Imaging Tumor Cell Migration in Glioblastoma Tissue Using Time-Lapse Scanning Confocal Microscopy

Abstract

Source: Parker, J. J., et al. A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs. J. Vis. Exp. (2017)

This video showcases the imaging of glioblastoma tissue slices expressing green fluorescent protein (GFP) via confocal microscopy. High-resolution images are captured at consistent intervals to track tumor cell movement and direction.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Tumor Cell Labeling Via Green Fluorescent Protein Expressing Retrovirus

NOTE: Time-lapse microscopy for analysis of tumor cell migration requires stable, long-term fluorescent labeling of cells within the slice culture. Use of retrovirus is suggested because it selectively infects dividing cells, thereby enriching fluorescent labeling within the tumor cell population as opposed to microglia or other cell types present within the slice. Standardization of infection suggests that a viral titer of 10⁴ CFUs/µL results in sufficient green fluorescent protein expression for the tracking and analysis of cell migration. Increased viral titer, use of non-selective virus (i.e. adenovirus, lentivirus), or other means of labeling all cells may preclude identification of clear cell boundaries during migration, thus complicating analysis. Use of alternative fluorescent markers can be utilized and optimized as needed.

1. Obtain retrovirus for infection of tumor slices either via standard protocols5,14 or from a commercially available source. Dilute the viral supernatant by adding the appropriate volume into an unsupplemented neuronal medium to attain a viral titer of 10⁴ CFUs (colony-forming units) /µL.
2. Between 7 - 10 days of culture infect the tumor slice cultures of interest with 5 - 10 µL of virus (10⁴ CFUs/µL). Place the virus gently dropwise onto the surface of each tissue slice. Decrease supernatant volume added if the slice floats on the surface of the insert. Return plates containing slice cultures to an incubator.
3. Assess the slices for labeled tumor cells beginning at 24 h after viral infection. This time delay will vary depending on viral incorporation and fluorescence gene expression kinetics. For the viral constructs used here, 72 h was required to observe a robust fluorescent signal with a standard epifluorescence microscope.​
   NOTE: Rarely, slices display a predominantly peripheral distribution of virally labeled cells, which can complicate confocal imaging. To avoid this complication, attempt to reduce slice thickness and thickness variation across the slice. If the slice culture has a thicker region near the center, this may prevent adequate nutrient penetration, thus limiting the population of actively dividing tumor cells. A qualitative assay to screen for this complication is the addition of a tetrazolium dye reagent (i.e. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) to the slice culture media. Areas of the slice that do not turn blue after adding the reagent indicate a lack of metabolic activity and compromised slice health.

2. Time-Lapse Single Photon Laser Scanning Confocal Imaging of Tumor Cell Migration

NOTE: After successful transduction and health of the culture is confirmed, cells may be imaged under control conditions, followed by an equal period of imaging under treatment conditions. Using this protocol, cells were successfully imaged and tracked for 12 hours in each condition. However, shorter or longer periods of imaging and environmental manipulation may also be informative.

1. Loading the Microscope
   1. Before imaging, place 1 mL of fresh slice media into a glass-bottom dish.
   2. Allow the media in the glass-bottom dish to equilibrate in an incubator for 15 min.
      ​NOTE: At this point soluble labeling agents, such as fluorescently conjugated lectins (i.e. Isolectin IB4 for microglia labeling) or ligand conjugated quantum dots, can be added to the slice culture media for identification of cellular subpopulations during imaging.
   3. Transfer the insert to be imaged to a glass-bottom dish using sterile forceps in a laminar flow hood. Transport the dish to the microscope stage.
   4. Maintain slice cultures at 37 °C and 5% CO₂ atmosphere in a sealed microscope stage-top incubator. Utilize sterile H₂O humidification of the incubation chamber if available to prevent excessive media evaporation (especially important for longer imaging experiments).
   5. Utilize a confocal microscope with a long working distance 10X air objective and laser excitation for single-photon and/or multi-photon.​NOTE: Ensure that the microscope objective lens provides adequate working distance. As a result of the added height from the stage-top incubator, glass bottom dish, and tissue culture insert, long working distance objectives are critical.
   6. Secure the glass bottom plate, remove the plastic Petri dish cover, and cover with a gas-permeable membrane.
2. Image Acquisition
   1. Use the microscope to visually inspect the slice, and locate a suitable field with adequate density of fluorescently labeled tumor cells between the slice edge and the center. Avoid imaging fields at the edge of the slice, due to increased susceptibility of tissue shifts during imaging. Tissue slice harps may be employed to limit this shift.
   2. Use imaging software in multidimensional analysis mode to set the first (bottom) and last (top) Z-stack boundaries, such that all positions to be imaged contain a visible fluorescent cellular signal. Imaging through 150 - 200 µm of the slice, with a constant Z-step of 10 µm, provided adequate resolution for tracking cell paths (this can be adjusted for individual imaging needs).
   3. If the microscope has a motorized stage, ensure adequate time is allowed for each Z-stack acquisition. Set the time interval between acquisitions to equal scan time per position x number of positions to image (i.e,. imaged tumor regions).​
      NOTE: For simultaneous excitation of the green and red fluorophores, utilize a dual-line laser line-scanning program, with simultaneous 488 nm and 633 nm excitation. The wavelength of laser excitation selected will vary according to the individual fluorescent proteins expressed.
   4. Maintain uniform laser power and confocal pinhole settings between the imaged tumor regions. Utilize the lowest laser power setting needed to clearly demarcate the tumor cell bodies and processes to limit phototoxicity. Specific imaging parameter units (i.e. power and confocal pinhole settings) will vary according to microscope and laser source specifications.
   5. Compensate for potential media evaporation by adding 2 - 3 "buffer" Z-stack steps in the focal planes advancing towards the objective. This effectively prevents the tissue from leaving the range of Z-stack acquisitions in the vertical plane.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM High Glucose	Invitrogen (Gibco)	11960-044
Neurobasal-A Medium, minus phenol red	Invitrogen (Gibco)	12349-015
B-27 Supplement (50X), serum free	Invitrogen (Gibco)	17504-044
Penicillin-Streptomycin (10,000 U/mL)	Invitrogen (Gibco)	15140-122
GlutaMAX Supplement	Invitrogen (Gibco)	35050-061
L-Glutamine (200 mM)	Invitrogen (Gibco)	25030-081
HEPES (1 M)	Invitrogen (Gibco)	15630-080
Nystatin Suspension	Sigma-Aldrich	N1638-20ML	10,000 unit/mL in DPBS, aseptically processed, BioReagent, suitable for cell culture
UltraPure Low Melting Point Agarose	Invitrogen (Gibco)	16520-050	Melts at 65.5 C, Remains fluid at 37 C, and sets rapidly below 25 C.
Isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 647 Conjugate	Thermo Fisher (Molecular Probes)	I32450	Used in media to label Microglia/Macrophages
pRetroX-IRES-ZsGreen1 Vector	Clonetech	632520
Retro-X Concentrator	Clonetech	31455	Binding resin for non-ultracentrifugation concentration of viral supernatants
pVSG-G Vector	Clonetech	631530	part of the Retro-X Universal Retroviral Expression System
GP2-293 Viral packaging cells	Clonetech	631530	part of the Retro-X Universal Retroviral Expression System
Cyanoacrylate Glue (Super Glue)	Sigma-Aldrich	Z105899	Medium-viscosity
Peel-A-Way Embedding Mold (Square - S22)	Polysciences, Inc.	18646A-1	Molds for tumor sample embedding
Stainless Steel Micro Spatulas	Fisher Scientific	S50823	Bend instrument 45 degrees at the neck of the spoon blade
Curved Fisherbrand Dissecting Fine-Pointed Forceps	Fisher Scientific	08-875
Single Edge Razor Blade (American Safety Razors)	Fisher Scientific	17-989-001	Blade edge is 0.009" thick. Crimped blunt-edge cover is removed before loading onto vibratome.
Leica VT1000 S Vibratome	Leica Biosystems	VT1000 S
Hydrophilic PTFE cell culture insert	EMD Millipore	PICM0RG50	30 mm, hydrophilic PTFE, 0.4 µm pore size
35 mm Glass Bottom Dishes	MatTek	P35G-1.5-20-C Sleeve	20mm glass diameter. Coverslip glass thickness 1.5
LSM 510 Confocal Micoscope	Zeiss	LSM 510	10x Air Objective (c-Apochromat NA 0.45)
PECON Stagetop Incubator	PeCON Germany	(Discontinued)	Incubator PM 2000 RBT is a comprable product designed for use with Zeiss Microscopes.