Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Brain metastasis is a common and deadly complication of systemic malignancies. Most brain metastases originate from primary tumors of the lung, breast or skin, which collectively account for 67-80% of cases^1,2. Estimates of the incidence of brain metastasis vary between 100,000 to 240,000 cases, and these numbers may be underestimates because autopsy is rare for patients who died of metastatic cancer³. Patients with brain metastases have a worse prognosis and lower overall survival relative to patients without brain metastases⁴. Current treatment options for brai....

Protokół

The method described here has been approved by the Institutional Animal Care and Use Committee (IACUC) of the MD Anderson Cancer Center and complies with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The schematic workflow, with all steps included, is presented as Figure 1.

1. Cell preparation

NOTE: The MDA-IBC3 (ER^-/PR^-/HER2⁺) cell line, generated in Dr. Woodward's lab²⁴, was stably transduced with a luciferase-green fluorescent protein (Luc-GFP) plasmid.

1. Culture trans....

Wyniki

With the rationale that labeled cells facilitate monitoring and visualization of brain metastasis in preclinical mouse models, we tagged MDA-IBC3 cells with Luc and with GFP to monitor brain metastases and quantify the metastatic burden by using bioluminescence imaging and fluorescent stereomicroscopy. Injection of the labeled MDA-IBC3 cells into the tail veins of immunocompromised SCID/Beige mice resulted in high percentages of mice developing brain metastasis (i.e., 66.7% to 100 %)^16,.......

Dyskusje

The protocol includes several critical steps. Cells should be kept on ice for no longer than 1 hour to maintain viability. Alcohol cotton pads should be used to wipe the tails of the mice before injection, with care taken to not wipe too hard or too often to avoid damaging the tail skin. Ensure that no air bubbles are present in the cell suspension, to prevent mice from dying from blood vessel emboli. Maintain the angle of injection at 45° or less to avoid piercing the blood vessel in the tails and insert at least 1.......

Materiały

Name	Company	Catalog Number	Comments
Cell Culture
1000 µL pipette tip filtered	Genesee Scientific	23430
10 mL Serological Pipets	Genesee Scientific	12-112
Antibiotic-antimycotic	Thermo Fisher Scientific	15240062	1%
Centrifuge tubes 15 mL bulk	Genesee Scientific	28103
Corning  500 mL Hams F-12 Medium [+] L-glutamine	GIBICO Inc. USA	MT10080CV
Countess II Automated Cell Counter (Invitrogen)	Thermo Fisher Scientific	AMQAX1000
1x DPBS	Thermo Fisher Scientific	21-031-CV
Eppendorf centufuge 5810R	Eppendorf
Fetal bovine serum (FBS)	GIBICO Inc. USA	16000044	10%
Fisherbrand  Sterile Cell Strainers (40 μm)	Thermo Fisher Scientific	22-363-547
Hydrocortisone	Sigma-Aldrich	H0888	1 µg/mL
Insulin	Thermo Fisher Scientific	12585014	5 µg/mL
Invitrogen Countess Cell Counting Chamber Slides	Thermo Fisher Scientific	C10228
MDA-IBC3 cell lines	MD Anderson Cancer Center		Generated by Dr. Woodward's lab24
Luciferase–green fluorescent protein (Luc–GFP) plasmid	System Biosciences	BLIV713PA-1
microtubes clear sterile 1.7 mL	Genesee Scientific	24282S
Olympus 10 µL Reach Barrier Tip, Low Binding, Racked, Sterile	Genesee Scientific	23-401C
TC Treated Flasks (T75), 250mL, Vent	Genesee Scientific	25-209
Trypan Blue Stain (0.4%) for use with the Countess Automated Cell Counter	Thermo Fisher Scientific	T10282
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200114
Tail vein injection
C.B-17/IcrHsd-Prkdc scid Lyst bg-J - SCID/Beige	Envigo	SCID/beige mice
BD Insulin Syringe with the BD Ultra-Fine Needle 0.5mL 30Gx1/2" (12.7mm)	BD	328466
Plas Labs  Broome-Style Rodent Restrainers	Plas Labs 551BSRR	01-288-32A	Order fromThermo Fisher Scientific
Volu SolSupplier Diversity Partner Ethanol 95% SDA (190 Proof)	Thermo Fisher Scientific	50420872	70 % used
Imaging
BD Lo-Dose  U-100 Insulin Syringes	BD	329461
Disposable PES Filter Units 0.45 µm	Fisherbrand	FB12566501	filter system to sterilize the D-luciferin
D-Luciferin	Biosynth	L8220-1g	stock concentration = 47.6 mM (15.15 mg/mL); use concentration = 1.515 mg/mL
1.7 mL microtube amber	Genesee Scientific	24-282AM
Isoflurane	Patterson Veterinary	NDC-14043-704-06	Liquid anesthetic for use in anesthetic vaporizer
IVIS 200	PerkinElmer		machine for luciferase imaging, up to 5 mice imaging at the same time, with anesthesia machine
Plastic Containers with Lids	Fisherbrand	02-544-127
Tissue Cassettes	Thermo Scientific	1000957
Webcol Alcohol Prep	Covidien	6818
Stereomicroscope Imaging
Stereomicroscope AZ100	Nikon	model AZ-STGE	software NIS-ELEMENT
Formalin 10%	Fisher Chemical	SF100-4
TC treated dishes 100x20 mm	Genesee Scientific	25202