Immunology and Infection
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Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing
Chapters
Summary November 29th, 2018
We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.
Transcript
This method can help answer key questions in preclinical research, where drug response studies in tumor slice explants may serve diagnostic purposes. The main advantage of this technique is that slice explants can for a short window of time model the complex biology of native tissues including spatially distributed functions. Though this method can provide insight into the use of slices from murine lung tumors, it can also be applied to other tissue types including human tissues.
To begin, prepare the vibratome and perform a VibraCheck according to instructions provided in the manual. Then fill each well of the 24-well plate with one milliliter of Hank's Balanced salt solution supplemented with penicillin and streptomycin and keep the plate on ice. Insert 25-gauge needles in all four paws of a euthanized mouse placed on a styrofoam lid so that the chest is exposed and the animal is stretched.
Spray 70%alcohol on the surface of the exposed skin. Use scissors placed in an angled position to cut open the skin from the abdomen towards the chest and up to the neck region. Stretch the skin on either side using forceps and insert 25-gauge needles.
Cut open the abdominal region. Then cut open the rib cage and the diaphragm to expose the lung and heart. Expose the trachea by cutting away the surrounding tissue.
Dissect the lungs, together with the heart. Place the tissues into a 50-milliliter tube containing 30 milliliters of ice cold HBSS supplemented with Pen-Strep placed on ice and proceed to the next step as quickly as possible. Now transfer the tumor-bearing lungs into a 10-centimeter tissue culture plate.
Use sterile scissors and forceps to separate the lung lobes and select lobes with tumors on the surface for slicing. Place a lung lobe with a tumor tissue on a piece of filter paper to avoid slipping and cut out part of the normal lung or additional tumor tissue that surrounds the tumor using a sterile scalpel to generate a flat tissue piece surface. Dip the flat side in a drop of cyanoacrylate adhesive and mount it onto the vibratome specimen holder so that the tumor faces the blade in an upright position, and let it dry for two to three minutes.
Place the vibratome specimen holder into the metal buffer tray. Fill the tray with cold HBSS supplemented with Pen-Strep until the tissue is immersed in the buffer. Cover the buffer tray with the Plexiglas lid that is provided with the instrument and place the tray into a white ice bath and add ice to keep the tissue cool during slicing.
Attach the white ice bath to the vibratome. Then select suitable slicing settings and start slicing. Bring the vibratome blade to the slicing position and set the slicing window.
Use sterile forceps to collect the slices in a 24-well plate filled with one milliliter of HBSS supplemented with Pen-Strep per well and keep on ice. While keeping track of the slicing order, mark each well of the 24-well plate according to the experimental plan. Collect a tissue slice adjacent to the cultured slices as a zero hours or uncultured reference.
Collect at least three reference slices to represent the top, center and bottom of the tissue. And once slicing is completed, continue with fixing and processing. After that, prepare a six-well plate containing 2.5 milliliters of culture medium per well by placing titanium grids in it.
Make sure that no air bubbles are formed between the titanium grid and the medium. To load a slice onto the grid keep the six-well plate in an angled position so that a portion of the medium covers the grid. Then place the slice in the medium on the grid and use forceps to spread it.
After placing the slices, load the six-well plates onto the rotating incubation unit placed inside a humidified incubator maintained at 37 degrees Celsius with 95%02 and 5%CO2. Start the rotation cycle. For long-term cultivation replenish the culture medium every day by using sterile forceps to lift the grid containing the tissue slices and place it in an empty well of the six-well plate.
Replace 70%of the medium with fresh culture medium and place the grid back in. Continue the rotation cycle as previously. For drug treatment on the tumor slices add 2.5 milliliters of media with the diluted drug or vehicle control into the six-well plate.
Place the titanium grids into the wells and then place the slices onto the grids as done previously. After performing the treatments for 24 hours, continue with fixing and processing. To start fixing the desired slice carefully transfer it using forceps into a 10-centimeter plate containing a soaked filter paper filled with two to three milliliters PBS and let it float.
Then lift out the filter paper with a pair of forceps and place it in a histo cassette. Transfer the filter paper into a histo cassette. Add a drop of diluted hematoxylin on top of the tissue slice to mark the position of the slice for the subsequent steps.
Close the cassette, and transfer it into 4%neutral buffered formalin solution and leave overnight at four degrees Celsius. After washing and processing of the cassette as described in the protocol, open the histo cassette and use a scalpel to carefully lift the fixed slice from the filter paper. Discard the filter paper and transfer the slice into a mold containing liquid paraffin.
Use a flat weight to press the tissue against the bottom of the embedding mold to ensure even sectioning. Place the bottom part of the histo cassette on top of the mold. Add liquid paraffin on top of it.
And let the mold cool on a cold plate for 30 minutes. After that separate the cooled mold from the paraffin block. Use a microtome to prepare four-micrometer thin sections of the FFPE tissue slice blocks.
Trim away the excess of paraffin surrounding the tissue. To obtain even sections throughout the tissue adjust the angle of the block so that the surface of the block is horizontally oriented with respect to the blade. Using a thin brush collect sequential tissue sections of the paraffin-embedded tissue slice starting at the upper part of the glass slides.
Continue collecting the sections of the deeper tissue layers to the middle, followed by the bottom part of the glass slides. The collection of sections from different layers of each tissue slice to the object slides is done to enable capture of potential culture-induced gradients in viability, cell migration or biomarker expression. After collecting the sections continue with their processing and further analysis as described in the protocol.
Tissue slice thickness can influence the viability of cultured slices. In this case, 250-micrometer thick slices show increased necrosis gradients compared to 200-micrometer thick slices due to thicker slices becoming prone to deficiencies in oxygen or nutrient diffusion across the slices. However, 160-micrometer thin slices contain large necrotic areas probably caused by technical handling during positioning of the slices as these are fragile and tend to curl.
Analysis of NKX2-1, a marker of well-differentiated lung adenocarcinoma, shows that its expression is not significantly altered in cultured slices as compared to zero hour uncultured slices. Quantitative analysis of NKX2-1 expression further confirmed that the expression was not significantly altered in cultured versus uncultured slices, suggesting that the process of cultivation does not visibly affect the differentiation status of adenocarcinoma tissue. To test whether tumor tissue slices can be used to evaluate the efficacy of targeted drugs, they were treated with DMSO or titrated amounts of compounds.
When treated with one micromolar dactolisib which targets the mTOR pathway phosphorylation of 4EBP1 was inhibited. 0.5 micromolar selumetinib which targets the MAP kinase pathway was effective in inhibiting the phosphorylation of ERK1/2. After watching this video you should have a good understanding of how to work with and critically analyze slices freshly cut from precious tumor tissues.
Our former postdoc, Katja Narhi, significantly contributed to the establishment of this method in collaboration with colleagues of the IMI Predect Consortium Tissue Slice Platform.
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