricard, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+brain+tumours+in+adults.">Primary brain tumours in adults.</a> Lancet. 379, 1984-1996 (2012).</li><li>Prados, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+III+randomized+study+of+radiotherapy+plus+procarbazine,+lomustine,+and+vincristine+with+or+without+BUdR+for+treatment+of+anaplastic+astrocytoma:+final+report+of+RTOG+9404.">Phase III randomized study of radiotherapy plus procarbazine, lomustine, and vincristine with or without BUdR for treatment of anaplastic astrocytoma: final report of RTOG 9404.</a> Int J Radiat Oncol Biol Phys. 58, 1147-1152 (2004).</li><li>Piao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+resistance+to+anti-VEGF+therapy+is+associated+with+myeloid+cell+infiltration,+stem+cell+accumulation,+and+a+mesenchymal+phenotype.">Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype.</a> Neuro Oncol. 14, 1379-1392 (2012).</li><li>Zhai, H., Heppner, F. L., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/macrophages+promote+glioma+progression.">Microglia/macrophages promote glioma progression.</a> Glia. 59, 472-485 (2011).</li><li>Studwell, A. J., Kotton, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shift+from+cell+cultures+to+creatures:+in+vivo+imaging+of+small+animals+in+experimental+regenerative+medicine.">A shift from cell cultures to creatures: in vivo imaging of small animals in experimental regenerative medicine.</a> Mol Ther. 19, 1933-1941 (2011).</li><li>Ustione, A., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+introduction+to+multiphoton+microscopy.">A simple introduction to multiphoton microscopy.</a> J Microsc. 243, 221-226 (2011).</li><li>Ricard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+effects+of+synchrotron+irradiation+on+vasculature+and+tissue+in+healthy+mouse+brain.">Short-term effects of synchrotron irradiation on vasculature and tissue in healthy mouse brain.</a> J Synchrotron Radiat. 16, 477-483 (2009).</li><li>von Baumgarten, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bevacizumab+has+differential+and+dose-dependent+effects+on+glioma+blood+vessels+and+tumor+cells.">Bevacizumab has differential and dose-dependent effects on glioma blood vessels and tumor cells.</a> Clin Cancer Res. 17, 6192-6205 (2011).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2, 932-940 (2005).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. , 680 (2008).</li><li>Lathia, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+imaging:+live+imaging+of+glioma+by+two-photon+microscopy.">Brain tumor imaging: live imaging of glioma by two-photon microscopy.</a> Cold Spring Harb Protoc. 2013, (2013).</li><li>Winkler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+glioma+cell+invasion+in+vivo+reveals+mechanisms+of+dissemination+and+peritumoral+angiogenesis.">Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis.</a> Glia. 57, 1306-1315 (2009).</li><li>Newcomb, E., Zagzag, D., Van Meir, E. 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This approach allows the use of optical imaging methods to monitor over days and weeks the growth of an orthotopically implanted glioma. The same animal can subsequently be subjected to virtually any brain imaging modality during the course of the pathology; yet the two-photon microscopy specific preparation offers the unique opportunity to achieve subcellular resolution inside the brain of the living animal. Our protocol presents the advantage to enforce the tumor growth into the cerebral parenchyma rather than extra-du...

Glioblastoma multiforme appears as the most aggressive form of brain tumor in adults with a median survival of 12 months and a 5-years survival rate of 5%. Clinical management relies on surgery, radiotherapy and chemotherapy often used in combination. However, the effects of these treatments remain palliative1-3.
Up to now, most of neuro-oncology studies rely on techniques that are only able to provide a static view and performed on large cohorts of tumor bearing animals sacrificed at different time-points (see for example4,5). The recent development of follow-up methods based on intravital imaging allows studying glioma growth and the interactions between tumor cells and their pathophysiological microenvironment on the same animal over time. This opens the way to exclusive piece of information that was so far unachievable6. Transgenic animals expressing fluorescent tags in cells of interest may be used to study specific interactions between tumor cells and e.g. neurons in this paper.
Over the past decade, intravital two-photon microscopy7 has become a gold standard in fundamental neuro-oncology studies and preclinical trials8,9 for its ability to perform deep intravital observation of mouse brain (&#62;500 &#181;m below the dura-mater) with a micrometric spatial resolution10. Using intravital two-photon microscopy with orthotopical animal models implanted with a chronic cranial window11, it is possible to follow the tumor progression over time on the same mouse9,12.
One of the major drawbacks of these previously published animal models is however that they do not mimic the physical constraints that govern tumor growth as the dura-mater is not sealed after the injection of the cell suspension9,13,14. Glioma cells may leak in the extradural space transforming an orthotopic glioma model into a heterotopic one.
The animal model presented here consists in the injection of a spheroid of fluorescent glioma cells in the cerebral cortex at a depth of 200 &#181;m followed by the sealing of the dura-mater with a cross-linked dextran gel hemi-bead and histo-compatible glue. The tumor growth is then restricted to the brain parenchyma that maintains pathophysiological physical constraints. A chronic glass window implanted above the tumor allows an easy optical access for intravital two-photon microscopy. Using transgenic animals expressing fluorescent tags in cells of interest it is possible to perform a follow-up of the glioma growth over time and to study its interaction with its microenvironment (here with neurons and vasculature highlighted with fluorescent dextrans).

<table border="0" cellpadding="0" cellspacing="0" width="1337">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:425px;">Drill</td>
			<td style="width:383px;">Dremel (Germany)</td>
			<td style="width:156px;">398</td>
			<td style="width:375px;">any high quality surgical bone drill would suffice</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:425px;">Drill burr (#1/4 Carbide Round Burr)</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>501860 (#1/4)</td>
			<td>also sold by Harvard Apparatus</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Tissue scissors</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>14395</td>
			<td style="width:375px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Dumont tweezers M5S</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>501764</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Dental cement</td>
			<td>GACD (USA)</td>
			<td>12-565 &#38; 12-568</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Cyanoacrylate</td>
			<td>Eleco-EFD (France)</td>
			<td>Cyanolit 201</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Glass capillaries without filament</td>
			<td>Clark Electromedical Instruments (UK)</td>
			<td>GC100-15</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Microliter syringe (25 &#181;l)</td>
			<td>Hamilton (USA)</td>
			<td>702</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Micromanipulator</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>Kite-R</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">T derivation (3-way stopcock - Luer lock)</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>14035-10</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Stereotactic frame (mouse adaptor)</td>
			<td style="width:383px;">World Precision Instruments (USA)</td>
			<td>502063</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Glass coverslips</td>
			<td>Warner Instruments (USA)</td>
			<td>CS-5R (64-0700)</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Cross-linked dextran gel (Sephadex) G50 Coarse 100-300 &#181;m beads</td>
			<td>Available from various suppliers including Sigma (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Eye ointment</td>
			<td>TVM (France)</td>
			<td>Ocry-gel</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Fluorescence macroscope</td>
			<td>Leica MZFLIII (Germany)</td>
			<td></td>
			<td>also sold by other companies</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Two-photon microscope</td>
			<td>Zeiss LSM 7MP (Germany)</td>
			<td></td>
			<td>also sold by other companies (Nikon, &#8230;)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Infrared tunable femtosecond laser (Ma&#239;-Ta&#239;)</td>
			<td>Spectra Physics (USA)</td>
			<td></td>
			<td>also sold by other companies</td>
		</tr>
	</tbody>
</table>

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.

Once the surgical protocol is performed (Figure 1), animals can be observed by means of fluorescent microscopy over weeks until sacrifice. An inflammatory reaction may be observed after the surgery that disappears within one or two weeks. Tumor growth can be observed by various microscopy techniques including fluorescent macroscopy and two-photon microscopy (Figure 2). Example images depicted here were realized on a fluorescence macroscope and a two-photon microscope coupled to a femtose...

Watch this Scientific Journal Video about An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy at JoVE.com

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

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-orthotopic-glioblastoma-mouse-model-maintaining-brain-parenchymal

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Medicine

19.6K vistas.  Aix Marseille University.  Hemos establecido un modelo ortotópico de glioblastoma cortical en ratones para intravital microscopía de dos fotones que recapitula las limitaciones biofísicas normalmente en juego durante el crecimiento del tumor. Una ventana de cristal crónica reemplazando el cráneo por encima de la del tumor permite el seguimiento de la progresión del tumor con el tiempo por microscopía de dos fotones. 

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

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...

flexible organic electronic devices for pulsed electric field therapy of glioblastoma

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of...

live imaging of microtubule dynamics in glioblastoma cells invading the zebrafish brain

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

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...

two-photon intravital microscopy in mouse model of glioblastoma

Two-Photon Intravital Microscopy in Mouse Model of Glioblastoma

two-photon intravital microscopy of glioblastoma in a murine model

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...

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

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

https://cloudfront.jove.com/CDNSource/teasers/66304_b.jpg

intravital two-photon microscopy of the transplanted mouse lung

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

https://cloudfront.jove.com/CDNSource/teasers/66566_b.jpg

surgical procedure for preparing the orthotopic mouse model of glioblastoma

Surgical Procedure for Preparing the Orthotopic Mouse Model of Glioblastoma

long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon...

combination radiotherapy in an orthotopic mouse brain tumor model

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial...

intravital imaging of the mouse thymus using 2-photon microscopy

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development...

imaging cell interaction in tracheal mucosa during influenza virus infection using two-photon intravital microscopy

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory...

two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

deep-tissue three-photon fluorescence microscopy in intact mouse and zebrafish brain

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Three-photon microscopy enables high-contrast fluorescence imaging deep in living biological tissues, such as mouse and zebrafish brains, with high spatiotemporal...

multi-photon imaging of tumor cell invasion in an orthotopic mouse model of oral squamous cell carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented.  This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive...

intravital microscopy to analyze blood cell-endothelial interactions in an inflammation mouse model

In this video, we demonstrate the real-time visualization of blood cell-endothelial interactions in the mesenterial veins of a mouse model injected intraperitoneally with...

Intravital Microscopy to Analyze Blood Cell-Endothelial Interactions in an Inflammation Mouse Model

in vivo imaging of the mouse spinal cord using two-photon microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

an isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy...

two-photon microscopy for the study of tendons

Two-Photon Microscopy for the Study of Tendons

This article outlines the process of preparing, setting up, and imaging tendons using multiphoton microscopy. Additionally, it covers the application of SHG for analyzing collagen fibril alignment and the creation of a 3D representation of tendons. This methodology proves highly valuable in characterizing tendon cells and their ECM during injury and...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

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 ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...