A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Podsumowanie

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Streszczenie

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model's ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Wprowadzenie

The laboratory study of glioblastoma (GBM), is hindered by a lack of models that faithfully recapitulate the requisite pathologic characteristics of the human disease, namely tumor cell migration and invasion. Comparative studies of 2D and 3D in vitro invasion assays as well as 3D rodent slice culture models have uncovered mechanistically disparate cellular migration programs in these two contexts, potentially limiting the translatability of findings from 2D systems to the human disease^1,2,3. The organotypic tumor slice culture and imaging paradigm described here allows for the study of tumor cell migration within slices of ex vivo human tumor tissue obtained from surgical resection. Thus, slice cultures of surgically resected tumor tissue in conjunction with time-lapse confocal microscopy provide a platform to study tumor cell migration in the native microenvironment without tissue dissolution or culture passaging.

There is extensive literature employing rodent brain slice culture models of GBM generated from human tumor xenografts, retroviral-induced tumors, and cellular overlays to study tumor invasion^1,2,3,4,5. Recently, several groups have described the generation of organotypic slice cultures directly from human GBM tissue^6,7,8,9,10. However, there is marked variation among published protocols with regards to slicing technique and culture media. Further, the use of organotypic slice cultures has focused on static experimental endpoints that have included changes in cell signaling, proliferation, and death. The protocol described herein expands upon prior slice culture paradigms by incorporating time-resolved observation of dynamic tumor cell behaviors through time-lapse laser scanning confocal microscopy. Recent discovery of inter¹¹ and intratumoral^12,13 genetic variation in human GBM underlines the importance of linking this heterogeneity with tumor cell behaviors and its implications on tumor response to therapy. Here, we report a streamlined and reproducible protocol for use of direct slice cultures from a human cancer tissue to visualize tumor cell migration in near real-time.

Protokół

Before collection of patient tissue samples is initiated, informed consent must be obtained from each patient under an approved Institutional Review Board (IRB) protocol. The authors of this protocol received consent for the work described under approved IRB protocols at the University of Colorado Hospital and Inova Fairfax Hospital. Data collected from these slice cultures were not used to direct patient care decisions.

1. Pre-slicing Preparation

1. Prepare "tissue processing" media and "slice culture maintenance" media the day before planned tumor resection and tissue collection (or utilize previously generated media within 2 weeks). Add 5 mL of Penicillin-Streptomycin solution (10,000 U/mL) and 5 mL of 1 M HEPES to 500 mL of a High-Glucose DMEM to generate the tissue processing medium.
2. Prepare 250 mL of slice culture maintenance media using a base of neuronal medium (e.g., Neurobasal) without phenol red. Supplement this medium with 10 mM HEPES, 1x B-27 supplement, 400 µM L-glutamine, 600 µM L-alanyl-L-glutamine dipeptide, 60 U/mL penicillin, 60 µg/mL streptomycin, and 6 U/mL nystatin.
3. Store all media at 4 °C for no longer than 2 weeks.

2. Day of Surgery: Tissue Acquisition

1. On the day of tissue acquisition, prepare 50 - 100 mL of 1% and 2% (wt/vol %) solutions of low melting temperature agarose in tissue processing media using autoclaved glassware and sterile technique in a laminar flow hood. Both concentrations of agarose are needed to accommodate unpredictable variation in tumor tissue consistency.
2. Heat the agarose suspension, using a microwave, until gentle boiling is observed. Place the agarose solution in a 37 °C water bath to maintain in a liquid state until use.
3. Place 1 mL of slice culture maintenance media in each well of a 6-well plate.
4. Use a sterile forceps to place empty PTFE culture inserts in each well. Place the plate in a humidified, water-jacketed, tissue culture incubator with a 5% CO₂ atmosphere maintained at 37 °C.
5. Using a sterile Pasteur pipette in a laminar flow hood, bubble a mixture of 95% O₂/5% CO₂ gas into a flask containing ice cold tissue processing media for approximately 15 to 30 min, prior to tumor tissue acquisition. The use of supra-oxygenated media minimizes hypoxia in the bulk tissue during processing.
6. Prepare labeled 50 mL conical tubes (enough for desired number of individual tumor regions to be isolated) containing 20 mL aliquots of supra-oxygenated ice-cold processing media to transport tissue between the operating room and slicing facility.
7. In conjunction with a neurosurgeon, pre-operatively plan the tumor region for sample acquisition. Select a tumor region with contrast enhancement as demonstrated on the patient's clinical imaging (T1 post-gadolinium magnetic resonance imaging (MRI) sequences). Previous experience suggests this area yields viable tumor tissue as opposed to necrotic tissue.
   NOTE: Generation of slices from the surrounding (peritumoral) white matter was attempted; however, background autofluorescence and decreased tumor cell density limited the experimental utility of these slices.
8. Obtain tumor tissue towards the beginning of tumor resection. Avoid collection of tumor tissue exposed to extensive bipolar cautery, which may compromise tissue viability secondary to thermal injury. Tissue samples acquired during piecemeal tumor resection demonstrate enhanced viability when compared to tissue acquired from lengthy en bloc resections. This observation may relate to differential tumor tissue deprivation of blood flow as a result of surgical resection and extended time to acquisition.
9. If desired, label and store tumor tissue in separate conical tubes to isolate samples from distinct tumor regions. (i.e. superficial vs. deep tumor tissue). Precise tissue locations can be recorded if/when intraoperative surgical navigation is available.

3. Slice Culture Preparation

NOTE: This protocol requires the use of fresh unfixed human tissue. All samples are presumed to be infectious, and should be handled according to universal blood borne pathogen protocols. Appropriate personal protective equipment should be donned at all times. Forceps and scalpels should be exposed to 15 min of UV light prior to use. During use, intermittently spray the tools with 70% ethanol (EtOH), allowing time for the liquid to evaporate before use. The slicing process is performed in a semi-sterile fashion utilizing a horizontal laminar flow hood with filtered air.

1. Place tumor tissue pieces in a Petri dish with ice-cold processing media. To wash the tumor tissue and minimize adherent red blood cells, use a pipette to gently exchange and discard the media in the Petri dish three times.
2. Using a scalpel, cut tumor pieces into rectangular box shapes. Trim tissue pieces to approximate 3 mm x 3 mm x 10 mm. Carefully remove any attached vessels through excision or gentle traction with fine forceps.
3. Pipette 5 - 7 mL of 37 °C agarose into a small cube-shaped (~ 2 cm³) plastic embedding mold. Confirm temperature of agarose solution is no greater than 37 °C with a sterile thermometer before use.
4. Allow agarose to sit for approximately 1 min on a bed of ice.
5. Place 2 - 4 tumor tissue "strips" into agarose with long axis oriented vertically.
   NOTE: Tissue strips will tend to "sink" in the agarose before solidification. To avoid this complication, temporarily hold the tissue strip in vertical orientation until agarose is further solidified.
6. Keep the tissue containing agarose mold on a bed of ice for 2 - 5 min to facilitate solidification.
7. Remove mold from ice and gently remove the agarose block by cutting the sides of the form with a scalpel. Avoid placing excessive force on the block, which may fracture the agarose.
8. Use a generous drop of cyanoacrylate glue to affix the agarose block to the vibratome specimen plate. Let glue set for approximately 1 - 2 min.
9. Fill the vibratome reservoir with ice-cold processing media to submerge the agarose block affixed to the specimen plate.
10. During slicing, bubble 95% O₂ / 5% CO₂ gas mixture into the vibratome reservoir through a trimmed sterile plastic pipette.
11. Set the vibratome slice thickness to 300 - 350 µm.
12. Adjust blade advance speed and blade amplitude according to tissue consistency. "Stiffer" GBM tissue requires slower blade advance speeds and higher amplitude. Exact blade speed and amplitude settings will vary according to vibratome specifications.
    NOTE: Several issues commonly arise during slicing. If the tissue strip dislodges from the agarose block, attempt embedding the tissue in a higher concentration agarose. If tumor slices are thicker than desired (machine set), or are asymmetric, increase the amplitude and decrease speed of vibratome blade. To ensure tumor tissue is not stuck to or "dragged" by the blade holder, as slicing occurs, carefully position the microspatula between the blade holder and slice, as the blade moves.
13. Transfer tissue slices to Petri dish with ice-cold processing media using a stainless steel microspatula.
14. Obtain 6-well plates containing PTFE inserts and equilibrated slice culture media from incubator (as prepared in section 2, step 4).
15. Plate tissue slices using a stainless steel microspatula. Minimize direct contact and manipulation of tissue by using a small fine bristle paintbrush to generate a "fluid wave" to gently push the slice off the spatula and onto the culture insert.
    NOTE: Minimize the amount of processing media introduced on top of the tissue culture insert. If excessive amounts of media are transferred causing the slices to float, use a sterile Pasteur pipette to remove media.
16. Return the 6-well plates containing tumor slices to an incubator maintained at 37 °C with a 5% CO₂ atmosphere.
    NOTE: The entire embedding and slicing protocol should be completed within 90 min of tumor tissue acquisition from the operating room.

4. Slice Culture Maintenance

1. After 12 - 24 h, transfer the slice cultures by grasping each insert rim with a sterile forceps, and transfer to plates containing fresh slice culture maintenance media. Ensure the slice culture maintenance media aliquoted into each well equilibrates in the incubator for at least 15 min before transferring inserts.
2. Move inserts to new 6-well plates with fresh equilibrated slice culture media every 48 h.
3. If desired, aliquot old slice culture media with a pipette for immediate use in biochemical assays (i.e. ELISA) or freeze at -80 °C for future use.

5. Tumor Cell Labeling Via Green Fluorescence 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 protocols^5,14 or from a commercially available source. Dilute the viral supernatant by adding the appropriate volume into unsupplemented neuronal medium to attain a viral titer of 10⁴ CFUs/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 incubator.
3. Assess the slices for labeled tumor cells beginning at 24 h after viral infection (see section 6). This time delay will vary dependent on viral incorporation and fluorescence gene expression kinetics. For the viral constructs used here, 72 h was required to observe 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) 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.

6. 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 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 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 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.

7. Image Post-Processing and Tumor Cell Tracking

NOTE: Many confocal imaging systems are equipped with proprietary image-processing software. The processing steps discussed below comprise a general protocol, which can be performed across software platforms. Specific instructions will be given for the open-source platforms, NIH ImageJ and MTrackJ¹⁵.

1. Open a Z-stack file. In ImageJ, click "Image→Stacks→Z Project." Chose the first and last Z-stack to include, and chose "Max Intensity" as the projection type. The result is a rendering called a maximum intensity projection (MIP). Create an MIP from each set of Z-stack images captured at each region imaged.
2. Concatenate the MIPs from each region to create a time series. Click "Image→Stacks→Images to Stack."
3. Manually identify the location of the cell body "centroid" by selecting the visually approximated center point of the tumor cell body. This is accomplished by clicking on the cell body using the "add" track functionality of MTrackJ (an open-source plugin-in for NIH ImageJ).
4. Click to demarcate the cell body location in each frame of the series of images. This creates a unique "track" for each cell. Sequentially, mark the cell body locations of the next cell. Repeat this process until all cell migration paths are tracked.
5. Once a population of cells is tracked in a given tumor micro region, export all cell track coordinates using the "measure" function of MTrackJ. Save the file as an .xls format so the raw data can be analyzed using spreadsheet or user-generated software.
6. Use the coordinates recorded for each point along a cell's migration track, to perform quantitative analyses including derivation of migration speed, directionality, and other migration metrics, as described in Parker et al¹⁶.
   NOTE: All calculated distances and speeds are under-estimates of actual values. This is inherent in the transformation of three-dimensional images (and migration paths) to two-dimensional data through generation of maximum intensity projections.

Wyniki

Our group has successfully generated slice cultures from over 50 patients undergoing initial GBM resection. This slice generation, culture, retroviral-labeling, imaging, and migration analysis protocol has been streamlined into a reproducible workflow (Figure 1). Critically, these organotypic GBM slices demonstrate concordance with originating tumor tissue throughout culture, including maintenance of pathologic hallmarks and microglia up to 15 days in culture (Fig...

Dyskusje

Organotypic slice cultures from human cancer tissue provide an attractive and underutilized platform for pre-clinical translational experimentation. Understanding of population-level behaviors of tumor cells with regards to migration, proliferation, and cell death in the native tumor microenvironment is lacking. Critically, studying tumor response to therapy in a dynamic, time-resolved fashion at the level of cell behavior may shed light on novel mechanisms of treatment resistance. Human tumor slice cultures provide a li...

Materiały

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
Equipment
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	20 mm glass diameter. Coverslip glass thickness 1.5 mm
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.