Name: intracarotid cancer cell injection to produce mouse models of brain metastasis
Uploaded: 2017-02-08T14:00:00
Description: Watch this Scientific Journal Video about Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis Video at JoVE.com

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>
	<li>Gavrilovic, I. T., Posner, J. B. <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:+epidemiology+and+pathophysiology.">Brain metastases: epidemiology and pathophysiology.</a> J Neurooncol. 75 (1), 5-14 (2005).</li><li>Patchell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+management+of+brain+metastases.">The management of brain metastases.</a> Cancer Treat Rev. 29 (6), 533-540 (2003).</li><li>Barnholtz-Sloan, J. S., 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=Incidence+proportions+of+brain+metastases+in+patients+diagnosed+(1973+to+2001)+in+the+Metropolitan+Detroit+Cancer+Surveillance+System.">Incidence proportions of brain metastases in patients diagnosed (1973 to 2001) in the Metropolitan Detroit Cancer Surveillance System.</a> J Clin Oncol. 22 (14), 2865-2872 (2004).</li><li>Schouten, L. J., Rutten, J., Huveneers, H. A., Twijnstra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94 (10), 2698-2705 (2002).</li><li>Bendell, J. C., 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=Central+nervous+system+metastases+in+women+who+receive+trastuzumab-based+therapy+for+metastatic+breast+carcinoma.">Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma.</a> Cancer. 97 (12), 2972-2977 (2003).</li><li>Clayton, A. J., 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=Incidence+of+cerebral+metastases+in+patients+treated+with+trastuzumab+for+metastatic+breast+cancer.">Incidence of cerebral metastases in patients treated with trastuzumab for metastatic breast cancer.</a> Br J Cancer. 91 (4), 639-643 (2004).</li><li>Romond, E. 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=Trastuzumab+plus+adjuvant+chemotherapy+for+operable+HER2-positive+breast+cancer.">Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer.</a> N Engl J Med. 353 (16), 1673-1684 (2005).</li><li>Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+patient+perspective+on+brain+metastases+in+breast+cancer.">A patient perspective on brain metastases in breast cancer.</a> Clin Cancer Res. 13 (6), 1623-1624 (2007).</li><li>Lin, N. U., Bellon, J. R., Winer, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J Clin Oncol. 22 (17), 3608-3617 (2004).</li><li>Zhang, C., Yu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment+determinants+of+brain+metastasis+of+brain+metastasis.">Microenvironment determinants of brain metastasis of brain metastasis.</a> Cell Biosci. 1 (1), 8 (2011).</li><li>Fidler, I. J., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+selectivity+for+implantation+survival+and+growth+of+B16+melanoma+variant+tumor+lines.">Organ selectivity for implantation survival and growth of B16 melanoma variant tumor lines.</a> J Natl Cancer Inst. 57 (5), 1199-1202 (1976).</li><li>Zhang, 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=Microenvironment-induced+PTEN+loss+by+exosomal+microRNA+primes+brain+metastasis+outgrowth.">Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth.</a> Nature. 527 (7576), 100-104 (2015).</li><li>Zhang, S., 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=SRC+family+kinases+as+novel+therapeutic+targets+to+treat+breast+cancer+brain+metastases.">SRC family kinases as novel therapeutic targets to treat breast cancer brain metastases.</a> Cancer Res. 73 (18), 5764-5774 (2013).</li><li>Lim, E., Modi, K. D., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescent+imaging+of+mammary+tumors+using+IVIS+spectrum.">In vivo bioluminescent imaging of mammary tumors using IVIS spectrum.</a> J Vis Exp. (26), (2009).</li><li>Zhang, C., Zhang, F., Tsan, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta2+is+a+molecular+determinant+for+site-specific+melanoma+metastasis+in+the+brain.">Transforming growth factor-beta2 is a molecular determinant for site-specific melanoma metastasis in the brain.</a> Cancer Res. 69 (3), 828-835 (2009).</li></ol>

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 Video at JoVE.com

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

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.

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. ...

Research

JoVE Journal

Cancer Research

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

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis Video (Video) | JoVE

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

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis Video (Video) | JoVE

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer Video (Video) | JoVE

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel Video (Video) | JoVE

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion Video (Video) | JoVE

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis Video (Video) | JoVE

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis Video (Video) | JoVE

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma Video (Video) | JoVE

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...