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. G. <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+guide+screw+method+for+intracranial+xenograft+studies+in+mice.">A simple guide screw method for intracranial xenograft studies in mice.</a> Journal of Visualized Experiments. (55), (2011).</li><li>Ozawa, T., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+intracranial+brain+tumor+xenografts+with+subsequent+analysis+of+tumor+growth+and+response+to+therapy+using+bioluminescence+imaging.">Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging.</a> Journal of Visualized Experiments. (41), (2010).</li><li>Fink, J. R., Muzi, M., Peck, M., Krohn, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodality+Brain+Tumor+Imaging:+MR+Imaging,+PET,+and+PET/MR+Imaging.">Multimodality Brain Tumor Imaging: MR Imaging, PET, and PET/MR Imaging.</a> Journal of Nuclear Medicine. 56 (10), 1554-1561 (2015).</li><li>Borges, A. R., Lopez-Larrubia, P., Marques, J. B., Cerdan, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+imaging+features+of+high-grade+gliomas+in+murine+models:+how+they+compare+with+human+disease,+reflect+tumor+biology,+and+play+a+role+in+preclinical+trials.">MR imaging features of high-grade gliomas in murine models: how they compare with human disease, reflect tumor biology, and play a role in preclinical trials.</a> American Journal of Neuroradiology. 33 (1), 24-36 (2012).</li><li>Prabhu, S. S., Broaddus, W. C., Oveissi, C., Berr, S. S., Gillies, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+intracranial+tumor+volumes+in+a+rodent+brain+using+magnetic+resonance+imaging,+Evans+blue,+and+histology:+a+comparative+study.">Determination of intracranial tumor volumes in a rodent brain using magnetic resonance imaging, Evans blue, and histology: a comparative study.</a> IEEE Transactions on Biomedical Engineering. 47 (2), 259-265 (2000).</li><li>Borowsky, A. 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=Syngeneic+mouse+mammary+carcinoma+cell+lines:+two+closely+related+cell+lines+with+divergent+metastatic+behavior.">Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior.</a> Clinical &amp; Experimental Metastasis. 22 (1), 47-59 (2005).</li><li>Journal of Visualized Experiments. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Lab+Animal+Research.+Compound+Administration+I.">JoVE Science Education Database. Lab Animal Research. Compound Administration I.</a> Journal of Visualized Experiments. , (2020).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. 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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 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.

modeling-brain-metastases-through-intracranial-injection-magnetic

Research

JoVE Journal

Cancer Research

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

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

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. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

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

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

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

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

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

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

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

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

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

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

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 diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

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

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

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

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

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

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...