Name: modeling brain metastases through intracranial injection and magnetic resonance imaging
Uploaded: 2020-06-07T15:00:00
Description: Watch this Scientific Journal Video about Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging Video at JoVE.com

Representative data was funded through the National Cancer Institute (K22CA218472 to G.M.S.). Intracranial injections are performed in The Ohio State University Comprehensive Cancer Center Target Validation Shared Resource (Director &#8211; Dr. Reena Shakya) and MRI is completed in The Ohio State University Comprehensive Cancer Center Small Animal Imaging Shared Resource (Director &#8211; Dr. Kimerly Powell). Both shared resources are funded through the OSUCCC, the OSUCCC Cancer Center Support Grant from the National Cancer Institute (P30 CA016058), partnerships with The Ohio State University colleges and departments, and established chargeback systems.

All methods described herein have been approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University (P.I. Gina Sizemore; Protocol #2007A0120). All rodent survival surgery IACUC policies are followed, including use of sterile techniques, supplies, instruments, as well as fur removal and sterile preparation of the incision site.
1. Intracranial injection of breast cancer cells
NOTE: The method described herein utilized the DB7 murine ...

<ol>
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The utilization of intracranial injection followed by serial monitoring with MRI provides the unique ability to visualize tumor growth with tumor volume accuracy over time. The application of digital imaging analysis allows for interpretation of brain lesions for tumor volume, hemorrhage, necrosis, and response to treatment.
As with any procedure, there are key steps that must be followed for success. First, careful setup of the stereotactic devices is imperative to the success of this techniq...

Brain metastases are 10 times more common than adult primary central nervous system tumors1, and have been reported in almost every solid tumor type with lung cancer, breast cancer, and melanoma exhibiting the highest incidence2. Regardless of the primary tumor site, the development of brain metastasis leads to a poor prognosis often associated with cognitive decline, persistent headaches, seizures, behavioral and/or personality changes1,3,4,5. In terms of breast cancer, there have been many advances in the prevention and treatment of the disease. However, 30% of women diagnosed with breast cancer will go on to develop metastases, and of those with stage IV disease, approximately 7% (SEER 2010-2013) have brain metastasis6,7. Current treatment options for brain metastasis involve surgical resection, stereotactic radiosurgery and/or whole brain radiotherapy. Yet, even with this aggressive therapy, the median survival for these patients is a short 8-11 months7,8,9. These grim statistics strongly support the need for the identification and implementation of novel, effective therapeutic strategies. Thus, as with all cancers that metastasize to the brain, it is essential to properly model breast cancer associated brain metastasis (BCBM) in the laboratory to ensure significant advancements in the field.
To date, researchers have utilized a variety of methodologies to study mechanisms of metastasis to the brain, each with distinct advantages and limitations10,11. Experimental metastasis methods such as tail vein and intracardiac injection spread tumor cells throughout the body and can result in immense tumor burden at other metastatic sites depending on the cells injected. These results are then confounding if specifically studying metastasis to the brain. The intracarotid artery injection method is advantageous as it specifically targets brain-seeding of tumor cells but is limited as it can be technically difficult to perform. Orthotopic primary tumor resection is often considered the most clinically relevant model of metastasis as it recapitulates the entire metastatic cascade. Yet, this approach involves prolonged wait periods for spontaneous metastasis to occur with dramatically lower rates of brain metastasis compared to the other metastatic sites such as the lymph node, the lung and the liver. Often, animals must be removed from studies due to tumor burden at these other metastatic sites prior to the development of brain metastasis. Other methods involving brain tropic cell lines are effective at metastasizing to the brain; however, these models are limited in that they take time to develop and often lose their tropism with propagation. Given these limitations, researchers have routinely used the intracranial injection method to model cancer metastasis to the brain11,12,13,14 with varying methodologies15,16,17,18,19. It is acknowledged that this approach similarly has limitations, most importantly in that it does not allow for investigation of early metastatic steps including intravasation out of the primary tumor, penetrance through the blood brain barrier, and establishment within the brain. However, it does allow for researchers to test (1) what tumor derived factors mediate growth within the brain (e.g., genetic manipulation of an oncogenic factor in tumor cells), (2) how changes in the metastatic microenvironment alter cancer growth at this site (e.g., comparison between transgenic mice with altered stromal components) and (3) effectiveness of novel therapeutic strategies on growth of established lesions.
Given the potential utility of the intracranial injection model, it is absolutely necessary to reduce technical error during injection and to precisely monitor tumor growth over time. The method described herein involves continuous dosing of inhaled gas anesthesia, and direct implantation of tumor cells into the brain parenchyma using a stereotactic drill and injection stand. Administering gas anesthetic allows for fine tuning the depth and length of anesthesia as well as ensuring a quick and smooth recovery. A digitally controlled, automated drill and needle injection system enhances injection-site precision and reduces technical error often incurred by drilling and free-hand injection methods. The use of magnetic resonance imaging (MRI) further increases precision in monitoring tumor growth, tumor volume, tissue response, tumor necrosis, and response to treatment. MRI is the imaging modality of choice for soft tissues20,21. This imaging technique does not use ionizing radiation and is preferred over Computed Tomography (CT), especially for multiple imaging sessions during the course of a study. MRI has much greater range of available soft tissue contrast then CT or ultrasound imaging (USG) and presents anatomy in greater detail. It is more sensitive and specific for abnormalities within the brain itself. MRI can be performed in any imaging plane without having to physically move the subject as is the case in 2D USG or 2D optical imaging. It is important to mention that the skull does not attenuate the MRI signal as in other imaging modalities. MRI allows the evaluation of structures that may be obscured by artifacts from bone in CT or USG. An additional advantage is that there are many contrast agents available for MRI, which enhances the lesion detection limit, with relatively low toxicity or side effects. Importantly, MRI allows monitoring in real-time unlike histological evaluation at the time of necropsy, which is limited in deciphering tumor volume. Other imaging modalities, such as bioluminescent imaging, are indeed effective for early tumor detection and monitoring over time; however, this method requires genetic manipulation (e.g., luciferase/GFP tagging) of cell lines and does not allow for volumetric measurements. MRI is further advantageous as it mirrors patient monitoring and downstream volumetric analysis of the MR images is known to be strongly correlated to histologic tumor size at necropsy22. Serial monitoring with MRI screening also increases the clinical monitoring of neurologic impairments, should they arise.
Overall, the presented method of stereotactic intracranial tumor injection followed by serial MRI enables us to produce reliable, predictable, and measurable results to study mechanisms of brain metastasis in cancer.

<table>
	<tbody>
		<tr>
			<td>Surgical Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products</td>
			<td>19-027132</td>
			<td>Povidone-iodine, 7.5%</td>
		</tr>
		<tr>
			<td>Bone Wax</td>
			<td>Surgical Specialities</td>
			<td>903</td>
			<td>Sterile and malleable beeswax and isopropyl palmitate</td>
		</tr>
		<tr>
			<td>Buponorphine SR-Lab</td>
			<td>ZooPharm</td>
			<td>N/A</td>
			<td>Long acting injectable analgesic 5 mL (0.5 mg/mL) polymetric formulation</td>
		</tr>
		<tr>
			<td>Cotton tip applicators</td>
			<td>Puritan</td>
			<td>25-806 10WC</td>
			<td>Sterile long stemmed cotton tip applicators</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Puralube</td>
			<td>17033-211-38</td>
			<td>Lubricating petrolatum and mineral oil based ophthalmic ointment</td>
		</tr>
		<tr>
			<td>Handwarmers</td>
			<td>Hothands</td>
			<td>HH2</td>
			<td>Air-activated heat packs</td>
		</tr>
		<tr>
			<td>Ibuprofen</td>
			<td>Up &#38; Up</td>
			<td>094-01-0245</td>
			<td>100mg per 5mL in liquid suspension</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein INC</td>
			<td>1182097</td>
			<td>Liquid anesthetic for use in anesthetic vaporizer</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Integra Miltex</td>
			<td>4-410</td>
			<td>#10 disposable scalpel blade</td>
		</tr>
		<tr>
			<td>Skin Glue</td>
			<td>Vetbond</td>
			<td>1469SB</td>
			<td>Skin safe wounds adhesive</td>
		</tr>
		<tr>
			<td>Sterile Dressing</td>
			<td>TIDI Products</td>
			<td>25-517</td>
			<td>Individually packed sterile drapes</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Covidien</td>
			<td>SP5686G</td>
			<td>45cm swedged 5-0 monofilament polypropylene suture</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic Unit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Drill (Foredom)</td>
			<td>Kopf</td>
			<td>Model 1474</td>
			<td>Max of 38,000 RPM</td>
		</tr>
		<tr>
			<td>Mouse Gas Anesthesia Head Holder</td>
			<td>Kopf</td>
			<td>Model 923-B</td>
			<td>Mouth bar with teeth hole and nosecone</td>
		</tr>
		<tr>
			<td>Non-Rupture Ear Bars</td>
			<td>Kopf</td>
			<td>Model 922</td>
			<td>Ear bars suitable for mouse applications</td>
		</tr>
		<tr>
			<td>Stereotaxic Instrument</td>
			<td>Kopf</td>
			<td>Model 940</td>
			<td>Base plate, frame and linear scale assembly with digital readout monitor</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injector Needle and syringe</td>
			<td>Hamilton</td>
			<td>80366</td>
			<td>26 gauge needle, 51 mm needle length and 10 &#956;L volume syringe</td>
		</tr>
		<tr>
			<td>Legato 130A automated Syringe Pump</td>
			<td>KD Scientific</td>
			<td>P/N: 788130</td>
			<td>Programmable touch screen base with automated injector</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Low-Flow Digital Vaporizer</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td>Digital anesthesia machine</td>
		</tr>
		<tr>
			<td>SomnoSuite Starter Kit for mice</td>
			<td>Kent Scientific</td>
			<td>SOMNO-MSEKIT</td>
			<td>Includes induction chamber, 2x anesthesia syringes, 18&#34; tubing, plastic nosecone, 2x waste aneshesia gas canisters</td>
		</tr>
	</tbody>
</table>

modeling brain metastases through intracranial injection and magnetic resonance imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Figure 3 overviews the tumor volume quantification for a single mouse at two time points (day 7 and day 10) post-injection of murine mammary tumor cells. For this experiment, 50,000 DB7 cells were injected, and the animal&#8217;s brain was evaluated by MRI. For each scan, 30 slices (0.5 mm thickness) were captured. Evaluation of the 30 slices per scan revealed that at day 7 post-injection, 5 slices exhibited tumor burden (Figure 3A) and at day 10 post-injection,...

Watch this Scientific Journal Video about Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging Video at JoVE.com

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging Video (Video) | JoVE

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

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In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis Video (Video) | JoVE

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment Video (Video) | JoVE

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors Video (Video) | JoVE

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland Video (Video) | JoVE

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells Video (Video) | JoVE

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells Video (Video) | JoVE

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Longitudinal Intravital Imaging Through Clear Silicone Windows

Longitudinal Intravital Imaging Through Clear Silicone Windows Video (Video) | JoVE

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy Video (Video) | JoVE

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...