Bioluminescence Imaging to Assess Tumor Growth in an Organotypic Mouse Brain Slice

Abstract

Source: Ciraku, L., et. al. An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth. J. Vis. Exp. (2021).

This video demonstrates the imaging of luciferase-expressing tumor cells in an organotypic mouse brain slice. Luciferin is added to the medium, and the plate is placed inside the chamber of the in vivo imaging system. Parameters for image capture are adjusted, and the tumor's shape and size are defined. The bioluminescent signals emitted by the tumor cells are then recorded.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

Note: Nu/Nu athymic female mice (6-8 weeks old) were used in this study.

1. Intracranial injection of tumor cells

1. Sterilize all equipment (tweezers, scissors, suturing scissors, hand drill) under a dry cycle of an autoclave for up to 45 min in sterilization pouches, including a sterilization indicator. If surgeries are conducted on multiple animals, sterilize tools between animals using a heated bead sterilizer (up to 3x).
2. Anesthetize the mice according to the users' animal care protocol (e.g., isoflurane (4% for induction, 1-2% maintenance) and administer analgesia (subcutaneous, 0.1 mL of 0.1mg/kg buprenorphine SR-LAB) prior to the start of surgery. Ensure optimal anesthesia depth with a toe pinch.
3. Place the mice in a stereotaxic frame and immobilize the head using standard ear bars (Figure 1A). Then, apply ophthalmic ointment to the eyes.
4. Sterilize the surface of the head by repeated, alternating application (3x) of iodine swabs and 70% ethanol prior to incision.
5. Use a surgical scalpel to make a 1 cm midline incision through the skin at a slightly diagonal angle to the imaginary line that divides the mouse's brain into two symmetrical halves to expose the skull. Wipe any blood with a cotton swab.
6. Deflect the skin laterally to expose the injection site: 2 mm on the right, 1 mm forward with reference to the bregma +2 mediolateral (+2ML), +1 rostral/caudal (+1RC).
7. Use a bur bit (0.73 mm) to penetrate the skull +2ML, +1RC to the bregma and drill a hole using slight pressure and twisting motion.
8. Use a brain injection syringe to inject 5 µL of a 100,000 MDA-MB-231BR (stably expressing green fluorescent protein or GFP-Luciferase) cells/µL solution in sterile 1x phosphate buffer saline (PBS) by initially inserting the syringe 3.5 mm in depth into the brain.
9. Allow the syringe to settle for 2 min. Pull it up about 1 mm, and after 2 min, slowly inject the first half of the volume.
10. Wait for another 2 min before injecting the rest, and then slowly pull the syringe 3 min after the final injection.NOTE: Slow injection is empirical in allowing the volume to be well absorbed by the brain tissue and not create leakage after pulling the syringe, which may lead to cancer cells growing outside of the intended site.
11. Apply bone wax to the injection site on the skull and then use polypropylene sutures to suture the tissue. Mice will recover within 5 min after removal of anesthesia.
12. Monitor their behavior right after surgery for about 10 min, 3 hours later, and the following day to determine if special treatment, including saline injection, soft food, etc., is needed to help with quick recovery.
13. Monitor the tumor growth via bioluminescence imaging on the imaging system.
    1. For this, first, turn on the oxygen and isoflurane in the imaging system. Allow both to be distributed to a separate chamber outside of the imaging box.
    2. Then, turn on the software, initialize the instrument, and choose the appropriate option for visualizing and capturing the whole mouse body.
14. Place the mice in a separate chamber with isoflurane and ensure optimal anesthesia depth with a toe pinch. Inject the mice intraperitoneally with 200 µL of 30 mg/mL luciferin.
15. Transfer the mice stomach down to the imaging chamber's noselet supplied with oxygen and isoflurane. Lock the chamber, and choose 2 min time exposure to start imaging.
16. Use the ROI (region of interest) tool of the software to quantify the bioluminescent signal of the tumor.
17. To analyze the tumor size, choose the circle shape tool to fit it around the tumor shape and size. Measure the ROI and report the total readings.
18. Allow solid tumor growth for 12-14 days (or until luciferase reaches about 7.0 x 10⁶ units) (Figure 1B) before generating brain slices.NOTE: Increasing the number of cells to be injected will lead to faster tumor growth; therefore, the generation of brain slices can occur earlier than 12 days. On the other hand, a bigger tumor will lead to more GFP+ slices if allowed to grow for 14 days. After 14 days, the mice's health begins to deteriorate rapidly; hence, animal health monitoring should be increased to once daily after the 10-day time point, and any mice displaying signs of pain and/or discomfort should be used to generate the slices.

2. Generate brain slices

NOTE: Perform the steps after brain isolation in a sterile laminar flow hood. It is typically possible to generate 35-40 individual slices containing tumor cells (luciferase signal) from one mouse injected with MDA-MB-231BR cells (Figure 1C).

1. Sterilize all the surgical tools in an autoclave. Place the tissue slicer in the sterile laminar flow hood and clean all tools and instruments with 70% ethanol.
2. Following 12-14 days after intracranial MDA-MB-231BR cell injection, anesthetize the mice according to the user's animal care protocol as described in 1.2. Ensure optimal anesthesia depth with a toe pinch.
3. Remove and place their brains rapidly (within 45 s) into ice-cold (4 °C) sucrose-artificial cerebrospinal fluid or sucrose-ACSF composed of the following: 280 mM sucrose, 5 mM Potassium Chloride (KCl), 2 mM Magnesium Chloride (MgCl₂), 1 mM Calcium Chloride (CaCl₂), 20 mM glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or HEPES, 5 mM Sodium (Na+)-ascorbate, 3 mM thiourea, 2 mM Na+, 29 mM pyruvate; pH=7.3.
4. Using a sharp, sterile scalpel or razor blade, cut away excess parts of the brain that do not contain any tumor from all sides, including the bottom part of the brain.
5. Bring the shape of the brain to a non-perfect cube to sit flat on the ACSF-wetted filter paper on a 60 mm dish lid to facilitate slicing.NOTE: Extra tissue of the brain where the tumor is unlikely to have grown is removed prior to slicing to allow generation of mostly GFP+ slices and create a shape of the brain that is stable on the surface of the instrument and will not be perturbed by the vibrations caused by the slicing.
6. Place several sheets of pre-wet (with ACSF) filter paper circles onto the cutting platform and place the blocked tissue atop this.
7. Set the cut size on the instrument to 2 or 2.5 units on the provided ruler to slice the tissue into 200-250 µm sections.NOTE: Slices up to 350 µm or as thin as 100 µm are viable. For slices <200 µm, use a vibratome or compresstome.
8. Use a paintbrush (sable) to transfer brain slices into a dish containing sucrose ACSF and separate the slices under a light microscope using 27 G needles.
9. Identify GFP-positive slices under the fluorescent microscope and transfer them to a new 60 mm dish containing 2-3 mL of adult slice media (Neurobasal medium A, 2% B12 supplement, 1% N2 supplement, 1% glutamine, 0.5% glucose, 10 U/mL penicillin, and 100 µg/mL streptomycin) using a sterile 1 mL broken off pipette (wide opening).NOTE: The brain slices that contain tumor cells growing over the brain surface (maybe due to injection cell leakage, etc.) are excluded.
10. Transfer 3-5 slices onto each 30 mm, 0.4 µm pore size tissue culture insert in 6-well plates at a distance that does not allow growing into each other.
11. Remove the excess medium from the surface of the insert using a P1000 pipette, add 1 mL of adult mouse slice medium underneath each insert and equilibrate the media in the incubator prior to slicing.
12. Incubate the tissues at 37 °C, 5% CO₂, and 95% humidity for 24 h before imaging.

3. In Vivo Imaging System (IVIS) imaging of slices

NOTE: Perform luciferase and inhibitor addition steps in a sterile laminar flow hood.

1. Pipette 5 µL of 30 mg/mL luciferin solution into the medium underneath the inserts by lifting the insert with forceps and placing them back into the well after addition of luciferin to the media in the well.
2. Place the 6-well plate with the lid inside the imaging chamber of the instrument below the stationary camera and lock the chamber door.
3. Open the software and initialize the instrument.
4. Choose camera settings that allow visualization and capturing of only one well per image. Move the stage up (field of view or FOV of 5 cm) and place the well that must be imaged directly under the camera.
5. Set the imaging time to most appropriate depending on the intensity of the luciferase that the tumor will emit, which can vary from 10 s to 5 min. Start with setting it to 10 s and then increase if the signal is low but keep it consistent for all time points and conditions until the end of the experiment (Figure 1D).
6. Use the ROI tool (circle shape tool), fit it around the tumor shape and size, measure the ROI, and report the total readings to quantify the bioluminescent signal of each tumor on the slice.

Results

Figure 1. Intracranial injection, brain slice generation, and BCBM growth ex vivo. (A) Schemat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL syringe, slip tip	BD	309659
30 G1/2 Needles	BD	305106
6-well plates	Genessee	25-105
Automated microscope and LUMAVIEW software	Etaluma	LS720
B27 (GEM21)	Gemini Bio-Products	400-160
Beaker 50 mL	Fisher	10-210-685
Blunt sable paintbrush, Size #5/0	Electron Microscopy Sciences	66100-50
Bone Wax	ModoMed	DYNJBW25
Brain injection Syringe	Hamilton Company	80430
CaCl₂	Fisher Scientific	BP510-250
D-Luciferin Potassium Salt	Perkin Elmer	122799
Double edge razor blade	VWR	55411-060(95-0043)
Filter Paper (#1), quantitative circles, 4.25 cm	Fisher	09-805a (1001-042)
Fine sable paintbrush #2/0	Electron Microscopy Sciences	66100-20
Forceps	Fine Science Tools	11251-20
Glucose	Sigma Aldrich	G8270
Glutamine (200 mM)	Corning cellgrow	25-005-Cl
Hand Drill Set with Micro Mini Twist Drill Bits	Amazon	YCQ2851920086082DJ
HEPES, free acid	Fisher Scientific	BP299-1
Just for mice Stereotaxic Frame	Harvard Apparatus (Holliston, MA, USA).	72-6049, 72-6044
KCl	Fisher Scientific	S271-10
Large surgical scissors	Fine Science Tools	14001-18
MDA-MB-231BR cells	Kindly provided by Dr. Patricia Steeg	Ref 14
MgCl₂·6H₂O	Fisher Scientific	M33-500
Mice imaging device	Perkin Elmer	IVIS 200 system
Mice imaging software	Caliper Life Sciences (Waltham, MA, USA).	Living Image Software
Microplate Reader	Tecan Spark
Mounting solution	Invitrogen	P36935
MTS reagent	Promega CellTiter 96 Aqueous One Solution	(Cat:G3582)
N2 supplement	Life Technologies	17502-048
Neurobasal medium	Life Technologies	21103049
Nu/Nu athymic mice	Charles Rivers Labs (Wilmington, MA, USA)
Paraformaldehyde	Affymetrix	19943
Pen/Strep	Life Technologies	145140-122
Polypropylene Suture	Medex supply	ETH-8556H
Povidone Iodine Swab sticks	DME Supply USA	Cat: 689286X
Scalpel blade #11 (pk of 100)	Fine Science Tools	10011-00
Scalpel handle #3	Fine Science Tools	10003-12
Sodium Pyruvate	Sigma Aldrich	S8636
Spatula/probe	Fine Science Tools	10090-13
SS Double edge uncoated razor blades (American safety razor co (95-0043))	VWR	55411-060
Sucrose	Amresco	57-50-1
Surgical Scalpel	Exelint International	D29702
Tissue Chopper	Brinkman	(McIlwain type)
Tissue culture inserts	Millipore	PICMORG50 or PICM03050