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-free media once before trypsinization (0.25%) for 1 - 2 min at 37 &#176;C. Add into cells 10% FBS-containing DMEM/F12 media to quench the 0.25% trypsin.</li>
	<li>Centrifuge the cells at 80 &#215; g for 3 min. Wash cells in serum-free media twice to remove residual serum, count the cells with a hemocytometer and re-suspend in Hank&#39;s Balanced Salt Solution (HBSS) at 1 - 5 &#215; 106 cells/ml, depending on the cell line. Keep on ice until time of injection. 
	NOTE: MDA-MB-231 breast cancer cells and K1735 melanoma cells were used in this study, and both cell lines were used at 2 &#215; 106 cells/ml concentration. Keep the trypsinization duration as short as possible as overtreatment may influence the outcomes of metastasis assays.</li>
</ol>
2. Prepare Mice for Tumor Cell Injection
<ol>
	<li>Anesthetize mice by intraperitoneal (i.p.) injection of ketamine/xylazine cocktail (ketamine 100 mg/kg, xylazine 10 mg/kg).</li>
	<li>Confirm complete anesthesia by pinching the animal feet and observe lack of response.</li>
	<li>Place the mouse on a glass plate (used for casting protein gels), and secure with rubber bands.</li>
	<li>If necessary, remove the hair of neck by shaving or applying a small amount of hair removal product and wiping the hair off with paper towel. NOTE: If using nude mice, skip this step as they do not have body hair.</li>
	<li>Clean the neck skin by applying povidone-iodine and 70% alcohol.</li>
	<li>Place mouse on stage of the dissecting microscope</li>
	<li>Make an incision in the skin ~ 1 cm long with a surgical scalpel.</li>
	<li>Bluntly dissect the muscle with toothed forceps to expose the carotid artery underneath.</li>
	<li>Separate the carotid artery from the adjacent vagus nerve with surgical forceps.</li>
	<li>Using surgical forceps, separate some cotton from a cotton ball, moisten, and fashion into a smaller cotton ball. Place this buffer-moisturized cotton ball underneath the carotid artery of intended site of injection.</li>
	<li>Place a suture distal and proximal to the cotton ball and make loose knots. Tighten the proximal knot to block the blood flow into the injection site.</li>
	<li>Proceed to cancer cell injection if the carotid artery on the cotton ball appears fully pressurized with fresh red blood.</li>
</ol>
3. Cancer Cell Injection into Carotid Artery
<ol>
	<li>Vortex and draw 100 &#181;l of cancer cells into the syringe.</li>
	<li>Under the dissecting microscope, slowly insert the 31 G needle (with bevel up) into the lumen of the carotid artery sitting on top of the cotton ball.</li>
	<li>Slowly inject the cells from the syringe into the carotid artery. Successful injection can be observed under the microscope by the changing color of nearby blood vessels and musculature when buffered cancer cells are pushed into the circulation.</li>
	<li>When injection of the entire volume is complete, lift the distal carotid artery gently to prevent regurgitation.</li>
	<li>Quickly tighten the distal knot to complete the injection process.</li>
	<li>After completing the injection, tighten the ligatures and trim the excess silk suture with micro-scissors under the dissecting microscope. Move back the separated muscle to cover the wound site. Close the skin with two staples.</li>
</ol>
4. Surgical Recovery
<ol>
	<li>Move the operated animal onto a heating pad for recovery. It may take 30 - 60 min for the animals to regain consciousness. In the meantime, administer buprenorphine (0.05 mg/kg) via subcutaneous injection as post-surgical analgesia. &#160;Note: Analgesia should be provided according to your&#160;local IACUC approved protocol.&#160;</li>
	<li>Once the animals awaken and resume their normal behavior, return the mice to their housing location. Provide mice with gel food for several days post-surgery. Maintain a surgical record and keep it in the room.</li>
</ol>
5. Non-invasive In Vivo Imaging
<ol>
	<li>For cells labelled with luciferase reporter constructs, measure cancer cell injection efficiency using in vivo imaging system14. 
	NOTE: Imaging is not performed on the day of injection to avoid excessive stress to research animals.</li>
</ol>

<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 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 potential overdose, the ketamine/xylazine cocktail may be diluted 1:1 or 1:2 in physiological saline to provide more flexibility in adjusting the volume used for anesthesia. Alternatively, non-controlled drug tribromoethanol may also be used as anesthetic; however, we have found that the propensity of tribromoethanol to degrade makes the substance less reliable as an effective anesthetic than the ketamine/xylazine cocktail.
A steadily placed and fully pressurized carotid artery greatly facilitates the injection of cancer cells; therefore, we highly recommend spending time to carefully prepare the position of the artery under the microscope rather than proceeding with injection under suboptimal conditions.
More cancer cells will lodge within the vasculature on the side of carotid arterial injection. Sometimes a significant portion of the metastatic cells may migrate to the other side of the brain so that both hemispheres appear to bear significant amount of metastatic lesions in moribund animals.
Metastatic lesions may appear in either the parenchyma or the leptomeninges of the CNS, or in both locations. There appear to be unique tumor-microenvironment interactions that underlie these biological phenotypes15. Therefore, when a new cell line is to be tested for its capability to produce experimental brain metastasis, it is necessary to pay special attention to such phenotypes.
The internal and external carotid arteries supply blood into the brain parenchyma and leptomeninges, respectively. Accordingly, tumor cell deposition into the parenchyma or leptomeninges can be achieved by slight modification of this protocol. To allow cells only into leptomeninges, the internal carotid artery can be ligated by a suture prior to injection of tumor cells, thereby directing cells into the external carotid artery; on the other hand, ligation of the external carotid prior to injection will allow cells entry only into the internal carotid artery and the brain parenchyma.
Overall, the intracarotid injection approach produces data of higher reproducibility and lower variability compared to tail vein or intracardiac injections; it also avoids injection-induced CNS inflammation compared to the direct intracerebral injection method. Hence, this approach is currently the most valuable in quantitative studies of brain metastasis that aim to generate significant statistical conclusions using a limited number of animals. At the same time, all experimental models have limitations. Similar to other methods of hematogenous tumor cell introduction, the intracarotid injection only recapitulates later steps in the metastasis cascade after cancer cell dissemination into the circulation. Thus it is not an appropriate model to study, for example, the biological factors that determine the dissemination of primary cancer cells into the bloodstream.

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><tbody><tr><td>Ketamine hydrochloride/xylazine hydrochloride solution</td><td>Sigma-Aldrich</td><td>K113</td><td></td></tr><tr><td>Routine Stereomicroscope Leica M50</td><td>Leica</td><td>M50</td><td>The microscope is modular and highly configurable to fit particular space requirements.</td></tr><tr><td>Surgical disposable scapel</td><td>Integra Miltex</td><td>4-410</td><td>to make skin incisions</td></tr><tr><td>Tissue Forceps - 1 x 2 Teeth</td><td>Fine Science Tools</td><td>11021-12</td><td>to bluntly dissect mucles</td></tr><tr><td>Dumont #5 - Mirror Finish Forceps</td><td>Fine Science Tools</td><td>11252-23</td><td>to separate and prepare the carotid artery for injection</td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools</td><td>15025-10</td><td>to cut sutures</td></tr><tr><td>EZ Clip Kit</td><td>Stoelting</td><td>59020</td><td>for wound cloure</td></tr><tr><td>BD Insulin Syringe</td><td>Becton Dickinson</td><td>328438</td><td>for cell injection</td></tr><tr><td>IVIS Spectrum &lt;em&gt;In Vivo&lt;/em&gt; Imaging System</td><td>PerkinElmer</td><td>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 carotid artery. The second point to evaluate the injection quality is post-surgical non-invasive imaging. For this purpose, the cancer cells are typically labelled with a luciferase reporter ex vivo before injection. To minimize stress on the animals, in vivo imaging is usually performed 24 hr post-injection. A successful injection results in strong intracranial cancer cell load, and the mouse head is the only region with positive signals (Figure 2A).
Using in vivo imaging system to track the luciferase-labelled cancer cells, we observe an initial decline of signal intensity within days post-injection; yet, cancer cells capable of outgrowth in the CNS eventually manage to divide and produce increasing in vivo imaging system signals as brain metastases develop (Figure 2B). Intracarotid injection usually produces consistent in vivo imaging system signals with relatively low variability, which is a major advantage of using this approach to produce experimental brain metastasis. As growing tumor lesions displace brain tissues, the quality of health for animals with fast progressing brain metastasis can deteriorate rapidly, mirroring the clinical manifestation of human brain metastasis patients. Typical symptomatic behaviors indicative of the significant presence of brain metastasis include loss of weight, hunched posture, and incessant circling. At the experimental end point, the animal brains can be extracted for examination of biochemical parameters or for histological examinations. Except for melanotic metastasis lesions (Figure 3A), it is usually difficult to distinguish the tumor lesion from surrounding brain tissues prior to histological evaluation (Figure 3B). In such cases, it is advised to label tumor cells with a fluorescent reporter (e.g. GFP) for the convenience of isolating metastatic cells (Figure 3C). Alternatively, the entire brain can be dissociated into single cells and tumor cells separated by magnetic bead-conjugated antibodies.
<img alt="Figure 1" src="/files/ftp_upload/55085/55085fig1.jpg" /> 
Figure 1: Preparation for carotid artery injection. A) The anesthetized mouse is placed on a glass plate and secured with rubber bands. The site and length of the incision is labelled with a black line on the neck. B) After all preparatory surgical steps are complete, the exposed carotid artery is placed for support on a firm cotton ball, ready for cancer cell injection. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55085/55085fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55085/55085fig2.jpg" /> 
Figure 2: Non-invasive monitoring for brain metastasis outgrowth. A) Modified MDA-MB-231 breast cancer cells were labelled with luciferase reporter gene ex vivo. Starting from 24 hr post-carotid artery injection, brain metastasis development was monitored non-invasively by repeated in vivo imaging system imaging (scale bar = 50 mm). B) Quantification of in vivo imaging system imaging data indicates an initial decline of tumor burden post-carotid artery injection, followed by exponential outgrowth of brain metastasis until animal moribundity. Error bars reflect the standard error of the mean (SEM) of the total luminescence signal. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55085/55085fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55085/55085fig3.jpg" /> 
Figure 3: Harvesting Brain Metastasis Lesions. A) K1735 melanoma cells produce melanotic brain metastasis lesions in the syngeneic C3H mouse brain, which greatly facilitates the separation of brain metastatic lesions from the surrounding stroma but is limited to certain melanoma models. B) Dissociated mammary tumor cells from the transgenic MMTV-Neu/Pten-null mouse produces overt brain metastasis after implantation via carotid artery injection. The tumor/stroma margin is difficult to distinguish despite the obvious presence of angiogenic metastatic lesions. C) MDA-MB-231 breast cancer cells were pre-labelled with a green fluorescent protein (GFP) reporter ex vivo to facilitate extraction of brain metastasis lesions via surgical resection or cell sorting. Note that the majority of the metastatic lesions are concentrated on the same side of the brain where carotid arterial injection takes place. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55085/55085fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

26.4K Views.  The University of Texas MD Anderson Cancer Center.  Brain metastase is uitgegroeid tot een dringende onvervulde medische noodzaak als de incidentie is toegenomen, terwijl de therapeutische opties palliatieve zijn gebleven. Het creëren van experimentele diermodellen van de hersenen metastase via intracarotid arteriële injectie van kankercellen vergemakkelijkt mechanistische studies van de ziekte biologie en de evaluatie van nieuwe interventies regimes. 

Video: Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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 ...

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.

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 ...

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 ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Medicine

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 ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Common Carotid Artery Repair After Injection in a Mouse Model 

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

Author Spotlight: Advancing Glioblastoma Treatment Through Intraarterial Delivery Strategies for Oncolytic Viruses and MicroRNAs

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

Modeling Brain Tumor Metastasis via Internal Carotid Artery Injection of Cancer Cells

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/23284/modeling-brain-tumor-metastasis-via-internal-carotid-artery-injection'>Source: Lim, M., et al. Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells. J. Vis. Exp. (2022)</a>In this video, an anesthetized mouse undergoes a surgical procedure in which cancer cells are injected into the common carotid artery (CCA) to facilitate targeted delivery to the brain. The procedure involves precise suturing and manipulation of the carotid arteries to direct the injected cells toward the internal carotid artery (ICA), promoting tumor formation in the brain over time.

Source: Lim, M., et al. Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells. J. Vis. Exp. (2022)In this video, an anesthetized ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Tumors

Intracranial Orthotopic Model: A Procedure to Implant Cancer Cells in Mouse Brain

Source: Geisler, J. A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61272/modeling-brain-metastases-through-intracranial-injection-magnetic">Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging.</a> J. Vis. Exp. (2020)
This video demonstrates implantation of tumor cells in the mouse brain by performing intracranial injection.

Source: Geisler, J. A. et al. Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging. J. Vis. Exp. (2020)
This video ...

Cancers of the Nervous System

In vivo studies

Brain Metastasis Mouse Model: A Method to Generate Brain Metastasis Mouse Model by Intracarotid Cancer Cell Injection

Source: Zhang, C. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55085" target="_blank">Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis.</a> J. Vis. Exp. (2017)
This video demonstrates the establishment of a brain metastasis mouse model by intracarotid injection of fluorescently labeled cancer cells. The mouse model can be used to study the effects of novel therapeutics and the underlying physiological mechanisms.

Source: Zhang, C. et al. Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis. J. Vis. Exp. (2017)
This video demonstrates the ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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