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Cancer Research

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

Published: February 4th, 2021

DOI:

10.3791/62249

1Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, 2Morgan Welch Inflammatory Breast Cancer Clinic and Research Program, The University of Texas MD Anderson Cancer Center, 3Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center
* These authors contributed equally

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.

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.

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 cases1,2. 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....

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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.......

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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,.......

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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.......

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We thank Christine F. Wogan, MS, ELS, of MD Anderson’s Division of Radiation Oncology for scientific editing of the manuscript, and Carol M. Johnston from MD Anderson’s Division of Surgery Histology Core for help with hematoxylin and eosin staining. We are thankful to the Veterinary Medicine and Surgery Core at MD Anderson for their support for the animal studies. This work was supported by the following grants: Susan G. Komen Career Catalyst Research grant (CCR16377813 to BGD), American Cancer Society Research Scholar grant (RSG-19–126–01 to BGD), and the State of Texas Rare and Aggressive Breast Cancer Research Program. Also supported in part....

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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

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