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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S., Camphausen, K. A., Smith, Q. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+metastases+as+preventive+and+therapeutic+targets.">Brain metastases as preventive and therapeutic targets.</a> Nature Reviews Cancer. 11 (5), 352-363 (2011).</li><li>Valiente, M., 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=The+Evolving+Landscape+of+Brain+Metastasis.">The Evolving Landscape of Brain Metastasis.</a> Trends in Cancer. 4 (3), 176-196 (2018).</li><li>Wang, H., 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=The+prognosis+analysis+of+different+metastasis+pattern+in+patients+with+different+breast+cancer+subtypes:+a+SEER+based+study.">The prognosis analysis of different metastasis pattern in patients with different breast cancer subtypes: a SEER based study.</a> Oncotarget. 8 (16), 26368-26379 (2017).</li><li>Wang, R., 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=The+Clinicopathological+features+and+survival+outcomes+of+patients+with+different+metastatic+sites+in+stage+IV+breast+cancer.">The Clinicopathological features and survival outcomes of patients with different metastatic sites in stage IV breast cancer.</a> BMC Cancer. 19 (1), 1091 (2019).</li><li>Gong, Y., Liu, Y. R., Ji, P., Hu, X., Shao, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+molecular+subtypes+on+metastatic+breast+cancer+patients:+a+SEER+population-based+study.">Impact of molecular subtypes on metastatic breast cancer patients: a SEER population-based study.</a> Scientific Reports. 7, 45411 (2017).</li><li>Kim, Y. J., Kim, J. S., Kim, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+subtype+predicts+incidence+and+prognosis+of+brain+metastasis+from+breast+cancer+in+SEER+database.">Molecular subtype predicts incidence and prognosis of brain metastasis from breast cancer in SEER database.</a> Journal of Cancer Researchearch and Clinical Oncology. 144 (9), 1803-1816 (2018).</li><li>Gomez-Cuadrado, L., Tracey, N., Ma, R., Qian, B., Brunton, V. 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H., 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=Changes+in+BAI1+and+nestin+expression+are+prognostic+indicators+for+survival+and+metastases+in+breast+cancer+and+provide+opportunities+for+dual+targeted+therapies.">Changes in BAI1 and nestin expression are prognostic indicators for survival and metastases in breast cancer and provide opportunities for dual targeted therapies.</a> Molecular Cancer Therapeutics. 14 (1), 307-314 (2015).</li><li>Russell, 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=PTEN+expression+by+an+oncolytic+herpesvirus+directs+T-cell+mediated+tumor+clearance.">PTEN expression by an oncolytic herpesvirus directs T-cell mediated tumor clearance.</a> Nature Communications. 9 (1), 5006 (2018).</li><li>Thies, K. A., 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=Stromal+platelet-derived+growth+factor+receptor-beta+signaling+promotes+breast+cancer+metastasis+in+the+brain.">Stromal platelet-derived growth factor receptor-beta signaling promotes breast cancer metastasis in the brain.</a> Cancer Research. , (2020).</li><li>Kramp, T. R., Camphausen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+radiotherapy+in+an+orthotopic+mouse+brain+tumor+model.">Combination radiotherapy in an orthotopic mouse brain tumor model.</a> Journal of Visualized Experiments. (61), e3397 (2012).</li><li>Pierce, A. M., Keating, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+anatomically+accurate+and+reproducible+intracranial+xenografts+of+human+brain+tumors.">Creating anatomically accurate and reproducible intracranial xenografts of human brain tumors.</a> Journal of Visualized Experiments. (91), e52017 (2014).</li><li>Abdelwahab, M. G., Sankar, T., Preul, M. C., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+implantation+with+subsequent+3D+in+vivo+bioluminescent+imaging+of+murine+gliomas.">Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas.</a> Journal of Visualized Experiments. (57), e3403 (2011).</li><li>Donoghue, J. F., Bogler, O., Johns, T. 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The authors do not have any disclosures.

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 at JoVE.com

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

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.

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

This intracranial injection protocol is significant because it allows precise injections, day-to-day monitoring, and accurate tumor volume measurement for more effective study of mechanisms of brain metastasis. The main advantage of this technique is that it allows for serial monitoring of intercranial tumor growth. Demonstrating the procedure will be Jonathan Spehar, a graduate research associate from the Sizemore Laboratory, and Anna Bratasz, an imaging research scientist from the Ohio State University Small Animal Imaging Shared Resource.Before beginning the procedure, twist the stage lock on the drill to allow a drill bit adapter and sterile one millimeter drill bit to be inserted into the drill and manually tighten the bit locks to lock the drill. Attach the drill to the stereotactic frame and set the digital injector delivery rate to 0.4 microliters per minute, with a target of two microliters. After confirming a lack of response to pedal reflex, place the anesthetized mouse onto the frame and use the blunt end of a cotton tip applicator to position the teeth in the trough of the mouth bar.Press the left ear bar against the medial canthus of the left ear to stabilize the skull and use the screw on the stereotactic frame to lock the skull into place. Then secure the other side of the skull with the right ear bar in the same manner. To make a calvarial window, clean the scalp with three sequential alternating Betadine solution and 70%ethanol scrubs and use a sterile scalpel to make a three millimeter incision through the skin along the central median aspect of the cranium following the sagittal suture line.Identify and orient the sterile drill bit perpendicular to the bregma, making sure to reset the digital vernier scale to zero and move the drill bit two millimeters lateral to the sagittal suture and one millimeter anterior to the coronal suture. Move the skin away from the drill and using the highest speed and paying attention to the landmarks, carefully drill a hole, roughly 0.5 millimeters deep, through the calvaria, cooling the drill site with drops of sterile saline as necessary. Once the calvarial window has been made, carefully raise the drill and remove the drill from the stereotactic frame.For cancer cell injection, attach the automatic injector unit to the stereotactic apparatus and load the six to eight microliters of cells thoroughly resuspended in DPBS into a sterile Hamilton syringe. Load the syringe onto the injector and dispense a small volume of solution onto a disposable sterile drape to prime the needle for injection. Use a cotton tip applicator to wipe the syringe with 70%ethanol and align the needle tip to the center of the calvarial window until it nearly touches the exposed cerebrum.Reset digital vernier scale to zero and slowly insert the needle into a three millimeter depth into the brain. Leave the needle in the brain for at least 60 seconds before selecting run on the injector screen to begin the cell delivery to the injection site. When all of the cells have been delivered, allow the needle to rest in the brain for at least three minutes to allow the brain parenchyma to acclimate to the injection before retracting the needle from the brain at a rate of 0.75 millimeters per minute.For magnetic resonance imaging of the tumor burden, 10 to 20 minutes before imaging at the appropriate experimental time point, administer 100 microliters per 20 grams of body weight gadolinium-based contrast agents to the mouse by standard intraperitoneal injection. Anesthetize the mouse post-injection and place the anesthetized mouse on a heated holder. Place the holder into a 9.4 T magnet equipped with a mouse brain surface coil and obtain a localizer image.Then image the mouse brain using a T2 weighted rare sequence and post gadolinium-based contrast T1 weighted rare sequence. To measure the total tumor volume, open the MRI data file of interest in ImageJ and use the free hand selections tool to draw an outline around the tumor. Then open the analyze tab and select measure to obtain the area of the selected region.When all of the tumor containing slices for an individual mouse at that specific time point have been assessed, export the values into an appropriate data analysis program. Here, a representative overview of the tumor volume quantification for a single mouse at day 7 and day 10 post murine mammary tumor cell injection can be observed. The evaluation of 30 slices per scan revealed that for this analysis, at day seven post-injection, five slices exhibited a tumor burden.While at day 10, nine slices exhibited a tumor burden. The tumor area in each image was then measured as demonstrated, allowing the change in tumor volume to be tracked over time. Each cell line and each recipient mouse model is unique and therefore requires optimization of the number of cells injected and the MRI schedule to ensure imaging accuracy.Following this procedure, the brain can be harvested for essentially any downstream assay, including single cell isolation or histopathological analysis.

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Research

JoVE Journal

Cancer Research

6.9K Views.  The Ohio State University. This intracranial injection protocol is significant because it allows precise injections, day-to-day monitoring, and accurate tumor volume measurement for more effective study of mechanisms of brain metastasis. The main advantage of this technique is that it allows for serial monitoring of intercranial tumor growth. Demonstrating the procedure will be Jonathan Spehar, a graduate research associate from the Sizemore Laboratory, and Anna Bratasz, an imaging research scientist from the Ohio Stat...