Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

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Summary

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

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Comba, A., Dunn, P. J., Kish, P. E., Kadiyala, P., Kahana, A., Castro, M. G., Lowenstein, P. R. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion. J. Vis. Exp. (158), e60939, doi:10.3791/60939 (2020).

Abstract

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Introduction

Gliomas are the most aggressive primary tumors of the central nervous system. They are highly invasive and heterogeneous1. Analysis of cellular and molecular components of the tumor will reveal novel therapeutic targets.

Among different methods currently available, laser capture microdissection (LMD) of frozen brain tumor tissue is a cost-effective, reliable technique that allows the isolation of discrete anatomical areas or specific cell populations from tumor tissues to study their molecular profile2,3. LMD allows the analysis of mRNA gene expression profiles of selected single cells or multicellular structures4,5. LMD can be utilized to gain in-depth mechanistic knowledge about the molecular events that take place during tumor progression. Improvement in processing of tumor tissues is necessary to obtain optimal optical resolution of tissue morphology and RNA-quality6. Although paraformaldehyde fixation is the best option for morphological analysis, RNA quality is affected and degraded under these conditions, resulting in poor RNA quality for RNA-seq analysis. The use of frozen tissue sections avoids ice crystal formation, which could break cell membranes and produce holes within cells, and remains the best option for RNA-Seq analysis7.

Here, we describe an optimized section fixation and staining method to process frozen mouse brain tumor tissues for LMD. To prevent ice crystals from forming in the tissue, we perfused mice with a solution of 30% sucrose. This solution disrupts interactions between polar water molecules and prevents the formation of ice crystals, preserving the tissue morphology. Tissue staining is necessary to differentiate and obtain specific population of cells or anatomically distinct areas within the tumor. It is essential to fix and stain the tissue with innocuous dyes to maintain RNA integrity. It has been previously shown that staining tissue with hematoxylin/eosin (H&E) deteriorates RNA integrity8. We fixed and stained the tissue of interest with ethanol, Cresyl violet 4% and eosin Y 0.5% solutions. Cresyl violet is an acidophilic dye that stains the cell nucleus with a dark blue color. Eosin Y is a basophilic dye that stains basic components of the cells, providing a distinction between cytoplasm and other cellular structures8. Both dyes are soluble in ethanol and do not deteriorate RNA quality. To avoid tissue damage and maintain high optical resolution of the cellular structures, we mounted the tissue sections prior to LMD9.

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Protocol

All methods described here that use experimental animals have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.

NOTE: Glioma neurospheres generated from a GEMM or stable lines can be used for intracranial tumor engraftment in mice10 and processed for LMD and RNA sequencing. These cells constitutively express firefly luciferase and GFP proteins, which will be further utilized for tumor growth analysis and localization.

1. Generation of intracranial mouse glioma model from neurospheres derived from genetically engineered glioma models

  1. To generate a primary culture of neurosphere cells from a genetically engineered mouse (GEMM) model, use the protocol described previously10,11.
  2. Prepare neurosphere culture medium as described: 500 mL of Dulbecco's Modified Eagle Medium F-12 (DMEM/F12) supplemented with 10 mL of 50x B-27 and 5 mL of 100x N-2 neuronal culture supplements, 5 mL of 100x Antibiotic-Antimycotic, and 1 mL of Normocin. Before plating the neurospheres, add 20 ng/mL human recombinant EGF and FGF to the culture medium.
  3. Culture glioma neurospheres in a tissue culture incubator at 37 °C and 5% CO2 for 2-3 days prior to intracranial tumor engraftment.
  4. On the day of the surgery, collect the glioma neurospheres and spin them at 550 x g for 5 min at room temperature. Carefully remove the supernatant without disturbing the cell pellet.
  5. To dissociate the neurospheres, resuspend the cell pellet in 1 mL of cell detachment solution and incubate at 37 °C and 5% CO2 for 2-4 min. Following the incubation period, pipette the neurospheres up and down with a 1 mL micropipette to ensure a single cell suspension.
  6. Inactivate the cell detachment solution by diluting the neurosphere suspension with 10 mL of un-supplemented DMEM/F12 media. Spin the neurospheres at 550 x g for 5 min at room temperature. Carefully, remove the supernatant.
  7. Resuspend the cell pellet in 100 µL of DMEM/F12 media with no supplements. Make a 1:50 dilution of the cell suspension, and then add 50 µL of Trypan Blue to count viable cells. Use the following formula to determine the concentration of cells:
    cells/mL = [((∑cells counted per square) / # of squares counted) x 50 x 10,000 cells/mL]
  8. After determining the cell count, to achieve a target concentration of 30,000 cells/µL in 100 µL volume, spin the neurospheres down and resuspend them in the appropriate volume of DMEM/F12 containing no supplements.
  9. Place neurospheres in a pre-labeled 0.6 mL tube on ice.
  10. Prepare working solutions of anesthesia, analgesic, and anesthesia reversal prior to implantation: For ketamine/dexmedetomidine anesthesia, add 0.6 mL of 100 mg/mL ketamine hydrochloride and 0.8 mL of 0.5 mg/mL dexmedetomidine hydrochloride to sterile 8.6 mL of 0.9% NaCl containing vial. For buprenorphine analgesic, add 1 mL of 0.3 mg/mL buprenorphine to 9 mL of 0.9% NaCl containing vial. For atipamezole anesthesia reversal, add 1 mL of 5 mg/mL atipamezole to 9 mL of 0.9% NaCl containing vial.
  11. Use 6-8-week-old C57BL/6J female mice for intracranial tumor engraftment.
  12. In a room approved for rodent survival surgery, set up sterile supplies for intracranial tumor engraftment. Perform surgeries on a rodent stereotaxic frame equipped with a sterilized 10 µL Hamilton syringe with a removable 33G needle. Utilize a bead sterilizer to sterilize tools between surgeries.
  13. Anesthetize mice with a single intraperitoneal (i.p.) injection of the anesthetic solution prepared in step 1.11 (ketamine:75.0 mg/kg and dexmedetomidine:0.5 mg/kg). Approximately, a 250 µL volume of the anesthetic solution will be delivered to a mouse weighing 20 g. Ensure that the mouse is unresponsive to pedal reflex prior to proceeding.
  14. Once the mouse is in a deeply anesthetized state, apply sterile petrolatum ophthalmic lubricant to the eyes to prevent drying. Shave the fur on the mouse cranium. Apply 10% povidone-iodine topical solution to the shaved area in order to disinfect.
  15. Secure the mouse's skull in a stereotactic frame. First, carefully open the mouth with forceps and gently pull the tongue and move it to one side of the mouth to prevent choking. Keep the mouth open with the forceps and place the top incisors into the keyhole of the tooth bar on the stereotactic frame.
  16. Holding the mouse head by the ears, place the ear bars against the postorbital bones and secure them. Ensure that the mouse's cranium is level with the surgical tabletop. Secure the ear bars carefully not applying pressure against the skull. Next, carefully secure the nose bar.
  17. Using a size 15 scalpel blade, make an incision along the mouse head, exposing the cranium. Retract the skin at the incision site using Colibri retractors. Use a sterile applicator to remove all pericranial tissue.
  18. Identify the bregma and lower the needle of the Hamilton syringe directly over it. Using the frame, position the needle 1 mm anterior of the bregma and 1.5 mm lateral. Using a 26G needle, mark this spot by scoring the cranium.
  19. Use a cordless power drill equipped with a 0.45 mm drill bit to create a burr hole at the target site. Drill until reaching the underlying dura mater. Carefully extract the remaining bone in the burr hole with a 26G needle.
  20. Using a pipette, homogenize the cells thoroughly and draw up 7 µL of the neurosphere suspension into the syringe. To ensure that that syringe is working properly, dispense 1 µL of the suspension onto a 70% alcohol-soaked pad.
  21. Lower the needle to the surface of the dura mater. Lower the syringe 3.5 mm ventral and retract 0.5 mm. This will create a 0.5 mm space in the brain for the neurospheres to deposit when injected.
  22. Leave the needle in place for 2 min for pressure equilibration. Slowly and smoothly deliver 1 µL of the cells over the course of 1 min. Allow the cells to settle in for 6 min. Remove the syringe slowly and smoothly from the brain over the course of 2 min.
  23. Using sterile saline solution, wash the surface of the cranium three times. Dispense the excess cells in the syringe onto a 70% alcohol pad, and wipe the needle clean with another 70% alcohol pad. Rinse the syringe with sterile PBS to avoid clogging of the needle.
  24. Remove the retractors and carefully take the mouse out of the stereotactic frame. Close the incision using 3-0 nylon sutures, forceps, and a needle driver.
  25. Administer atipamezole (1.0 mg/kg), approximately 100 µL for a 20 g mouse. Administer buprenorphine (0.1mg/kg subcutaneous) subcutaneously, approximately 70 µL for a 20 g mouse. Place post-surgical mice into a clean recovery cage and monitor them until alert and active.

2. Animal perfusion and brain preservation

  1. To monitor tumor progression, determine in vivo bioluminescence using an in vivo imaging system until animals show signs of tumor burden. Euthanize mice when they reach a signal between 106 to 107 photon/s.
  2. Anesthetize mice displaying signs of tumor burden with intraperitoneal (i.p.) injection of ketamine (75.0mg/kg) and dexmedetomidine (0.5 mg/kg) solution. Deliver approximately 250 µL to a mouse weighing 20 g. Then, ensure that the mouse in unresponsive to pedal reflex.
  3. Using forceps, hold the skin above the peritoneal cavity, and using a large pair of dissection scissors make a "Y" incision by penetrating the peritoneal wall, puncture the diaphragm, and then cut the rib cage.
  4. Insert a blunt 20G cannula into the left ventricle of the mouse's heart. Then snip the right atrium of the heart to allow for exsanguination.
  5. Allow oxygenated Tyrode's solution (0.8% NaCl, 0.0264% CaCl2, 0.005% NaH2PO4, 0.1% glucose, 0.1% NaHCO3, 0.02% KCl) to flow through the mouse circulatory system until the liver and lungs have completely cleared due to the removal of blood (~ 5 min).
  6. Continue perfusing the animal with 30% sucrose solution dissolved in Tyrode's solution for an additional 15 min. To evaluate the success of the perfusion, confirm that the neck, tail and legs are rigid post 30% sucrose circulation.
  7. Using a small pair of dissection scissors, cut the scalp at the midline. Starting at the occipital bone and working forwards towards the snout. This will expose the cranium.
  8. Using a pair of rongeurs, break through the cranium beginning at the occipital bone and continue forward to totally expose the surfaces of the brain. Then turn the head side up and dissect the nerves at the base of the brain to release it from the cranium.
  9. Prepare 30% sucrose solution with RNase free water and filter it through a 40 µm nylon mesh filter to reduce RNA degradation.
  10. To maximize sucrose solution infiltration, place the dissected brains into 30% sucrose solution and store them at 4 °C overnight. Before further processing ensure that the brain reaches the bottom of the tubes containing the sucrose solution.

3. Cryopreservation of brains harboring glioma tumors

  1. Prior to cryopreserving the brains, prepare a jar filled with cold isopentane/2-methylbutane and place the jar into a container filled with liquid nitrogen. Let the solvent cool down.
  2. Remove the brain from the 30% sucrose solution and blot it dry on a filter paper.
  3. Label the cryomold with a permanent marker. Carefully, add approximately 5 mL of OCT (optimal cutting temperature compound) into the center of the cryomold avoiding air bubbles.
  4. Place the brain into cryomold containing OCT in the desired orientation. Fill the mold with OCT until the brain is fully submerged. Using clean forceps, quickly place the cryomold with OCT and the brain into the cold isopentane/2-methylbutane.
  5. Once the OCT solidifies (~30-40 s), remove the cryomold with the brain and place it in dry ice. Do not leave the mold containing the brain in 2-methylbutane past 2 min as this may cause cracks in solid OCT. Wrap the cryomold with the brain in aluminum foil and store it at -80 °C.

4. Sectioning frozen brain tumor tissues

  1. Label 2 µm polyethylene naphthalate (PEN) slides with the sample information. Tissue sections will be placed directly on these slides following sectioning.
  2. Set the temperature of the cryostat chamber between -20 to -24 °C. Before sectioning, place the sample block in the cryostat chamber and let it equilibrate to the temperature in the chamber for 30-60 min.
  3. Clean the cryostat chamber and the knife holder with 100% ethanol and spray the brushes to be used with RNase cleaning solution. Working inside the cryostat chamber, remove the mold and attach the OCT block containing the brain to the cryostat specimen disk with OCT. Place the block in the disk holder and align the block with the knife blade.
  4. Install a disposable blade into the sectioning holder.
  5. Section the brain at 10 µm thickness. Make sure there are no streaks or scratch lines in the tissue. Using a paintbrush, cautiously flatten and uncurl the tissue onto the cutting surface.
  6. Carefully mount the tissue containing the brain sections onto RNase free PEN glass slides. Flip the positive charged glass slides with the fingers in direction of the tissue and smoothly press the glass slide down towards the tissue section.
    NOTE: Temperature of hands will help the tissue attach to the glass.
  7. After mounting the brain sections onto the slides, keep slides in a box inside the cryostat chamber and then store them at -80 °C. Never keep the slides at room temperature.
    NOTE: Folding of the tissue, and tearing are common. For accurate posterior analysis, it is important to minimize these artefacts.

5. Fixation and staining of cryopreserved brain tissue sections

  1. To preserve RNA integrity, clean all instruments to be used with RNase cleaning solution. Proceed with the fixation and staining protocol inside a fume hood.
  2. Prepare the described fixative solutions in clean RNase free 50 mL tubes. Make all solutions with RNase free water on the day of the staining.
  3. Same day the laser microdissection will be performed, prepare 100%, 95%, 70% and 50% ethanol solutions. Keep the solutions in tightly closed tubes at room temperature.
  4. Prepare 4% Cresyl violet and 0.5% eosin Y in 75% ethanol solution. Vortex the solution vigorously for 1 min and filter them through a 0.45 µm nylon filter to eliminate traces of undissolved powder.
  5. Place the tissue slides into a container with 95% ethanol for 30 s. Transfer slides to the tube containing 75% ethanol; leave slides there for 30 s.
  6. Transfer slides to 50% ethanol and leave them there for 25 s. At this point, the OCT will be dissolved. Transfer the slide to 4% Cresyl violet solution for 20 s, and then transfer to 0.5% eosin Y solution for 5 s.
  7. Take the slide out of the dye solution and blot the slide dry with a filter paper. Then, place the slides in 50% ethanol for 25 s. Transfer the slides to 75% ethanol for 25 s. Transfer the slides to 95% ethanol for 30 s. Transfer the slides to 100% ethanol for 60 s.
  8. Rinse the slide with xylene. Transfer them to a container with xylene and wait 3 min.
  9. Prepare mounting medium (e.g., Pinpoint gum) in RNase-free water. To mount mouse brain sections, dilute the mounting medium in RNase-free water at a ratio of 1:10.
  10. Dry the slides on a RNase-free surface at room temperature for 10 s. Before the xylene dries, proceed to mounting the slides with the tissue sections.
  11. Gently disperse mounting solution on top of the tissue on the slide with a sterile and RNase-free thin paintbrush. Wait 10-20 s, and then immediately transfer the tissue slides to the microscope microdissection platform.
    NOTE: The ratio of mounting medium to RNase-free water used for mounting varies depending on the tissue of interest. The mounting medium/water ratio preserves glioma tissue morphology for laser microdissection without affecting the RNA integrity.

6. Laser capture microdissection

NOTE: A laser capture microdissection microscope needs to be utilized to laser microdissect specific areas of interest within the tumor tissue. To minimize the time for tissue laser microdissection, have the LMD microscope prepared before fixation and staining.

  1. To start the system, first turn on the power strip, followed by turning on the laser. Then, turn on the microscope controller and the computer. Start the LMD software.
  2. Under Microscope control, select 10x magnification. Under Laser control, set the laser parameters for tissue dissection. Set a laser frequency of 120 Hz for best cutting results. Always set the laser current to 100%.
  3. For accurate laser microdissection, set the speed at 10 and an aperture setting at 2.0-10.0 µm. Set the power to 53. Having the laser power at a higher setting may cause glass etching.
  4. Load the tissue collector that will capture the tissue following dissection. Click the second unload button. Remove the empty collector and place the DNase/RNase free 0.5 mL PCR flat head tubes containing 30 µL of lysis buffer into the collector. Place the collector back into the machine and click Continue on the software to proceed.
  5. Load the processed (fixed and stained) specimen onto the microscope. First, click unload on the LMD software. Next, mount the sample on the slide holder and place the slide holder onto the stage. Click Continue on the software to proceed.
  6. Under Cut Shapes windows, select Draw + Cut. Use the microscope controls to find the area of interest. Draw the region of interest (ROI) and select a destination collector tube. It is possible to draw multiple ROIs to micro dissect different areas from a single slide at the same time.
  7. Click Start Cut to proceed to tissue micro dissection. After sectioning the areas of interest remove the collector tubes from the holder and place the tubes on dry ice. Transfer the RNA tissue samples collected to -80 °C for long term storage.

7. RNA isolation of micro-dissected glioma tissue

  1. For RNA extraction from LMD use an RNA isolation kit optimized for small samples and low RNA yield (see Table of Materials). Follow the manufacture instructions. Carry out all the isolation steps at room temperature (25 °C). To maintain RNA quality, work rapidly. Prepare all the solutions as indicated by the manufacturer.
  2. Adjust the sample volume to 350 µL with lysis buffer with 1% β-mercaptoethanol. Vortex the sample for 40 s in order to reduce sample viscosity and increase RNA elution spin column efficiency.
  3. Transfer the entirety of the sample to a gDNA eliminator spin column placed in a 2 mL collection tube. Centrifuge the tube for 30 s at 8,000 x g. Save the flow-through and ensure that no liquid is left on the column following centrifugation.
  4. Add 350 µL of 70% ethanol to the flow-through and mix well by pipetting up and down. Transfer the sample, to an RNA elution spin column placed in a 2 mL collection tube. Close the lid gently, and centrifuge for 15 s at 8,000 x g. Discard the flow-through, saving the column.
  5. Add 700 µL of RNA washing buffer 1 (20% ethanol, 900 mM GITC, 10 mM Tris-HCl pH 7.5) to the RNA elution spin column. Close the lid gently, and centrifuge for 15 s at 8,000 x g to wash the spin column membrane. Discard the flow-through.
  6. After centrifugation, carefully remove the RNA elution spin column from the collection tube so that the column does not contact the flow-through.
  7. Add 500 µL of the second RNA washing buffer (ethanol 80%, NaCl 100 mM, Tris-HCl 10 mM pH 7.5) to the spin column. Close the lid of the column and spin 8,000 x g for 20 s to wash the column membrane. Dispose the flow-through.
  8. To wash the RNA elution column, add 500 µL of 80% ethanol, close the lid of the column and centrifuge at 8000 x g for 2 min. Throw out the tubes with the elution solution.
  9. Use a new collection tube, open the lid of the spin column and centrifuge at 8000 x g for 5 min to dry the column. Discard the elution tube.
  10. Place the RNA elution spin column in a new 1.5 mL collection tube. Add 12 µL of RNase-free water warmed at 37 °C directly to the center of the spin column membrane. Wait for 4 min and centrifuge for 1 min at full speed to elute RNA.

8. RNA quality control, library preparation and RNA-Seq analysis

  1. Following RNA extraction and purification, amplify RNA and create a cDNA library using a special kit suitable for RNA isolation at pico-molar concentrations following manufacturer instructions (see Table of Materials). Follow manufacturer's instructions. Clean workstation in order to avoid contamination with other PCR products and nuclease degradation of samples.
  2. If RNA has a RIN value greater than 4, meaning the RNA is of good quality or only partially degraded, proceed with the fragmentation step. Prepare all items on ice.
  3. Create a master mix as indicated (Table 1). Create reaction mixture in a nuclease-free thin-wall 0.2 mL PCR tube. Incubate the tubes at 94 °C in a hot-lid thermal cycler. Fragmentation incubation time depends on the quality of the RNA. RIN ≥ 7: 4 min, RIN 5-6: 3 min, RIN 4-5: 2 min.
  4. Following incubation, place the tubes on a PCR chiller rack that was previously cooled at -20 °C and let it sit for 2 min.
  5. For each tube of RNA sample, prepare first strand synthesis reaction master mix (Table 2). Incubate the tubes in a hot-lid thermal cycler under conditions described in Table 3. cDNA products can be frozen at -20 °C until for two weeks before proceeding to the next step.
  6. Create the PCR master mix as indicated in Table 4. Place the tubes in a hot lid thermal cycler and run the PCR reaction under the settings indicated in Table 5.
  7. Allow the beads, which will be used to purify the DNA, to warm to room temperature. Once warmed, add 40 µL of the beads to each sample. Vortex the tubes to mix and spin down briefly to collect liquid at bottom of the tube. Let the tubes incubate at room temperature for 8 min to allow the DNA to bind to the beads.
  8. Place the tubes on a magnetic separation device and let sit for about 5 min, or until the solution becomes completely clear. Keeping the tubes on the separation device, use a pipette to remove the supernatant carefully without disturbing the beads.
  9. Keeping the tubes on the separation device, add 200 µL of freshly made 80% ethanol to the beads to wash without disturbing the beads. Wait for 30 s before removing the 80% ethanol. Repeat this step.
  10. Briefly spin down the tubes and place the tubes back on the separation device. Remove any residual 80% ethanol without disturbing the beads.
  11. Let the tubes air dry with the caps open for 5 min. Do not let it sit longer as the beads will over dry.
  12. Keeping the tubes on the separation device, add 52 µL of nuclease-free water to cover the beads. Remove the tubes from the separation device and pipette up and down until all the beads are resuspended. Incubate at 5 min at room temperature.
  13. Place the tubes back on the separation device until solution becomes clear, about 1 min. Transfer 50 µL of the resulting supernatant to the wells of an 8-well strip. Add 40 µL of new beads to each sample. Vortex thoroughly to mix. Allow beads to bind to DNA by incubating at room temperature for 8 min.
  14. During this incubation period, begin thawing the components to be used for any rRNA present in the samples. Once thawed, place them on ice. Also, pre-heat a thermal cycler to 72 °C.
  15. Place the samples on the magnetic separation device until the solution clears (about 5 min). Aliquot 1.5 µL of probes per sample to a chilled PCR tube. Place the tube in the preheated thermal cycler under the settings of 72 °C for 2 min and 4 °C.
  16. Keeping the sample tubes in the magnetic separation device, remove the supernatant with a pipet without disturbing the beads. Then add 200 µL of freshly made 80% ethanol to the beads to wash without disturbing the beads. Wait 30 s before removing the 80% ethanol. Repeat this step.
  17. Briefly spin down the tubes and place the tubes back on the separation device. Remove any residual 80% ethanol without disturbing the beads. Let the tubes air dry with the caps open for 2 min. Do not let sit longer as the beads will over dry.
  18. Prepare master mix for all samples by combining the following components in the order presented (Table 6).
  19. Mix the master mix by vortexing and add 22 µL of the mix to the dried beads of each sample and mix thoroughly to resuspend. Let it incubate at room temperature for 5 min.
  20. Spin down the tubes and place them on the magnetic separation device for 1 minute or until the samples become clear. Transfer 20 µL of the supernatant, without disturbing the beads to new PCR tubes. Place the tubes in a pre-heated thermal cycler under settings in Table 7.
  21. Prepare a PCR master mix for enough for reactions for each sample. Add the following components to the master mix in the indicated Table 8. Add 80 µL of the PCR master mix to each of the sample tubes from step 8.20 and place in the thermal cycler at the following settings (Table 9).
  22. Allow beads, which will be used to purify the DNA, to warm to room temperature. Once warmed, add 100 µL of the beads to each sample. Let the tubes incubate at room temperature for 8 min to allow the DNA to bind to the beads.
  23. Place the tubes on a magnetic separation device and let sit for about 5 min, or until the solution becomes completely clear.
  24. Keeping the tubes on the separation device, use a pipette to remove the supernatant carefully without disturbing the beads.
  25. Keeping the tubes on the separation device, add 200 µL of freshly made 80% ethanol to the beads to wash without disturbing the beads. Wait for 30 s before removing the 80% ethanol. Repeat this step.
  26. Briefly, spin down the tubes and place the tubes back on the separation device. Remove any residual 80% ethanol without disturbing the beads.
  27. Let the tubes air dry with the caps open for 5 min. Do not let sit longer as the beads will overdry. Pipette 20 µL of Tris Buffer to the dried pellet. Remove the tube from the separation device and mix thoroughly with a pipet to resuspend the beads. Let it incubate at room temperature for 5 min.
  28. Place the tubes on the separation device for 2 min, or until solution becomes clear. Acquire the supernatant and transfer it to new tubes. Then store the collected solution at -20 °C.
  29. Check the final libraries need for quality and quantity control. Pool the samples, clustered on and sequence, as paired-end 50 nt reads, according to manufacturer's recommended protocols.

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Representative Results

Our laboratory has generated a genetically engineered mouse models (GEMMs) using the sleeping beauty transposase system (Figure 1A). This system incorporates specific genetic alterations into the genome of neural progenitor cells in neonatal mice. These altered progenitor cells form endogenous glioma tumors. Plasmid sequences used to generate the tumors were: (1) pT2C-LucPGK-SB100X for Sleeping Beauty transposon & luciferase expression, (2) pT2-NRASSV12 for NRAS expression, (3) pT2-shp53-GFP4 for p53 knock-down and GFP protein expression, and (4) pT2-shATRx-GFP4 for ATRX knock-down. Plasmids were injected into the lateral ventricle of 1-day old neonatal pups as described previously11. Plasmid uptake and tumor formation was monitored via in vivo bioluminescence imaging system. Once tumor-bearing mice displayed signs of tumor burden, they were sacrificed. Tumors were either used to generate neurosphere cultures or directly cryo-preserved for LMD processing (Figure 1A).

Cell cultures started from the GEMM were used to generate a translatable glioma model (Figure 1B). Glioma neurospheres derived from GEMM tumors were cultured and implanted intracranially into the striatum of immune-competent mice. Single cell suspensions obtained from neurospheres culture were used to generate glioma tumors by intracranial implantation as described before by our laboratory10,11,12. This methodology allows the careful quantification of the number of cells to be implanted per mouse (30,000 cells/1 µL/mouse). This protocol permits the reproducibility of the results between different experimental implantations. However, implantation of neurospheres could be an alternative option to generate mouse glioma tumors and subsequent LMD and RNA-Seq analysis. Nevertheless, this method is considered to be more accurate. Tumor progression was monitored by in vivo bioluminescence spectrum imaging system. Mice displaying signs of tumor burden were transcardially perfused and the brain was cryo-preserved for LMD processing (Figure 1C).

Tissue sections were laser microdissected to characterize the transcriptome of multicellular structures within gliomas. The perfusion, freezing and embedding procedures described were optimized to preserve tissue morphology and obtain good quality RNA after laser microdissection. Different perfusion approaches were evaluated in order to acquire tissues with superior morphology and RNA integrity (Table 10). To dissect the areas of interest, it was necessary to stain the tissue with innocuous dyes for RNA (Figure 2). We observed that perfusing tumor bearing mouse with Tyrode's solution for 5 min, and then 30% sucrose for 15 min, followed by overnight storage of the dissected brain in 30% sucrose preserves morphology and RNA integrity of the tumor tissue (Figure 3D). Perfusion with 30% sucrose solution prevented ice-crystal formation within the tissue. Although, paraformaldehyde tissue fixation resulted in high quality tissue morphology, RNA integrity was negatively affected (Figure 3B). Other approaches such as Tyrode's solution for 5 min or Tyrode's solution for 5 min + 30% sucrose solution for 15 min (Figure 3A and C) did not affect the RNA quality. Under these conditions, brains were not preserved in 30% sucrose overnight; we observed reduced resolution in tissue morphology.

We performed various staining techniques followed by RNA integrity quality control analysis. We observed that 4% Cresyl violet and 0.5% eosin Y staining was sufficient to identify glioma multicellular structures and maintain RNA integrity. Cresyl violet is an acidophilic dye that stains the nucleus of cells with a dark blue color. Eosin Y is a basophilic dye that stains basic components of the cells.

We observed that if the tissue was not mounted with mounting medium, the sections became dehydrated and the morphology deteriorated (Figure 4A). To maintain high quality tissue morphology, we mounted the tissue with mounting medium. We observed that 15% mounting medium dissolved in water (30 µL in 200 µL of water) maintained high quality tissue morphology (Figure 4B).

In Figure 5A, areas of glioma heterogeneity are shown. Images were acquired using a laser capture microdissection microscope preceding dissection. Red lines depict areas with abundant mesenchymal cells (elongated cells). Blue lines depict areas with no mesenchymal cells (rounded cells) (Figure 5A, middle image). Figure 5B shows images of laser micro-dissected tumor areas (red lines) and normal brain tissue areas (blue lines). ROI selection is made to dissect areas of interest (Figure 5A and B, lower images). We can observe in these images the success in the dissection of the selected areas.

RNA extraction was performed using a commercial kit. It was determined that a total area of dissected tissue between 2.5 x 106 - 7 x 106 µm2 was required for mRNA extraction and cDNA library preparation for subsequent RNA sequencing. RNA quality control determined a RIN between 6 to 7 in glioma tissue after laser microdissection. A RIN of 6 was determined to be appropriate for cDNA library preparation. Following RNA extraction, a kit for RNA isolation at picomolar concentrations was utilized to generate a cDNA library suitable for next generation sequencing.

0.25 ng - 10 ng (1-8 µL) of RNA
4 µL of the 5x First-Strand Buffer
1 µL of the Oligos Mix V2
Nuclease-free water to final volume of 13 µL

Table 1: RNA quality control and library preparation: Master Mix preparation for step 8.3.

4.5 µL Mix V2
0.5 µL RNase inhibitor
2 µL Reverse Transcriptase
Final volume of 7 µL
Add the 7 µL to the RNA sample tube

Table 2: RNA quality control and library preparation: Master Mix preparation for step 8.5.

42 °C for 90 min
70 °C for 10 min
4 °C hold

Table 3: RNA quality control and library preparation: Thermocycler conditions for step 8.5.

2 µL nuclease-free water
25 µL 2x PCR Buffer
1 µL DNA Polymerase for each reaction
Mix gently and spin down briefly
Add 28 µL of the master mix to each of the sample tubes
Add 1 µL of each 5' and 3' primer to each tube
Mix the reactions gently and spin down briefly

Table 4: RNA quality control and library preparation: Master Mix preparation for step 8.6.

1 cycle
94 °C for 1 min
5 cycles
98 °C for 15 s
55 °C for 15 s
68 °C for 30 s
1 cycle
68 °C for 2 min, 4 °C hold

Table 5: RNA quality control and library preparation: Thermocycler conditions for step 8.6.

16.8 µL nuclease-free water
2.2 µL 10x R Buffer
1.5 µL R v2
1.5 µL R-Probes v2

Table 6: RNA quality control and library preparation: Master Mix preparation for step 8.18.

37 °C 60 min
72 °C 10 min
4 °C hold

Table 7: RNA quality control and library preparation: Thermocycler conditions for step 8.20.

26 µL nuclease-free water
50 µL CB PCR Buffer
2 µL PCR2 Primers v2
2 µL DNA Polymerase

Table 8: RNA quality control and library preparation: Master Mix preparation for step 8.21.

1 cycle
94 °C for 1 min
X cycles
98 °C for 15 s
55 °C for 15 s
68 °C for 30 s
1 cycle
68 °C for 2 min, 4 °C hold

Table 9: RNA quality control and library preparation: Thermocycler conditions for step 8.21.

Step 1 Step 2 Step 3
Method 1 Tyrode's 15' -- --
Method 2 Tyrode's 5' Sucrose 30% 15' --
Method 3 Tyrode's 5' 4% PFA 10' --
Method 4 Tyrode's 5' 30% Sucrose 15' Brain in 30% Sucrose Overnight

Table 10: Methods for different perfusion approaches.

Figure 1
Figure 1: Mouse glioma models used for laser microdissection. (A) Sleeping beauty GEMM used to develop de novo glioma. Images display tumor progression: (i) plasmid injection into the lateral ventricle of neonates, (ii) tumor burden. (B) Generation of transplantable glioma model using glioma cell cultures. Neurosphere cells cultured from GEMM are implanted intracranially into the striatum. (C) Depiction of cryo-sectioning of the brain embedded in coronal orientation following perfusion. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Schematic representation of the method used to stain cryo-preserved tissue sections with Cresyl violet and Eosin. Slides used for staining were handled with tools cleaned with RNase-free water. All solutions were prepared with RNase/DNase free water on day of staining. Cresyl violet stains nuclei violet and eosin Y stains cytoplasm pink. Dyes were dissolved in 70% ethanol. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Comparison of various perfusion approaches to preserve tissue morphology and RNA integrity. H&E images represent comparative morphology of brain tissue that was subjected to perfusion under different methods: (A) Tyrode's solution for 15 min, (B) Tyrode's solution for 5 min, 4% PFA for 10 min, (C) Tyrode's solution for 5 min, 30% sucrose for 15 min, and (D) Tyrode's solution for 5 min, 30% sucrose for 15 min with overnight immersion in 30% sucrose. RNA quality assessment is displayed for each perfusion method. Plots depict 18s and 28s rRNA peaks and the initial marker peak. The ratio of 18s and 28s rRNA is used to determine RNA quality. A gel image of the RNA fragments is displayed to the right of the plots. RNA quality was assessed using the RIN values. RNA quality was high in methods A, C, and D. Tissue morphology was superior in methods C and D. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Representative images of mounted and non-mounted glioma tissue. (A) Representative images of tissue stained with H&E. This tissue was left unmounted and became dehydrated displaying poor morphology with breaks in the tissue. (B) Representative images of tissue stained with H&E and mounted with mounting medium. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Representative images of glioma tissue used for laser microdissection. (A-B) Representative images of tissue stained with H&E and areas of multicellular structures selected for LMD. (A) On the left, areas of elongated cells (red) and control areas (blue) were selected for LMD. (B) On the right, areas of collective invasion (red) and control areas (blue) were selected for LMD. (C) Representative RNA quality control for laser microdissected areas. A total area of 6.5 x 106 µm2 was microdissected. The analysis shows RNA concentration of 2,346 pg/µL and RNA Integrity Number (RIN): 6.8. Please click here to view a larger version of this figure.

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Discussion

Understanding the molecular mechanisms underlying glioma heterogeneity and invasion are of critical importance to uncover novel therapeutic targets13. In this manuscript, we describe a detailed and optimized method to analyze the molecular landscape of glioma heterogeneity and invasion using laser capture microdissection (LMD) followed by transcriptomic analysis.

Laser capture microdissection (LMD) can be used to identify different areas or single cells within the tumor, providing a specific sample to further analyze the molecular pattern maintaining the spatial context of the tumor14. This technique is reliable and low-priced compared with other methods used to analyze the spatial transcriptome in solid tumors15. We analyzed glioma heterogeneity and invasion in genetically engineered mouse tumor models or intracranial implantable models using LMD. These models recapitulate the salient characteristics of human gliomas, allowing the study of glioma heterogeneity and invasion10,12.

Maintaining high-quality tumor tissue morphology and RNA integrity is one of the limitations for LMD. To improve tissue morphology, we perfused mice with Tyrode's solution for 5 minutes, followed by 30% sucrose dissolved in the same solution. Once the brain was dissected, we preserved it in 30% sucrose dissolved in RNase/DNase-free water overnight or until the brain reached the bottom of the storage container. These steps significantly improve the morphology of the tissue and reduced the formation of ice-crystals in the tissue during cryo-preservation. Although human glioma tissue was not used for this protocol, incubation of human samples in 30% sucrose solution overnight could be a feasible methodology to improve cryosections morphology. Another possible limitation for LMD is the preservation of RNA integrity post-staining8. Although other research teams have performed laser microdissection on glioma tissue followed by RNA-seq analysis they do not illustrate any RNA and/or morphology nor do they comment on morphological quality, or particular controls for glioma frozen sections16. In this protocol accurate morphological identification was essential in order to laser microdissect precise macrocellular structures within gliomas. We observed that fixing glioma tissue with ethanol solution and staining it with 4% Cresyl violet and 0.5% eosin Y dissolved in ethanol-maintained RNA quality and enabled the microscopic identification of single cells and multicellular structures. We demonstrated that mounting glioma sections with mounting solution is a critical step which prevents cracking and fissure formation in the tissue. Please note that the mounting medium needs to be prepared in water, as using the mounting medium dissolved in ethanol will result in poor tissue morphology. Laser microdissection has to be performed as fast as possible in an RNase-free environment. We also recommend to section up to 2.5 x 106 µm2 total tumor/tissue area in order to obtain appropriate amounts of RNA, both for RNA quality control and transcriptomic analysis.

LMD enables the analysis of the molecular signaling pathways that regulate glioma heterogeneity and invasion. This analysis could reveal novel potential targets for diagnosis, prognosis and future translational development in preclinical glioma models.

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Disclosures

All authors of this paper declare no potential conflicts of interest.

Acknowledgments

Work was supported by National Institutes of Health, (NIH/NINDS) Grants: R37-NS094804, R01-NS105556, R21-NS107894 to M.G.C.; (NIH/NINDS) Grants R01-NS076991, R01-NS096756, R01-NS082311 to P.R.L.; (NIH/NIBI): R01-EB022563; (NIH/NCI) U01CA224160; the Department of Neurosurgery, Rogel Cancer Center at The University of Michigan, ChadTough Foundation, and Leah's Happy Hearts Foundation to M.G.C. and P.R.L. RNA Biomedicine Grant F046166 to M.G.C. National Institutes of Health, UL1 TR002240 for Michigan Institute for Clinical and Health Research (MICHR), Postdoctoral Translational Scholars Program (PTSP) grant, Project F049768 to A.C. University of Michigan Forbes Cancer Research Institute, a Physician-Scientist Award from Research to Prevent Blindness, Inc. (RPB), grant R01 EY022633 from the NEI of the NIH (AK), and an unrestricted grant from RPB to the Department of Ophthalmology and Visual Sciences. This research utilized the Vision Research Core (P30 EY007003), and the Cancer Center Research Core (P30 CA046592). AK is supported by the Mrs. William Davidson Emerging Scholar Award from the A. Alfred Taubman Medical Research Institute.

Materials

Name Company Catalog Number Comments
Accutase Cell Detachment Solution Biolegend 423201
Animal-Free Recombinant Human EGF Peprotech AF-100-15
Antibiotic-Antimycotic (100X) Gibco 15240062
B-27 Supplement (50X), serum free Gibco 17504044
Buffer RLT Qiagen 79216
Corning PCR Tubes Sigma Aldrich CLS6530
Cresyl Violet Acetate Sigma Aldrich C5042
DMEM/F12 - Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 Gibco 11330057
Eosin Y Sigma Aldrich E4009
HiSeq 4000 Illumina N/A
Laser Microdissection (LMD) System Leica LMD7000
N-2 Supplement (100X) Gibco 17502048
Normocin - Antimicrobial Reagent Invivogen ant-nr-1
Peel Away Disposable Embedding Molds Electron Microscopy Sciences 70182
PEN Membrane Glass Slide (2 µm) Lieca 1150518
Pinpoint Solution Zymo Research D3001-1
Recombinant Human FGF-basic Peprotech 100-18B-1MG
Research Cryostat Leica CM3050s
RNaseZap RNase Decontamination Solution Fisher Scientific AM9780
RNeasy Plus Micro Kit Qiagen 74034
SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian Takara Bio 634411
Tissue-Plus O.C.T. Compound Fisher Scientific 23-730-571

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References

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