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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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,

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
100µm cell strainerCorningCLS431752
30G Microlance needleBD23748
31G Ultra-Fine II insulin syringeBD326103
Angled forcepsProscitechT67A-SSFine pointed, angled without serrations, 18mm tip, length 128 mm
Animal heat mat
Antibiotic and antimycoticThermoFisher Scientific15240062
Autoclave bags
BT-474 (HTB-20) breast cancer cell lineATCCHTB-20
Buprenorphine (TEMGESIC)
Countess cell counterThermoFisher ScientificC10227
Diet-76AClearH2O72-07-5022
Dissection microscope
Ear puncher
Electric clippers
Fine angled forcepsProscitechDEF11063-07Angled 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) 1/100inCole ParmerEW-06419-00
Foetal bovine serumThermoFisher Scientific26140079
Hank's Balanced Salt Solution without calcium and magnesiumThermoFisher Scientific14170120
HydrogelClearH2O70-01-5022
Isoflurane
Kimwipes Low lint disposable wipersKimberly Clark- KimwipesZ188964
Mashed mouse chow
Meloxicam (METACAM)
Nose coneFashioned out of a microfuge tube
PAA ocular lubricant (Carbomer 2mg/g) Bausch and lomb
Povidone-iodine solutionBetadine2505692
PPE (glove, mask, gown, hairnet)
RetractorsKent ScientificSURGI-5001
RPMI 1640 MediaThermoFisher Scientific11875093
Silk suture 13mm 5-0, P3, 45cmEthiconJJ-640G
Sterile normal salineThermoFisher ScientificTM4469
Sticky tape
Surgical boardA chopping board wrapped with autoclavable bag.
Surgical scissorsProscitechT104Tip Dimensions (LxD): 38x7mm, Length 115mm
Suture forcep/ Curved Brophy forcepsProscitechT113CCurved, Rounded narrow 2 mm tip, with serrations, length 165 mm
Suture needle holder (Olsen Hegar needle holder)ProscitechTC1322-180length 190 mm, ratchet clamp
Syringe driver with foot pedal/ UMP3 Ultra micro pumpWorld Precision InstrumentsUMP3-3
T75 tissue culture flaskThermoFisher Scientific156499
Thread
Trigene II surface disinfectantCeva
Trypan Blue and Cell Counting Chamber SlidesThermoFisher ScientificC10228
TrypLE Express dissociating mediumThermoFisher Scientific12605010

References

  1. Nayak, L., Lee, E. Q., Wen, P. Y. Epidemiology of brain metastases. Current Oncology Reports. 14 (1), 48-54 (2012).
  2. . Australian Institute of Health and Welfare. Cancer in Australia. , (2017).
  3. Maher, E. A., Mietz, J., Arteaga, C.....

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