This research is supported by funding from the Intramural program of the NIH.

I. Cell Preparation
<ol>
	<li>The cells are grown to confluence in DMEM with 10% FBS about one 80% confluent 225 cm3 flask for every 5 mice to be implanted.</li>
	<li>Aspirate media off of the cells; wash with 10 ml of PBS without Calcium or Magnesium. Aspirate PBS off cells.</li>
	<li>Trypsinize cells with 3 ml of trypsin, wait 10-15 min, and neutralize the trypsin with 15 ml of media.</li>
	<li>Pipet all cells and media into a 50 ml conical. Two large flasks fit into one 50 ml conical.</li>
	<li>Spin cells at 1600 rpm for 7 min at 4 &deg;C. Completely aspirate supernatant. Resuspend cells in 10 ml of cold PBS, and rinse cells by gently pipetting up and down. Make sure the cell pellet is mixed uniformly with the PBS. The suspension should be cloudy.</li>
	<li>At this time a cell count is done using a coulter counter. The cells are centrifuged again at 1600 rpm for 7 min at 4 &deg;C.</li>
	<li>After the 2nd centrifugation of the cells, aspirate all PBS and resuspend in 1 ml of PBS.</li>
	<li>The cells are spun in the 1ml of PBS at 3000 rpm for 10 min at 4 &deg;C. The PBS is aspirated, and PBS is added to adjust the final concentration to 1x106 per 5 &mu;l for U87 cells, other cell lines may require a different number and concentration of cells for optimal tumor growth.</li>
	<li>After the final volume and concentration is obtained the cells are kept on ice in an eppendorf tube.</li>
</ol>
II. Xenograft
<ol class="ualpha">
	<li>Preparing the mouse
	<ol>
		<li>The mice are anesthetized using an injection mix of Ketamine/Xylazine/Saline. The mixture is 120 mg/kg of Ketamine, 16 mg/kg of Rompun and Saline. The mice are weighed individually and given 0.1 ml per 10 g based on their weight. The anesthetic is given with 26G needle attached to 1 ml syringe IP. It will take approximately 15 min for the mice to reach a sleeping phase for no pain response. You can check the pain response of the mice by pinching their footpad.</li>
		<li>After the mouse is fully anesthetized, eye ointment is administered to their eyes to prevent them from drying out.</li>
		<li>The mouse is placed on the bed of the stereotaxic with the front upper teeth in the notch on the mouth bar. The bed is then placed in the stereotaxic.</li>
		<li>The nose guard is placed over the mouse's nose to prevent movement of the head, and the ear bars are gently place in the ear canals of the mice. Be careful not to do either placement of the nose bar or ear bars too tight, as the skull may fracture and/or prevent the mouse from breathing.</li>
		<li>Make sure that the head of the mouse is level using the tick marks on the stereotaxic is helpful during this process.</li>
		<li>To prep the mouse, alcohol and providine iodine is applied liberally on the head of the mouse alternating for a total of 3 application of alcohol and 3 applications of iodine. One final application of an alcohol pad with 70% is applied to the head.</li>
	</ol>
	</li>
	<li>Surgical procedure
	<ol>
		<li>All instruments in the procedure should be sterilized either by autoclaving or soaking instruments in 70% ethanol before beginning the procedure.</li>
		<li>An incision is made on top of the skull behind the right eye diagonally back to the left posterior of the skull. The incision should be about 10 mm to 15 mm long.</li>
		<li>Using a Q-tip, slowly separate the incision to expose the skull and wipe away the membrane that is between the skull and the skin. Use a Q-tip to blot any blood and watch for excessive bleeding.</li>
		<li>Expose the top of the skull to visualize the bregma. Using the Hamilton syringe point, position it over the bregma. The correct position of the hole to be drilled in the skull is 2 mm right and 1 mm anterior of bregma (figure 2). A surgeon's felt-tipped marker, that is purchased sterile, is used to mark the location of the hole to be drilled. Please note that the hole is located close to the anterior suture line.</li>
		<li>Next, drill a small hole in the skull at the marked position using the point of a 22G needle in a pin vase or fed through an 18G needle.</li>
		<li>Load the 10 &mu;l glass Hamilton syringe with 5 &mu;l of cells. The Hamilton syringe is 10 &mu;l with a blunt 30G to 33G fixed needle attached to it. Be sure to mix the cells before loading them, either by pipetting them or using a 1ml syringe with a 25G needle attached. After the Hamilton syringe is loaded, place it back in the arm to line it up with the hole in the skull.</li>
		<li>Lower the bunt end of the needle to the surface of the skull, once the needle is level with the skull; lower the needle until it is at the depth of 3 mm below the surface of the skull.</li>
		<li>After the needle is lowered, it shall remain there for 2 minutes to avoid any major trauma from the needle affecting the brain. Implanting the cells is at a rate of 1 &mu;l per minute for a total of 6-8 minutes, be careful of any backflow. After the last of the cells have been implanted, leave the needle in place for another 2 minutes, to allow all the cells to settle in.</li>
		<li>Remove the needle from the brain, and clean the hole with a Q-tip. Proceed to clean the needle and syringe with 50% Actone, 100% EtOH, and PBS, 3x in each solution and in that order.</li>
		<li>The skin on the skull is ready to be sutured at this point. Using 5-0 PBS close the incision with about 3 stitches.</li>
		<li>Allow the mouse to wake up on a heat pad, and reapply eye ointment if needed. The mouse can be returned to the cage after it shows movement on its own (i.e. Lifting up the head or moving the shoulders).</li>
	</ol>
	</li>
</ol>
III. Treatment of the IC implant By Radiation and Drug
<ol class="ualpha">
	<li>Radiation treatment
	<ol>
		<li>Depending on the cell line, treatment of the mice can take place 5 to 7 days post-implant. Our department uses an orthovoltage radiotherapy unit for radiation treatment.</li>
		<li>Imaging such as BLI (Bioluminescence Imaging), MRI or CT can be used to visualize the tumor prior to randomization (figure 3A).</li>
		<li>The mice are anesthetized with the Ketamine/Xylazine/saline cocktail as before, and eye ointment is applied to their eyes.</li>
		<li>The mice are positioned in a custom made radiation jig that is configured like a pie wheel, in which the heads of the mice point towards the center. The bodies of the mice are shielded from the radiation with lead.</li>
		<li>Drug treatment can be given either before or radiation through different delivery routes, i.e. SQ, IP and IV.</li>
	</ol>
	</li>
</ol>
IV. Results
The mouse should wake up from the procedure within 45-60 minutes after anesthesia injection. If the mouse has difficultly waking up after the procedure and time has exceeded 1&frac12; hr after the procedure, the mouse may not recover and euthanasia may be an effective alternative. If there is excessive bleeding or the skull becomes cracked at any point during the procedure euthanasia is advised. If all goes well and depending on the cell line, a tumor should start forming within 2-4 weeks after the procedure has take place. When using intracranial xenograft models, therapeutic efficacy is usually measured as survival post-implantation5. However, as this is an IC model signs and symptoms will occur prior to death. Some of the physical signs the mouse may exhibit are a hunched posture, loss of body weight, and inactivity. Some of the neurological signs may be seizures, head tilt, and loss of balance. Figure 3B shows an HE stain of a tumor at mortality. A Kaplan-Meier plot is used to evaluate the data (figure 4). The number of days of survival at 50% are calculated and compared to the control. If the combination group lived longer than the addition of the individual radiation and drug arms the combination is was successful.
<img alt="Figure 1" src="/files/ftp_upload/3397/3397fig1.jpg" /> 
Figure 1. The Stoelting Stereotaxic equipment.
<img alt="Figure 2" src="/files/ftp_upload/3397/3397fig2.jpg" /> 
Figure 2. A diagram of the location of the drill hole in the mouse skull.
<img alt="Figure 3A" src="/files/ftp_upload/3397/3397fig3A.jpg" /> 
Figure 3A. BLI imaging after GBM implantation.
<img alt="Figure 3B" src="/files/ftp_upload/3397/3397fig3B.jpg" /> 
Figure 3B. H and E of GBM tumor at mortality.
<img alt="Figure 4" src="/files/ftp_upload/3397/3397fig4.jpg" /> 
Figure 4. Results of U87 IC implants injected with PARP inhibitor 7 days post surgery.
&emsp;

<ol>
	<li>Bleau, A. M., Holland, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+the+mouse+genome+to+hunt+human+alternations.">Trapping the mouse genome to hunt human alternations.</a> Proc. Nat. Acad. Sci. U.S.A. 104, 7737-7738 (2007).</li><li>McGrady, B. J., McCormick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+intracranial+invasion:+morphological+observations+on+central+nervous+system+invasion+by+melanoma+cells.">A murine model of intracranial invasion: morphological observations on central nervous system invasion by melanoma cells.</a> Clin. Exp. Metastasis. 10, 387-393 (1992).</li><li>Fei, X., Zhang, Q., Dong, J., Diao, y., Wang, Z., Li, R., Wu, Z., Wang, A., Lan, Q., Zhang, S., Huang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+clinically+relevant+orthotopic+xenograft+mouse+model+of+metastatic+lung+cancer+and+glioblastoma+through+surgical+tumor+tissues+injection+with+trocar.">Development of clinically relevant orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma through surgical tumor tissues injection with trocar.</a> Journal of Experimental &#38; Clinical Cancer Research. 29, 84-84 (2010).</li><li>Fujita, M., Zhu, X., Sasaki, K., Ueda, R., Low, K., Pollack, I., Okada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+STAT3+Promotes+the+Efficacy+of+Adoptive+Transfer+Therapy+Using+Type-1+CTLs+by+Modulation+of+the+Immunological+Microenvironment+in+a+Murine+Intracranial+Glioma.">Inhibition of STAT3 Promotes the Efficacy of Adoptive Transfer Therapy Using Type-1 CTLs by Modulation of the Immunological Microenvironment in a Murine Intracranial Glioma.</a> J. Immunol. 180, 2089-2098 (2008).</li><li>Candolfi, M., Curtin, J. F., Nichols, W. S., Muhammad, A. K. M. .. . G., King, G., Pluhar, G. D., McNiel, G. E., Ohlfest, E. A., Freese, J. R., Moore, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathlogical+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathlogical characterization and tumor progression.</a> J. Neurooncol. 85, 133-148 (2007).</li><li>Kaye, A., Morstyn, G., Gardner, I., Pyke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+xenograft+glioma+model+in+mouse+brain.">Development of a xenograft glioma model in mouse brain.</a> Cancer Research. 46, 1367-1373 (1986).</li><li>Zhao, Y., Xiao, A., diPierro, C., Carpenter, J., Abdel-Fattah, R., Redpath, G., Lopez, M., Hussaini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Extensive+Invasive+Intracranial+Human+Glioblastoma+Xenograft+Model.">An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model.</a> American Journal of Pathology. 176, 3032-3049 (2010).</li><li>Learn, C., Grossi, P., Schmittling, R., Xie, W., Mitchell, D., Karikari, I., Wei, Z., Dressman, H., Sampson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Analysis+of+Intracranial+Tumors+in+a+Murine+Model+of+Glioma+Demonstrate+a+Shift+in+Gene+Expression+in+Response+to+Host+Immunity.">Genetic Analysis of Intracranial Tumors in a Murine Model of Glioma Demonstrate a Shift in Gene Expression in Response to Host Immunity.</a> J. 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We have nothing to disclose.

Malignant gliomas, such as glioblastoma multiforme, represent the most common primary brain tumors and have a dismal prognosis4. Survival of patients affected by GBM has remained virtually unchanged during the last decade (i.e., 9-12 months post-diagnosis) despite advances in surgery, radiation, and chemotherapy5. The features of an appropriate model for the study of glioma treatment should include a reproducible tumor location and growth of the cells as a relatively discrete tumor; the cells should...

<table border="1">
	<tbody>
		<tr>
			<td>Lab Standard Stereotaxic Instrutment</td>
			<td>Stoelting Mouse and Rat Adaptor</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>100% EtOH</td>
		</tr>
		<tr>
			<td>70% EtOH</td>
			<td>50% Acetone</td>
		</tr>
		<tr>
			<td>6" Q-tips</td>
			<td>Alcohol swabs</td>
		</tr>
		<tr>
			<td>Hamilton syringe 80008</td>
			<td>Ethicon 5-0 PD</td>
		</tr>
		<tr>
			<td>1701 SN 31-33G/1"/PT3 needle</td>
			<td>1 ml syringes</td>
		</tr>
		<tr>
			<td>25G needles</td>
			<td>Heating pad</td>
		</tr>
		<tr>
			<td>Artificial Tears Ointment</td>
			<td>Very fine tipped permanent marker</td>
		</tr>
	</tbody>
</table>

combination radiotherapy in an orthotopic mouse brain tumor model

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.

Watch this Scientific Journal Video about Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model at JoVE.com

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 model.

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

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16.3K Views.  National Cancer Institute. 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.

Video: Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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 ...

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

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 ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

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  ...

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 ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.