Name: modeling brain metastasis by internal carotid artery injection of cancer cells
Uploaded: 2022-08-02T15:00:00
Description: Watch this Scientific Journal Video about Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells at JoVE.com

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>

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			<td>Sterile normal saline</td>
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			<td>Surgical board</td>
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			<td>A chopping board wrapped with autoclavable bag.</td>
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			<td>Surgical scissors</td>
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			<td>Suture needle holder (Olsen Hegar needle holder)</td>
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			<td>Thread</td>
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			<td>12605010</td>
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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 ...

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