Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

All mouse experiments described in this study were performed by following the guidelines from the Finnish National Board of Animal Experimentation, and were approved by the Experimental Animal Committee of the University of Helsinki and the State Provincial Office of Southern Finland (License number ESAVI/9752/04.10.07/2015).

1. Preparations Prior to Slicing

1. Keep the following materials ready: vibratome specimen holder, vibratome buffer tray, vibratome ice bath, 10 cm culture plate, 24-well plate, 10 mL pipette, pipette boy, waste bag, 70% EtOH in a 50 mL tube to disinfect the instruments, and tissue glue.
2. Prepare the vibratome: wipe the blade holder with 70% EtOH, attach a new blade, and perform a vibrocheck according to instructions provided in the manual (http://photos.labwrench.com/equipmentManuals/10103-3895.pdf).
   NOTE: It is important to perform the vibrocheck step, as it minimizes the vertical deflection of the blade and ensures good quality slices.
3. Fill each well of the 24-well plate with 1 mL of Hanks Balanced Salt Solution (HBSS) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (HBSS + P/S); keep the plate on ice.
4. Prepare F12 culture medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM glutamax, 22 mM glucose, and 10% fetal bovine serum (FBS).
   NOTE: Tumor slice culture medium and growth factor supplements can vary depending on the tumor tissue²⁵.
5. Prepare the required amount of F12 medium for drug treatment, using the same composition as described in step 1.4, but omitting the FBS.
   NOTE: Since growth factors in the serum may affect oncogenic signaling in cultured slices, serum-free medium is recommended for short-term drug perturbation studies on tumor slices. If longer-term slice cultures are analyzed, it is important to first evaluate the effect of serum-free medium on tissue viability and tumor-specific marker expression in untreated slice cultures.

2. Collection of Tumor-bearing Lungs

1. Euthanize a tumor-bearing mouse by cervical dislocation when it shows symptoms of labored breathing and loss of body weight.
   NOTE: CO₂-mediated euthanasia is known to induce hypoxic conditions in the lungs, which may have an effect on slice viability or oncogenic activities via eliciting a hypoxic response.
2. Stretch the euthanized mouse onto a Styrofoam lid by inserting 25 G needles in all four paws, so that the chest is exposed. Spray 70% EtOH on the surface of the exposed skin.
3. 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 G needles. Cut open the abdominal region, followed by the rib cage and diaphragm, to expose the lung and heart.  Expose the trachea by cutting away the surrounding tissue. Keep the scissors in an angled position to avoid tissue damage.
4. Dissect the tumor-bearing lungs together with the heart and place them into a 50 mL tube containing 30 mL of ice-cold HBSS + P/S; keep the tube ice-cold and proceed to the next step as quickly as possible.
   NOTE: Delays in processing of lung tumors may alter oncogenic functions, for example signaling pathway activities.

3. Generation of Precision-cut Lung Tumor Slices

1. Transfer the tumor-bearing lungs in HBSS + P/S into a 10 cm tissue culture plate. Separate the lung lobes using sterile scissors and forceps, and select lobes with tumors on the surface for slicing.
   NOTE: Tumors >3 mm in size are suitable for slicing. Since normal lung tissue surrounding the big tumor compromises the slicing due to differences in tissue stiffness, tumor tissue undergoing slicing should be separated away from the normal tissue, such that a cleared tumor region faces the vibratome blade.
2. Place the selected lung lobe with a tumor tissue on a piece of filter paper to avoid slipping, and cut a part of the normal lung or additional tumor tissue that surrounds the tumor away with a sterile scalpel to generate a flat tissue piece surface.
   1. Dip the flat slide in a drop of cyanoacrylate adhesive. Mount this side onto the vibratome’s specimen holder so that the tumor faces the blade in an upright position. Let the glue dry for 2-3 min.
      NOTE: The normal lung tissue glued to the specimen holder does not interfere with tumor tissue slicing, and slicing is stopped before the normal tissue is reached. The tumor tissue sometimes bends due to the spongy texture of the normal lung tissue glued to the specimen holder, compromising its upright position. If this happens, glue a piece of additional normal lung support tissue next to the pre-mounted normal lung tissue to retain the tumor in an upright position (Figure 1A iii).
3. Place the specimen holder into the metal buffer tray and fill it with cold HBSS + P/S until the tissue is immersed in the buffer, and cover the buffer tray with the plexiglass lid that is provided with the instrument. Place the metal buffer tray onto the white ice bath and add ice so to keep the tissue cool while slicing.
4. Attach the white ice bath to the vibratome. Select suitable slicing settings: amplitude ranging between 2.5–2.8, slicing speed between 0.10-0.14 ms, and a cutting thickness ranging between 160–250 µm. Bring the vibratome blade to the slicing position, set the slicing window and start slicing.
   NOTE: Slicing settings need to be adjusted according to the hardness of the tissue. Hard tissue is easier to slice than softer tissue, and softer tissues requires slicing with lower speed (0.1–0.12 ms) and higher amplitude (2.6-2.8). A 4–5 mm large tumor typically provides 15–20 slices of 200 µM thickness. For short-term cultivation, murine tumors can be sliced under semi-sterile conditions outside the laminar hood. However, clinical tumors should always be sliced inside a class II biosafety laminar hood to avoid exposure to possible infectious agents in the human tissue.
5. Using sterile forceps, collect the slices in 1 mL of HBSS in separate wells of a 24-well plate held on ice, closely keeping track of the order in which slices are sliced. Mark each well of the 24-well plate according to the experimental plan. For example, mark sequential wells of a 24-well plate as culture time points or as 0 h, vehicle control (C), drug treated (T) (Figure 1B ii).
   NOTE: Do not disturb or pull the tumor tissue while collecting a slice, as this will alter the tumor’s orientation with respect to the angle of the blade, leading to inconsistencies in the thicknesses of the subsequent slices.
6. When all slices are collected, transfer them onto titanium grids (2-3 slices per grid) placed in a 6-well plate containing 2.5 mL of culture medium per well. Make sure that no air bubbles are formed between the titanium grid and the medium.
   1. To load a slice on to the grid, keep the 6-well plate in an angled position so that a portion of the medium covers the grid, and place the slice in the medium on the grid; use forceps to spread the slice if it curls. Load the 6-well plates onto the rotating incubation unit placed inside a humidified incubator maintained at 37 °C with 95% air and 5% CO₂, and start the rotation cycle (Figure 1C i-ii).
      NOTE: Metallic grids and 6-well plates need to be accurately weight balanced before turning on the rotating unit. It is important to position the slices in the middle of the grid so that they fully alternate between the air and liquid phases during the rotation cycles. Slices that are placed too low or high are not appropriately exposed to oxygen or nutrients (Figure 1C i), which can compromise tissue viability. It is important to follow the position of the slice on the grid during cultivation, as the slice can occasionally slide down. If this happens within 1–2 h of culture onset, correct its position and note down that this sample may be damaged. Slices that are mispositioned for an extended period of time should be discarded, as tissue viability is significantly affected by improper oxygenation and nutrient supply.
7. Collect a tissue slice adjacent to the cultured slices as a 0 h, uncultured, reference. Collect at least three reference slices to represent the top, middle and center of the tissue being sliced. Fix the 0 h slices immediately and process as described in 5.1.
   NOTE: If the number of slices is limited, e.g., if multiple compound treatments or technical replicates are done, comparisons of each treated sample with its neighboring 0 h sample can be difficult. In such cases, use the nearest 0 h slice (at least 400–600 µm apart) to assess relative tissue viability or expression of relevant markers at culture onset.
8. For long-term cultivation, replenish the culture medium every day. Lift the grid containing the tissue slices using sterile forceps, and place it in an empty well of the 6-well plate; replace 70% of the medium with fresh culture medium, and place the grid back in the medium. Continue the rotation cycle as explained in step 3.6.

4. Treatment of Tumor Slices with Small Molecule Inhibitors

1. Prepare the required concentrations of compounds in treatment medium. Typically, a 10-fold higher drug concentration compared to the IC50s measured in cell cultures is required to achieve target inhibition in tissue slices. To avoid unspecific cytotoxicity, test a range of concentrations to obtain minimally effective concentration for each compound. Here, 0.1–1 µM of the PI3K/mTOR inhibitor dactolisib and 0.05–0.5 µM of the MEK inhibitor selumetinib were tested on murine NSCLC slices.
2. Add 2.5 mL of media with the diluted drug or DMSO or other vehicle control into the 6-well plate. Place the titanium grids into the wells.
3. Place the tissue slices onto the grids as described in step 3.6.
4. Perform vehicle or drug treatments for 24 h. Proceed with tissue fixation and processing of the slices into paraffin blocks as described in section 5.
   NOTE: Duration of drug treatment can be optimized depending on the objective of the experiment, taking into consideration the ability of tissue slices to retain in situ tissue functions analyzed during the culture period.

5. Fixation and Processing of the Tissue Slices

1. Carefully lift the uncultured 0 h reference or cultured slice onto a filter paper soaked in PBS.
   1. To do this, add 2-3 mL of PBS on top of a filter paper placed in a 10 cm plate. Place the slice on top of the filter paper and lift out the filter paper using a pair of forceps.
   2. Transfer the filter paper into a histocassette, and add a drop of diluted hematoxylin (1:1 in deionized water) on top of the tissue slice to visibly mark the position of the slice during the subsequent processing steps (Figure 1D).
   3. Close the cassette, and transfer it into 4% neutral buffered formalin solution. Fix the tissues overnight at 4 °C.
      NOTE: While placing the slice on top of the filter paper, make sure that the top section of the slice is facing upwards; this is necessary to follow the top, middle, and bottom section of a slice during sectioning and analysis as described in step 6.3.
2. The next day, transfer the cassettes into 70% EtOH, and immediately proceed with the paraffin-embedding tissue processing step.
3. Prior to tissue processing, wash the histocasettes in 100% EtOH 2x for 10 min each. In this case, use a microwave station for tissue processing. Select the program used for 1 mm tissue thickness and follow the instructions provided in the manual (https://www.totaltissuediagnostics.com/images/MM073-005_-_KOS__Operator_Manual.pdf).
   NOTE: When using other tissue processing machines, use the program suitable for thin tissue samples.
4. For paraffin-embedding, open a histocassette and use a scalpel to carefully lift the slice from the filter paper. Discard the filter paper, and transfer the slice into a mold containing liquid paraffin.
   1. Press the tissue against the bottom of the embedding mold, for instance with a flat weight, to ensure even sectioning. Place the bottom part of the histocasette on top of the mold, add liquid paraffin on top of it. Let the mold cool on a cold pate for 30 min, and separate the mold from the paraffin block.
      NOTE: As an alternative to horizontal embedding, it is possible to embed the tumor slice vertically by positioning the slice in an upright position. Vertical sections readily permit analysis of gradients in viability or functional marker expression on a single section²⁵. Gradient analysis with horizontally-embedded slices requires paraffin sectioning as described in step 6.2.

6. Processing and Analysis of Formalin-fixed and Paraffin-embedded (FFPE) Tissues

1. Prepare 4 µm thin sections of the FFPE tissue slice blocks using a microtome. When sectioning, adjust the angle of the block so that the surface of the block is horizontally oriented with respect to the blade; this is necessary to obtain even sections throughout the tissue.
2. To enable capture of a potential culture-induced viability gradient, cell migration across the slices, or gradients in biomarker expression across cultured slices, collect sections from the top, center and bottom layers of each of the tissue slice on object slides as explained below.
3. Collect sequential tissue sections of the paraffin-embedded tissue slice first on the upper part of the glass slides. Continue collecting the sections of the deeper tissue layers to the middle followed by bottom part of the glass slides (Figure 1E).
   NOTE: To accommodate three sections on a glass slide, trim away the excess of paraffin surrounding the embedded tissue slice.
4. Allow the sections to dry overnight at 37 °C, and proceed with H&E staining or immunohistochemistry as described below.
   NOTE: Loss of antigenicity can occur when FFPE sections are stored at high temperatures or for extended periods of time. Paraffin sections are recommended to be stored at 4 °C, and IHC analyses should be carried out within 6 months after sectioning.
   1. For H&E staining, deparaffinize and rehydrate the paraffin sections as follows: xylene 3x for 5 min, 100% EtOH 3x for 1 min, 96% EtOH 2x for 1 min, 70% EtOH 1 x 1min, and deionized water 2x for 1 min.
   2. Incubate the sections in freshly filtered hematoxylin solution for 10 min, and wash under running tap water for 5 min. Dip the sections in acid alcohol (1% HCl in 70% EtOH) for 2 times, and wash under running tap water for 5 min followed by incubation with 0.5% eosin for 2 min.
   3. Following the eosin step, dehydrate the sections by immersing the slides in alcohol and xylene solutions, as follows: 96% EtOH 2x for 15 s, 100% EtOH 3x for 30 s, xylene 3x for 1 min. Finally, embed the sections in a xylene-based mounting medium.

7. Analysis of Tissue Viability and Biomarker Expression

1. Acquire high resolution images by generating whole slide scans of H&E-stained slides using a scanner. To assess tissue viability, take snapshots from the tissue scans representing the top, middle and bottom sections of the slices.
   1. Using photo manipulator software, manually draw masks on the necrotic areas of the tissues, followed by quantification of necrotic regions using MATLAB.
   2. Calculate the relative viability of the slices cultivated for different time points compared to the nearest 0 h slice as done in our previous study (Närhi et al., Figure S2B and C)²⁶. Similarly, assess potential intra-slice viability gradients by quantifying viability in the top, middle and bottom section of a cultured slice, and calculate the relative viability of each section to its closest 0 h slice.
      NOTE: While the mere cultivation of murine NSCLC slices induces necrotic cell death, biological responses to ex vivo culture conditions vary depending on the tumor tissue. For example, gradients in HIF1α, ER, and macrophages marked by F4/80 were detected in filter-supported slice cultures derived from a breast cancer model²⁵. Therefore, H&E- as well as IHC-based analyses of cultured slices need to be considered for each tissue type.
2. Perform IHC on the paraffin sections. The following IHC protocol is a starting point, and requires further optimization for other antibodies. Briefly, deparaffinize and rehydrate the paraffin sections as follows: xylene 3x for 5 min, 100% EtOH 3x for 1 min, 96% EtOH 2x for 1 min, 70% EtoH 1x for 1 min, and deionized water 2x for 1 min.
   1. To expose the antigenic epitopes, perform heat-mediated antigen retrieval using 10 mM citric acid at pH 6 in a PT module, followed by blocking with 1% Bovine Serum Albumin (BSA) and 10% Normal Goat Serum (NGS) in 1x PBS for 30 min at ambient temperature (21-23 °C).
   2. Incubate the primary antibody diluted in 1% BSA and 5% NGS in 1x PBS, either for 1–2 h at ambient temperature or overnight at 4 °C. Incubate with anti-rabbit secondary antibody, for 30 min at ambient temperature, followed by detection using 3,3′-Diaminobenzidine (DAB).
   3. Sections are counterstained with hematoxylin (diluted to 1:10 in deionized water) for 30 s, and wash under tap water for 5 min, followed by dehydration by immersing the slides in alcohol and xylene solutions, as follows: 70% EtOH 1x, 96% EtOH 2x, 100% EtOH 3x, xylene 3x (1 min each step). Finally, embed the sections in a xylene-based mounting medium.
3. Acquire whole slide scans of IHC-stained slides,  using an image viewer export them as TIFF images at a magnification that captures the whole tissue region to be analyzed, and perform the quantifications using ImageJ.
   NOTE: As an alternative to the scanner, microscopic images at 20X or 40X magnification can be acquired for quantifications. Image magnification can be chosen depending on the marker to be quantified; high magnification images are recommended for nuclear staining, while cytoplasmic or membrane markers can be quantified using 20X images.
   1. For quantification of nuclear markers, in this case NKX2-1 expression, convert each image to 16-bit images using Fiji-ImageJ, and upload images for image analysis.
   2. Customize the image analysis pipeline depending on the analysis. In this protocol, use the following pipeline for the quantification of NKX2-1 positive nuclei: Identify Primary Objects | Measuring Object Intensity | Filter Objects (minimal value=0.0025) | Calculate Math. The results are represented as percentages of DAB-stained nuclei of the total number of nuclei.