The authors have no acknowledgements.

Ethics statement: All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Texas MD Anderson Cancer Center.
1. Prepare Cancer Cells for Injection
<ol>
	<li>Seed the cancer cells one or two days before injection. Use DMEM/F12 media supplemented with 10% FBS, unless a specialty medium is indicated in literature for a particular cell line.</li>
	<li>On the day of surgical operation, harvest cells when they reach 70 - 80% confluence by first washing with serum-...

<ol>
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The authors have nothing to disclose.

The most critical steps for successful carotid arterial injection of cancer cells are: 1) deep anesthesia of mouse in preparation for surgical operation; 2) steady placement of carotid artery on top of the cotton support; 3) tight ligation of carotid artery after successful injection.
Deep anesthesia of ~ 30 min is usually necessary for steady surgical performance under the dissecting microscope. We use commercially ready-to-use ketamine/xylazine cocktail for mouse anesthesia. To avoid potenti...

The metastasis of cancer to the central nervous system (CNS) is a devastating disease, and can involve either the brain parenchyma or the leptomeninges (&#34;brain metastasis&#34; refers to both in this article). It is the predominant intracranial malignancy, outnumbering primary gliomas by &#62; 10:11,2. Lung cancer, breast cancer, and melanoma are the top three major neoplastic diseases that produce high incidences of brain metastasis3,4. In recent years, impressive advances in novel cancer therapies have dramatically prolonged survival and improved quality of life for many cancer patients. However, upon recurrence, the incidence of brain metastasis is rising rapidly. For example, the anti-HER2 antibody trastuzumab (Herceptin) has demonstrated significant clinical efficacy in patients with HER2+ breast cancer; yet a disturbing trend has emerged in these patients: up to 1/3 of those whose extracranial systemic disease initially benefited from trastuzumab treatment later develop brain metastasis5,6,7. Sadly, patients with brain metastasis are refractory to almost all current treatments, usually experiencing a traumatic deterioration of quality of life, and their one-year survival after diagnosis is only ~ 20%8. Current therapies for brain metastasis (including steroids, cranial radiotherapy, and surgical resection in selected patients) are merely palliative, not curative9. Therefore, brain metastasis is emerging as the next imposing challenge in this era of novel cancer therapies. To address the unmet challenge patients face every day in the clinic, we urgently need to better understand the underlying mechanism of brain metastasis and use this knowledge to develop novel therapies.
Successful colonization of the brain by metastatic cancer cells requires performance of a series of functions mediated by the intricate interaction of multiple biological players, such as the bypass of the blood brain barrier (BBB) and escape from the brain&#39;s intrinsic immune defensive mechanisms10, none of which is recapitulated in an ex vivo or in vitro system. Hence, proper and faithful in vivo models are critical for studies of brain metastasis. A conventional in vivo metastasis assay introduces cancer cells via tail vein injection, leading the majority of cells to become lodged in the lung. Brain metastatic lesions are rarely produced in these models before animal death caused by tumor burden in the lungs11. Direct intracerebral injection of cancer cells produces consistent tumor outgrowth in the CNS and is widely used in the studies of primary gliomas. However, such injection compromises the BBB and causes traumatic injury at the site of injection, both major points of concern as to the physiological relevance of this model. Another frequently used cancer cell introduction route, intracardiac injection, is easy to administer and does produce experimental metastasis to the CNS. However, concurrent metastases to organ sites other than the CNS are always produced and may cause animal mortality11; therefore, the high degree of variability of this model makes it inappropriate for quantitative evaluation of biological mechanisms or therapeutic agents with a limited number of animals.
Here we describe the procedures to produce experimental brain metastasis via the injection of cancer cells into the common carotid artery. We have used this approach to dissect individual genes&#39; contributions to the metastatic cascade of brain metastasis and to evaluate efficacy of therapeutic interventions12,13. The major advantages of this approach are the high degree of reproducibility and low degree of variability; the major disadvantage is the sophistication and dexterity required to perform the microsurgery.

<table border="0" cellpadding="0" cellspacing="0" width="1156">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Ketamine hydrochloride/xylazine hydrochloride solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:151px;">K113</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Routine Stereomicroscope Leica M50</td>
			<td style="width:151px;">Leica</td>
			<td style="width:151px;">M50</td>
			<td>The microscope is modular and highly configurable to fit particular space requirements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Surgical disposable scapel</td>
			<td style="width:151px;">Integra Miltex</td>
			<td style="width:151px;">4-410</td>
			<td>to make skin incisions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Tissue Forceps - 1x2 Teeth</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11021-12</td>
			<td>to bluntly dissect mucles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Dumont #5 - Mirror Finish Forceps</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11252-23</td>
			<td>to separate and prepare the carotid artery for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Spring Scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">15025-10</td>
			<td>to cut sutures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">EZ Clip Kit</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:151px;">59020</td>
			<td>for wound cloure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BD Insulin Syringe</td>
			<td style="width:151px;">Becton Dickinson</td>
			<td style="width:151px;">328438</td>
			<td>for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">IVIS Spectrum In Vivo Imaging System</td>
			<td style="width:151px;">PerkinElmer</td>
			<td style="width:151px;">124262</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

There are two points at which the quality of injection can be evaluated. The first chance is by the operator&#39;s observation of changing blood vessel colors during injection. Leakage from poor injections can be easily observed under the dissecting microscope. The steady and secure placement of the mouse body (Figure 1A) and its carotid artery (Figure 1B) on the supporting cotton ball are critical factors for smooth and successful injection into the caro...

Watch this Scientific Journal Video about Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis at JoVE.com

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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. ...

The overall goal of this microsurgical injection of cancer cells through the carotid artery is to produce consistent in vivo experimental brain metastasis models in mice. So this method can help answer key questions in the brain metastasis field, such as whether a new therapeutic may show efficacy in treating brain metastasis. So the main advantage of this technique is that it allows the production of in vivo experimental brain metastasis with high reproducibility and low variability.Generally individuals new to this technique will struggle initially because a fair amount of practice is needed to maintain a steady hand to inject cancer cells into the carotid artery of these mice under the microscope. Begin this procedure by seeding the cancer cells with DMEM F12 media supplemented with 10%FPS and culturing them for one to two days before injection. On the day of surgical operation, remove the cells from the incubator and harvest them when they reach 70%to 80%confluence by washing with serum-free media.Next, incubate the cells with 0.25%trypsin for one to two minutes at 37 degrees Celsius. Afterward, remove them from the incubator and add 10%FBS containing DMEM F12 media to the cells to quench chipsenation. Centrifuge the cells at 80 times G for three minutes.Then, remove the medium and wash the cells in serum-free twice to remove residual serum. Next, count the cells with a hemocytometer and resuspend them in HBSS. Then, keep the cells on ice until the time of injection.In this procedure, anesthetize the mouse with ketamine/xylazine cocktail by intraperitoneal injection. Confirm complete anesthesia by pinching the animal's feet and observe the lack of response. Next, remove the neck hair by shaving or using a small amount of hair removal product.Then, place the mouse on a glass plate and secure it with rubber bands. Clean the skin by applying 70%alcohol in povidone iodine. Now, make an incision in the skin about one centimeter long with a surgical scalpel.Then, bluntly dissect the muscle with surgical forceps to expose the carotid artery underneath. Separate the carotid artery from the adjacent vagus nerve with surgical forceps. Afterward, place two sutures.One distal and the proximal to the cotton ball and make loose knots. Tighten the proximal knot to block the blood flow into the injection site. After that, moisten a small cotton ball with buffer and place it underneath the carotid artery of the intended site of injection.Then, proceed to cancer cell injection if the carotid artery on the cotton ball appears fully pressurized with fresh red blood. In this step, vortex and draw 100 microliters of cancer cells into the syringe. Under the dissecting microscope, slowly insert the 31 gauge needle with bevel up into the lumen of the carotid artery, sitting on top of the cotton ball.Slowly inject the cells from the syringe into the carotid artery. Successful injection can be observed by the changing color of nearby blood vessels and musculature when the buffered cancer cells are pushed into the circulation. When the injection is complete, lift the distal ligature gently to prevent regurgitation.Then, quickly tighten the distal knot to complete the injection process. After completing the injection, tighten the ligatures and trim the excess silk suture with micro scissors. Move back the separated muscle to cover the wound site and close the skin with two staples.For recovery, transfer the animal onto a heating pad. Then, administer buprenorphine via subcutaneous injection as post-surgical analgesia. Once it regains consciousness and resumes normal behavior, return the mouse to its housing location.Provide the animal with gel food for several days post-surgery. These images show that the modified MDA MB231 breast cancer cells were labeled with loose foraise reporter gene ex-vivo. Starting from 24 hours post-carotid artery injection, brain metastasis development was monitored non-invasively by repeated in vivo imaging system imaging.Quantification of imaging data indicates an initial decline of tumor burden post-carotid artery injection, followed by an exponential outgrowth of brain metastasis until animal's more abundity. So what is mastered this technique can be done in less than 10 minutes for each mouse injection if it's performed properly. So while performming this procedure, the single most important thing to remember is you have to ensure, study and secure placement of the carotid artery on top of the cotton ball prior to proceeding to the injection of the cancer cells.Following this procedure, other methods can be performed such as therapeutic treatments to answer other questions. For example whether a new drug would be effective inhibiting brain metastasis outgrows. After its development, this technique paved the way for researchers in the brain metastasis field to explore basic or translational questions in vivo experimental model in mice.So after watching this video you should have a good understanding of how to inject the cancer cells into the carotid artery of mice to produce a in vivo experimental brain metastasis model.

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Research

JoVE Journal

Cancer Research

25.6K Views.  The University of Texas MD Anderson Cancer Center. The overall goal of this microsurgical injection of cancer cells through the carotid artery is to produce consistent in vivo experimental brain metastasis models in mice. So this method can help answer key questions in the brain metastasis field, such as whether a new therapeutic may show efficacy in treating brain metastasis. So the main advantage of this technique is that it allows the production of in vivo experimental brain metastasis with high reproducibility and low variability.G...