This research was funded by The Australian National Health and Medical Research Council (NHMRC), grant number APP1162560. ML was funded by a UQ postgraduate research scholarship. We would like to thank everyone who assisted with animal husbandry and in vivo imaging of the animals. We thank the Royal Brisbane and Women&#39;s Hospital for donating aliquots of zirconium for this study.

All studies were conducted within the guidelines of the Animal Ethics Committee of The University of Queensland (UQCCR/186/19), and the Australian Code for the Care and Use of Animals for Science Purpose.
1. Preparation of cancer cells for injection
NOTE: In this study, the human breast cancer cell line, BT-474 (BT474), was used. BT474 was cultured in complete growth medium comprising RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% in...

Brain metastasis is a complex process of cancer cells spreading from their primary site to the brain. Different animal models are available that mirror certain stages of this multi-step process and there are physiological and practical considerations to designing preclinical metastasis studies41,42. Most published studies investigating the use of nanomedicine for brain metastasis treatment have used intracardiac43,<sup class="xr...

Brain metastasis is a prevalent malignancy associated with a very poor prognosis1,2. The standard of care for brain metastasis patients is multimodal, consisting of neurosurgery, whole brain radiotherapy and/or stereotactic radiosurgery depending on the patients&#39; general health status, extracranial disease burden, and the number and location of tumors in the brain3,4. Patients with up to three intracranial lesions are eligible for surgical resection or stereotactic radiosurgery, while whole-brain radiation therapy is recommended for patients with multiple lesions to avoid the risk of surgery-related infection and edema5. However, whole brain radiotherapy can inflict damage on radiosensitive brain structures, contributing to poor quality of life6.
Systemic therapy is a non-invasive alternative and logical approach to treat patients with multiple lesions7. However, it is less considered due to the long standing notion that systemic therapies have poor efficacy because the passive delivery of cytotoxic drugs via the bloodstream cannot achieve therapeutic levels in the brain without the risk of unsafe toxicity8. This paradigm is starting to change with the recently U.S. Food and Drug Administration (FDA)-approved systemic therapy (tucatinib with trastuzumab and capecitabine indicated for metastatic HER2+ breast cancer brain metastasis)9,10,11,12 and the update in treatment guidelines to include consideration of systemic therapy options for brain metastasis patients13,14.
In this context, developments in the field of molecular targeted therapy, immunotherapy, and alternative drug-delivery systems, such as a targeted nano-drug carrier, can potentially overcome the challenges of brain metastasis treatment15,16,17,18. In addition, chemical and mechanical approaches to improve drug delivery via permeabilization of the brain-tumor barrier are also being investigated19,20. To study and optimize such approaches to be fit for purpose, it is crucial to use preclinical models that not only mirror the complex physiology of brain metastasis but also allow for objective analysis of intracranial drug response.
Broadly, the current approaches to model brain metastasis in vivo involve intracardiac (left ventricle), intravenous (usually tail vein), intracranial, or intracarotid (common carotid artery) injection of cancer cells in mice21,22,23,24,25,26,27. Apart from tumor engraftment strategies, genetically engineered mouse models where tumor formation is triggered by the removal of tumor suppressor genes or activation of oncogenes are useful for tumor modeling. However, only a few genetically engineered mouse models are reported to produce secondary tumors and even fewer that reliably produce brain metastases28,29,30.
Engraftment methods such as intracardiac (left ventricle) and intravenous (usually tail vein) injection mimic the systemic dissemination of cancer. These models typically produce lesions in multiple organs (e.g., brain, lungs, liver, kidneys, spleen) depending on the capillary bed that traps most tumor cells during their circulatory &#39;first pass&#39;31. However, inconsistent rates of brain engraftment will require more animals to achieve the sample size for the desired statistical power. The number of tumor cells that eventually get established in the brain via these intracardiac and intravenous injection methods is variable. Hence, brain metastasis tumor burden can vary between animals and the difference in progression can make standardizing the experimental timeline and interpretation of results a challenge. The extracranial tumor burden can lead to non-brain metastasis mortality, rendering these models unsuitable for evaluating intracranial efficacy. Brain-tropic cell lines have been established using artificial clonal selection processes to reduce extracranial establishment, but take rates have been inconsistent, and the clonal selection process can reduce the heterogeneity normally found in human tumors32.
Brain-specific engraftment methods such as the intracranial and intracarotid injection allow for more consistent and efficient brain metastasis modeling. In the intracranial method33, cancer cells are typically injected into the frontal cerebral cortex, which generates quick and reproducible tumor outgrowth with low systemic involvement. While the procedure is well tolerated with low mortality33, the caveats are that it is a relatively crude approach that rapidly introduces a (localized) bolus of cells in the brain and does not model early brain metastasis pathogenesis. The needle damages brain tissue vasculature, which then causes localized inflammation5,34. From experience, there is a tendency for tumor cell injectate to reflux during removal of the needle, leading to leptomeningeal involvement. Alternatively, the intracarotid method delivers cells into the common carotid artery with brain microvasculature as the first capillary bed to be encountered, modeling survival in circulation, extravasation, and colonization24. In agreement with others25, our experience with this method found that it can result in facial tumors due to unintentional delivery of cancer cells via the external carotid artery to capillary beds in these tissues (unpublished data). It is possible to prevent facial tumors by first ligating the external carotid artery before common carotid artery injection (Figure 1). In the rest of the article, this method is referred to as the &#39;internal carotid artery injection&#39;. From experience, the internal carotid artery injection method consistently generates brain metastasis with very few systemic events and has been successful in generating brain metastasis models of different primary cancers (e.g., melanoma, breast, and lung cancers) (Figure 1). The disadvantages are that it is technically challenging, time-consuming, invasive, and requires careful optimization of cell numbers and a monitoring timeline. In summary, both the intracranial and internal carotid artery injection methods produce mouse models suitable for evaluating therapeutic impact on brain tumor-related survival benefit.
This protocol describes the internal carotid artery injection method to produce a mouse model of brain metastasis with almost no systemic involvement and therefore suitable for preclinical evaluation of drug distribution and efficacy of experimental therapeutics.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64216/64216fig01.jpg" /> 
Figure 1: Schematic representation of internal carotid artery injection protocol for brain metastasis. Internal carotid artery injection with external carotid artery ligation can reliably produce a brain metastasis model from various primary cancers. In this protocol, three ligatures are placed on the carotid artery (annotated as L1-L3 in the figure). <a href="https://www.jove.com/files/ftp_upload/64216/64216fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100&#181;m cell strainer</td>
			<td>Corning</td>
			<td>CLS431752</td>
			<td></td>
		</tr>
		<tr>
			<td>30G Microlance needle</td>
			<td>BD</td>
			<td>23748</td>
			<td></td>
		</tr>
		<tr>
			<td>31G Ultra-Fine II insulin syringe</td>
			<td>BD</td>
			<td>326103</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled forceps</td>
			<td>Proscitech</td>
			<td>T67A-SS</td>
			<td>Fine pointed, angled without serrations, 18mm tip, length 128 mm</td>
		</tr>
		<tr>
			<td>Animal heat mat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic and antimycotic</td>
			<td>ThermoFisher Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave bags</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BT-474 (HTB-20) breast cancer cell line</td>
			<td>ATCC</td>
			<td>HTB-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (TEMGESIC)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counter</td>
			<td>ThermoFisher Scientific</td>
			<td>C10227</td>
			<td></td>
		</tr>
		<tr>
			<td>Diet-76A</td>
			<td>ClearH2O</td>
			<td>72-07-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ear puncher</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electric clippers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine angled forceps</td>
			<td>Proscitech</td>
			<td>DEF11063-07</td>
			<td>Angled 45&#176;, Tip smooth, Tip width: 0.4 mm, Tip dimension: 0.4 x 0.3 mm, length 9cm</td>
		</tr>
		<tr>
			<td>Fine tubing for cannula, Tubing OD (in) 1/32, Tubing ID (in) 1/100in</td>
			<td>Cole Parmer</td>
			<td>EW-06419-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution without calcium and magnesium</td>
			<td>ThermoFisher Scientific</td>
			<td>14170120</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>ClearH2O</td>
			<td>70-01-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes Low lint disposable wipers</td>
			<td>Kimberly Clark- Kimwipes</td>
			<td>Z188964</td>
			<td></td>
		</tr>
		<tr>
			<td>Mashed mouse chow</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (METACAM)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nose cone</td>
			<td></td>
			<td></td>
			<td>Fashioned out of a microfuge tube</td>
		</tr>
		<tr>
			<td>PAA ocular lubricant (Carbomer 2mg/g)</td>
			<td>&#160;Bausch and lomb</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Betadine</td>
			<td>2505692</td>
			<td></td>
		</tr>
		<tr>
			<td>PPE (glove, mask, gown, hairnet)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Kent Scientific</td>
			<td>SURGI-5001</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>ThermoFisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk suture 13mm 5-0, P3, 45cm</td>
			<td>Ethicon</td>
			<td>JJ-640G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile normal saline</td>
			<td>ThermoFisher Scientific</td>
			<td>TM4469</td>
			<td></td>
		</tr>
		<tr>
			<td>Sticky tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical board</td>
			<td></td>
			<td></td>
			<td>A chopping board wrapped with autoclavable bag.</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Proscitech</td>
			<td>T104</td>
			<td>Tip Dimensions (LxD): 38x7mm, Length 115mm</td>
		</tr>
		<tr>
			<td>Suture forcep/ Curved Brophy forceps</td>
			<td>Proscitech</td>
			<td>T113C</td>
			<td>Curved, Rounded narrow 2 mm tip, with serrations, length 165 mm</td>
		</tr>
		<tr>
			<td>Suture needle holder (Olsen Hegar needle holder)</td>
			<td>Proscitech</td>
			<td>TC1322-180</td>
			<td>length 190 mm, ratchet clamp</td>
		</tr>
		<tr>
			<td>Syringe driver with foot pedal/ UMP3 Ultra micro pump</td>
			<td>World Precision Instruments</td>
			<td>UMP3-3</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 tissue culture flask</td>
			<td>ThermoFisher Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trigene II surface disinfectant</td>
			<td>Ceva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue and Cell Counting Chamber Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express dissociating medium</td>
			<td>ThermoFisher Scientific</td>
			<td>12605010</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Comparing common carotid artery injection with or without external carotid artery ligation 
When cancer cells were injected via the common carotid artery without first ligating external carotid artery24, facial tumors were found in 77.8% of the grafted mice (n = 7/9 animals). An example of facial tumor is illustrated in Supplementary Figure 3. The method described in this protocol prevents unintended facial metastasis by ligating the externa...

Watch this Scientific Journal Video about Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells at JoVE.com

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

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 ...

So why is this protocol significant? Well, brain metastases is very challenging to treat and this protocol produces a clinically-relevant mouse model with minimal extracranial tumors. This technique allows objective analysis of drug distribution and response from intracranial tumors.The main advantage of this technique is that the model is highly reproducible and the brain metastases develop in a relatively short time after injection. The disease progression is similar amongst animals, making it easier for researchers to standardize the experiments, treatment timelines, and endpoints. My advice for beginners is to practice using the forcep and syringe while looking through the dissection microscope, getting familiarized with the anatomical landmarks around the neck and practicing the protocol in the presence of a vet.Begin by seeding BT-474 cancer cells at a seeding density of two times 10 to the six cells into a T-75 flask using 10 milliliters of complete growth media and culture till the cells reach a confluency of 70 to 80%before injection. On the day of injection, discard growth media and wash the cell monolayer with PBS twice and add five milliliters of pre-warmed cell culture dissociation reagent. Incubate at 37 degrees Celsius for five minutes or until cells have detached.After five minutes, remove the flask from the incubator and gently tap it to aid cell detachment. Add five milliliters of complete growth media containing 10%FBS to quench the dissociation reagent activity. Gently resuspend the cells in the solution by pipetting to reduce cell clumps.Transfer the cell suspension into a 50-milliliter tube and centrifuge at 180 G for three minutes at room temperature. Decant the supernatant and resuspend the cell pellet in 10 milliliters of HBSSS without calcium and magnesium to minimize cell clumping. Centrifuge the cell suspension at 180 G for three minutes at room temperature Decant the supernatant to remove residual serum or dissociation reagent and resuspend the cell pellet in three milliliters of HBSS.Place a 100-micrometer cell strainer on a fresh 50-milliliter conical tube and pass the cell suspension through to remove cell clumps. Calculate the number of viable cells using trypan blue and dilute the cell suspension with HBSS to a cell concentration of 2.5 times 10 to the six cells per milliliter. Keep the tube horizontal on ice and gently rock the tube periodically to minimize clumping.The cell suspension can be stored on ice for a maximum of six hours. Ear-punch mouse for identification and use electric clippers to shave the fur from the neck region. Clean excess hair from the exposed skin using tape.Apply ocular lubricant to the eyes to prevent drying. Secure the mouse gently by hooking the upper incisor teeth using thread taped to the surgical board, followed by taping the front and hind legs. This step extends the body and keeps the neck straight during the procedure.For pre-operative skin preparation, wipe the neck with a topical antiseptic such as povidone iodine to reduce skin microflora load and remove loose hair. Clean from the center of the skin, working outward to prevent recontamination of the incision site. Repeat the process using 70%ethanol and perform three alternating rounds of iodine and ethanol for disinfection.Check for reflex via the pinch test to ensure the anesthesia death before continuing the procedure. Lay a sterile surgical drape over the animal. Under the dissection microscope, make a vertical 15-millimeter incision along the midline at the mouse's neck region using scissors, starting from five millimeters below the jaw to the thoracic inlet.Using two pairs of angled forceps, part the skin and underlying salivary glands and apply retractors to keep the trachea exposed. Using two pairs of fine angled forceps, bluntly dissect the muscle and fat tissue adjacent to the trachea to expose the right carotid sheath. The carotid sheath is the fibrous layer covering the common carotid artery, vein, and vagus nerve, and this bundle can be visualized by the bright red common carotid artery.Clear a segment of the common carotid artery caudal to the carotid bifurcation of the surrounding fascia and separate it from the vagus nerve and veins. Isolate and clear the carotid bifurcation from the surrounding nerves and fascia. Position fine forceps under the external carotid artery and pass a 5-0 silk suture under the artery.Knot and tighten the suture and cut excess line. Position fine forceps under the common carotid artery and pass a 5-0 silk suture under the artery. Tie a knot and tighten the suture at a position proximal to the proposed injection site.Cut the excess suture, leaving about 10 millimeter of the line. Cut and moisten a strip of low-lint disposable wipers about 10-by-5 millimeters. Fold the strip and place it underneath the carotid artery at the proposed site of injection.This will support the vessel during the injection. On the common carotid artery rostral to the proposed injection site, place a third ligation with a loose knot. This is tightened only after the injection.Gently agitate the cell suspension and draw 200 microliters of cell suspension into an insulin syringe with a 31-gauge needle. Load the syringe into the syringe driver connected to an activating foot pedal. Attach a fine cannula with a 31-gauge needle to the syringe and prime the line.Check whether the carotid artery is well-positioned and pressurized. Using two fine-angled forceps, one gently tensioning onto the end of the first ligature and the other holding the 31-gauge needle, slowly insert the needle with the bevel up into the lumen of the blood vessel, taking care not to puncture it. Slowly inject 100 microliters of cell suspension prepared earlier into the common carotid artery at 10 microliters per second.This will deliver 2.5 times 10 to the fifth cells into the blood vessel. Successful injection can be visualized via the clearing of blood from the carotid blood vessel. Gently lift and tighten the loose ligature immediately after withdrawing the needle to prevent backflow and bleeding.Trim excess sutures and remove the piece of moistened low-lint disposable wipers. Using a P-200 pipette, rinse the surgical cavity twice with 150 to 200 microliters of sterile water or saline. After checking for bleeds, reposition the soft tissue, salivary glands, and skin over the carotid artery and trachea.Close the skin layer of the incision using a suture needle-holder, forceps, and an absorbable or non-absorbable 6-0 monofilament suture in a continuous pattern. Inject 50 micrograms per kilogram of buprenorphine and one milligram per kilogram of meloxicam via subcutaneous injection as post-surgical pain relief. Move the animal to a warm and clean cage to recover from anesthesia.Lectin was injected into the mice's common carotid artery with or without external carotid artery ligation. A reduction in lectin was observed in the cheek tissues when the external carotid artery was ligated. The results depicted that the ligation did not impact brain delivery.Tumor progression was monitored using the luciferase-expressing, HER2-amplified breast cancer cell line. The BT474 brain metastasis model showed an increase in the bioluminescent signals from week five post-internal carotid injection and progressively grew thereafter. From weeks five to eight, heterogeneous signal intensity indicated an intracranial tumor with complex fluid perfusion.Gadolinium concentration within the tumor region increased as it leaked from the blood circulation into tumor tissue. The accumulation of nanomedicine and brain metastases was confirmed in the BT-474 mice by higher nanomedicine levels detected in the tumor region compared to the uninvolved brain regions. As the BT-474 brain metastasis model progressed, animals began to lose up to 20%body weight, making the median survival time nine weeks post-injection.Histology results depicted that the tumors were located unilaterally, matching the site of carotid injection where the tumors mostly appeared as solid, solitary masses. Pockets of empty spaces were frequently observed and consisted of necrotic cells, while smaller outgrowths were also present in some animals. The most important thing to remember when attempting the protocol is to prepare and position the carotid artery correctly before inserting the needle, and then to apply proper tension during insertion of the needle.This model is useful for researchers exploring drug delivery to the brain and the efficacy of the new therapeutics for treatment of brain metastasis.

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Abstract