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 brain metastases are largely palliative and fail to improve survival outcomes for most patients⁵. Thus, brain metastasis remains a challenge, and the need remains pressing to better understand the mechanisms of brain metastasis progression to develop more effective therapies.

The use of experimental models has provided important insights into specific mechanisms of breast cancer metastatic progression to the brain and allowed evaluation of the efficacy of various therapeutic approaches^{6,7,8,9,10,11,12,13,14,15,16}. However, very few models can accurately and fully recapitulate the intricacies of brain metastasis development. Several experimental in vivo models have been generated via inoculation of cancer cells into mice by different routes of administration, including orthotopic, tail-vein, intracardiac, intracarotid arterial, and intracerebral injections. Each technique has advantages and disadvantages, as reviewed elsewhere³. None of these mouse models, however, can fully replicate the clinical progression of brain metastasis.

Brain metastases are particularly common in patients with inflammatory breast cancer (IBC), a rare but aggressive variant of primary breast cancer. IBC accounts for 1% to 4% of breast cancer cases, but it is responsible for a disproportionate 10% of breast cancer-related deaths in the United States^17,18. IBC is known to rapidly metastasize; indeed, one-third of IBC patients have distant metastasis at the time of diagnosis^19,20. Specific to brain metastasis, patients with IBC have a higher incidence of brain metastasis than do patients with non-IBC²¹. Recently, we demonstrated that the MDA-IBC3 cell line, derived from the malignant pleural effusion fluid of a patient with ER^-/PR^-/HER2⁺ IBC that recapitulates IBC characteristics in mouse xenografts, has an enhanced propensity to develop brain metastases rather than lung metastases in mice when injected by tail-vein, making this cell line a good model for studying the development of brain metastasis¹⁶.

Herein we describe the procedures to generate brain metastasis via tail-vein injection of MDA-IBC3 cells and to evaluate the metastatic burden via stereofluorescent microscopy and luciferase imaging. This method has been used to discover key mediators of breast cancer metastasis to the brain and to test the efficacy of therapeutic interventions^16,22,23. The disadvantage of this technique is that it does not recapitulate all the steps in the brain metastatic process. Nevertheless, its major advantages include robustness and reproducibility, involvement of the relevant metastasis biology of intravasation, traversing the lungs and extravasation into the brain, and its relative simplicity in terms of technique.

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 transduced cells in Ham's F-12 media supplemented with 10% fetal bovine serum (FBS), 1 µg/mL hydrocortisone, 5 µg/mL insulin, and 1% antibiotic-antimitotic.
2. Plate MDA-IBC3-Luc-GFP cells at 37 °C and 5% CO₂ in a T-75 flask until confluent. Change media every 3 days before cells are confluent enough to passage.
3. Harvest cells by removing media, washing each flask with 10 mL of 1x Dulbecco's (i.e., calcium- and magnesium-free) phosphate-buffered saline (DPBS), and adding 2 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Incubate for 3-5 min at 37 °C until cells detach.
4. Add 5 mL of complete media to the flask to collect the cells and transfer the entire contents to a 15 mL centrifuge tube (optimized to 50 mL centrifuge tube depending on the total number of cells needed). Centrifuge at 290 x g for 5 min to pellet cells.
   NOTE: Complete media is Ham's F-12 media supplemented with 10% fetal bovine serum (FBS), 1 µg/mL hydrocortisone, 5 µg/mL insulin, and 1% antibiotic-antimitotic.
5. Discard the supernatant. Then wash the cells with 10 mL of 1x DPBS. Centrifuge at 290 x g for 5 min to pellet the cells and discard the supernatant carefully. Resuspend the cells with 1 mL of fresh 1x DPBS and mix well by pipetting up and down.
6. To make single-cell suspensions, filter cells through 40 µm sterile cell strainers.
7. Calculate cell density by using an automated cell counter.
   NOTE: To reduce errors in counting, create different cell dilutions and count them separately, and calculate the average value to determine the concentration of cells (number of cells/mL).
   1. Collect 2 µL of cell suspension + 8 µL of 1x DPBS to make a 1:5 dilution sample.
   2. Collect 1 µL of cell suspension + 9 µL of 1x DPBS to make a 1:10 dilution sample.
   3. Add 10 µL of 0.4% trypan blue stain. Mix the sample mixture well by pipetting it up and down a few times.
   4. Gently pipette 10 µL of the sample into the half-moon-shaped sample loading area of the counting chamber slides. Make sure there are no bubbles inside; wait for 30 s to allow the cells to settle in the chamber before counting.
8. Dilute the cells with 1x DPBS to a density of 5 x 10⁵ or 1 x 10⁶ cells per 100 µL. Transfer cell suspension into 1.7 mL microcentrifuge tubes for tail-vein injection. Place cells on ice until injection to maintain viability.

2. Tail vein injection

1. Use 4- to 6-week-old female athymic SCID/Beige mice.
2. Prepare a 30 G syringe to draw 100 µL of cell suspension. Remove all air bubbles.
3. Place mouse in a rodent restrainer (diameter about 1 inch) to facilitate tail-vein injection and prevent injuries from movement during the injection process.
4. Use alcohol cotton pads (made with 70% ethanol) to gently wipe the mouse's tail 3-4 times and hold for 5-10 s to make the tail vein more clearly visible.
5. Insert the syringe into the tail of the mouse at an angle of 15-30°. Slowly push the cell suspension into the tail vein.
6. Remove the syringe gently and use the cotton pad to hold the tail for few seconds to help stop any bleeding.
7. Return the mice to their cages and check them 2-4 h after injection to ensure that no adverse effects occur.

3. Evaluation of brain metastasis burden

1. Prepare diluted D-luciferin solution.
   ​NOTE: Stock concentration is 47.6 mM (15.15 mg/mL), and the concentration for use is 1.515 mg/mL.
   1. Add 5 mL of 1x DPBS to the D-luciferin bottle.
   2. Place 2.5 mL of the solution into each of two 50 mL conical tubes.
   3. Add another 5 mL of 1x DPBS to the D-luciferin bottle again to rinse it. Put 2.5 mL into each 50 mL conical tube.
   4. Place another 5 mL of 1x DPBS into the bottle and rinse it again.Repeat the rinsing process twice.
   5. Add 1x DPBS to each 50 mL tube to a total of 66 mL (about 33 mL per tube). There should be about 33 mL of the solution in each tube.
   6. Mix the tubes well and then combine both into a sterile filtration unit (150 mL PES filter 0.45 µm).
   7. Filter the solution and then aliquot the filtered solution into sterile amber 1.7 mL microtubes.
2. Detecting brain metastases by luciferase imaging.
   1. Thaw D-luciferin by keeping on ice, and vortex before injection into the mice.
   2. Clean the area of injection with alcohol cotton pads and inject 100 µL of D-luciferin per mouse intraperitoneally by using a 0.5 mL insulin syringe, one syringe for each cage.
   3. After 10 min, anesthetize the mice with isoflurane (2% O₂- 2.5% isoflurane) for 5 min using the veterinary vaporizer connected to the small animal induction chamber.
   4. Turn on the in vivo imaging system. While the mice from the first cage are under anesthesia, inject the D-luciferin into the mice in the next cage.
   5. Put mice into the in vivo imaging system machine and obtain ventral and dorsal images. Select an exposure time of 2 min, and in the field view choose option E (whole body). Press Acquire. Next, save the image (Figure 2).
      ​NOTE: If the image shows saturation of luminescence, reduce the exposure time.
3. Detecting brain metastases by GFP imaging.
   1. Brain tissue preparation.
      1. About 8-10 weeks post injection of cells or when the mice are moribund due to brain metastasis burden, euthanize the mice by inhalation of an isoflurane overdose followed by cervical dislocation, in accordance with protocols approved by the IACUC.
      2. After euthanasia with isoflurane followed by cervical dislocation, spray the mice with a 70% ethanol solution and remove the fur from the head area and ears with scissors. Next, make a cut in the cervical area of the neck (being careful to not decapitate the mouse). Cut the skull and pull it out. Next, carefully remove the brain.
      3. Place the collected brains into tissue cassettes.
      4. Transfer the cassettes to a container with cold 1x DPBS.
         ​NOTE: Brain samples should not be kept in 1x DPBS for more than 1 h. If several brain samples are to be collected at the same time, euthanize only a few mice at a time (10 per round).
   2. Stereomicroscopic imaging.
      1. Transfer the whole brain onto a 100 cm tissue dish lid.
      2. Turn on the stereomicroscope and the UV light, at position 3 with lens 0.5x, to allow visualization of the whole brain as shown in Figure 3A (red square).
      3. Press Live view (Figure 3A, green square).
      4. Focus the view while the brain is in the ventral position.
      5. Select GFP (if cells are GFP-labeled) or TXRED (if cells are RFP-labeled) (Figure 3A, yellow square) in the software, select the proper filter on the microscope, and take a picture.
         ​NOTE: Keep the SOLA light source setting at about 30%; if the GFP is too bright, reduce until the proper signal is observed.
      6. Without moving the sample, turn on the bright light and select BF (Figure 3A, yellow square). Take a picture.
      7. Repeat the previous steps with the brain in the dorsal position.
         ​NOTE: Depending on the size, the same GFP positive brain metastasis lesion can appear in both ventral and dorsal positions. In such a case, avoid duplicate quantification of the same lesion. Save the images with a TIFF extension (which keeps all the information in the picture file).
      8. Save the images with a TIFF extension (which keeps all the information in the picture file).
      9. Repeat steps for all samples.
      10. Convert the TIFF images into JPEG so that the files can be opened with any computer that does not have the associated software installed (File | Import/Export | convert files).
      11. To merge the fluorescent image with the bright field image, open both images and go to File | Merge channels. Select the proper components, green for GFP and brightfield for BF, and press Ok. Save the merged image.
      12. To quantify brain tumor area, use the fluorescent image. Select Measure | Manual Measurement | Area. Select automatic selection (Figure 3B, green square), and move the arrow to the GFP-positive area and click to automatically create a selection. Click again to confirm the selection and then all measurements will show (Figure 3B, red square). If more than one GFP-positive area is present, repeat the procedure.
   3. After brain images are obtained, proceed to routine tissue fixation protocols using 10% neutral buffered formalin.
      1. Once the brains are fixed, proceed to standard histological sectioning followed by hematoxylin and eosin staining to confirm the presence of brain metastasis and immunohistochemical staining to detect selected markers.
         NOTE: Immunocytochemistry was performed at the Pathology Core Laboratory at UT MD Anderson Cancer Center that has standardized protocol for immunohistochemical staining of known markers.

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