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