This research work received financial support from Innovative Medicines Initiative Joint Undertaking grant agreement no 115188, the University of Helsinki Doctoral Programme in Biomedicine scholarships (A.S.N.), and the Sigrid Juselius and Orion-Farmos Foundations (E.W.V.). We sincerely thank our PREDECT tissue slice platform consortium members (www.predect.eu), namely John Hickman, Heiko van der Kuip, Meng Dong, Emma Davies, Simon Barry, Wytske van Weerden and Hanneke van Zoggel, for collaborative development of the slice technology. We thank Taija af H&#228;llstr&#246;m and Siv Knaappila for support in setting up the rotator system, and Riku Turkki for support with the MATLAB analysis.&#160;Jouko Siro is thanked for capturing the Figure 1 photographs. We thank the FIMM WebMicroscope team for scanning histological slides, and the Laboratory Animal Centre for husbandry support.

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;g/mL streptomycin (HBSS + P/S); keep the plate on ice.</li>
	<li>Prepare F12 culture medium supplemented with 100 U/mL penicillin, 100 &#181;g/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.</li>
	<li>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.</li>
</ol>
2. Collection of Tumor-bearing Lungs
<ol>
	<li>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.</li>
	<li>Stretch the euthanized mouse onto a Styrofoam lid by inserting 25&#160;G needles in all four paws, so that the chest is exposed.&#160;Spray 70% EtOH on the surface of the exposed skin.</li>
	<li>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.&#160; Expose the trachea by cutting away the surrounding tissue. Keep the scissors in an angled position to avoid tissue damage.</li>
	<li>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.</li>
</ol>
3. Generation of Precision-cut Lung Tumor Slices
<ol>
	<li>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 &#62;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.</li>
	<li>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.
	<ol>
		<li>Dip the flat slide in a drop of cyanoacrylate adhesive. Mount this side onto the vibratome&#8217;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).</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Attach the white ice bath to the vibratome. Select suitable slicing settings: amplitude ranging between 2.5&#8211;2.8, slicing speed between 0.10-0.14 ms, and a cutting thickness ranging between 160&#8211;250 &#181;m.&#160;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&#8211;0.12 ms) and higher amplitude (2.6-2.8). A 4&#8211;5 mm large tumor typically provides 15&#8211;20 slices of 200 &#181;M 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.</li>
	<li>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&#8217;s orientation with respect to the angle of the blade, leading to inconsistencies in the thicknesses of the subsequent slices.</li>
	<li>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.
	<ol>
		<li>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 &#176;C with 95% air and 5% CO2, and start the rotation cycle (Figure 1C i-ii). 
		NOTE: Metallic grids and&#160;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&#8211;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.</li>
	</ol>
	</li>
	<li>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&#8211;600 &#181;m apart) to assess relative tissue viability or expression of relevant markers at culture onset.</li>
	<li>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.</li>
</ol>
4. Treatment of Tumor Slices with Small Molecule Inhibitors
<ol>
	<li>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,&#160;0.1&#8211;1 &#181;M of the PI3K/mTOR inhibitor dactolisib and 0.05&#8211;0.5 &#181;M of the MEK inhibitor selumetinib were tested on murine NSCLC slices.</li>
	<li>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.</li>
	<li>Place the tissue slices onto the grids as described in step 3.6.</li>
	<li>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.</li>
</ol>
5. Fixation and Processing of the Tissue Slices
<ol>
	<li>Carefully lift the uncultured 0 h reference or cultured slice onto a filter paper soaked in PBS.
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Close the cassette, and transfer it into 4% neutral buffered formalin solution. Fix the tissues overnight at 4 &#176;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.</li>
	</ol>
	</li>
	<li>The next day, transfer the cassettes into 70% EtOH, and immediately proceed with the paraffin-embedding tissue processing step.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Processing and Analysis of Formalin-fixed and Paraffin-embedded (FFPE) Tissues
<ol>
	<li>Prepare 4 &#181;m 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Allow the sections to dry overnight at 37 &#176;C, and proceed with H&#38;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 &#176;C, and IHC analyses should be carried out within 6 months after sectioning.
	<ol>
		<li>For H&#38;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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
7. Analysis of Tissue Viability and Biomarker Expression
<ol>
	<li>Acquire high resolution images by generating whole slide scans of H&#38;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.
	<ol>
		<li>Using photo manipulator software, manually draw masks on the necrotic areas of the tissues, followed by quantification of necrotic regions using MATLAB.</li>
		<li>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 (N&#228;rhi 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 HIF1&#945;, ER, and macrophages marked by F4/80 were detected in filter-supported slice cultures derived from a breast cancer model25. Therefore, H&#38;E- as well as IHC-based analyses of cultured slices need to be considered for each tissue type.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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 &#176;C).</li>
		<li>Incubate the primary antibody diluted in 1% BSA and 5% NGS in 1x PBS, either for 1&#8211;2 h at ambient temperature or overnight at 4 &#176;C. Incubate with anti-rabbit secondary antibody, for 30 min at ambient temperature, followed by detection using 3,3&#8242;-Diaminobenzidine (DAB).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Acquire whole slide scans of IHC-stained slides,&#160; 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.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bruin, E. C., McGranahan, N., Swanton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+intratumor+heterogeneity+unravels+lung+cancer+evolution.">Analysis of intratumor heterogeneity unravels lung cancer evolution.</a> Molecular and Cellular Oncology. 2 (3), e985549 (2015).</li><li>Travis, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2015+World+Health+Organization+Classification+of+Lung+Tumors:+Impact+of+Genetic,+Clinical+and+Radiologic+Advances+Since+the+2004+Classification.">The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification.</a> Journal of Thoracic Oncology. 10 (9), 1243-1260 (2004).</li><li>Junttila, M. R., de Sauvage, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+tumour+micro-environment+heterogeneity+on+therapeutic+response.">Influence of tumour micro-environment heterogeneity on therapeutic response.</a> Nature. 501 (7467), 346-354 (2013).</li><li>Kredel, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Culture+of+Intracranial+Tumors+with+a+Note+on+the+Meningiomas.">Tissue Culture of Intracranial Tumors with a Note on the Meningiomas.</a> The American Journal of Pathology. 4 (4), 337-340 (1928).</li><li>Wright, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+investigation+of+the+relation+between+the+clinical+and+tissue+culture+response+to+chemotherapeutic+agents+on+human+cancer.">Further investigation of the relation between the clinical and tissue culture response to chemotherapeutic agents on human cancer.</a> Cancer. 15, 284-293 (1962).</li><li>Roller, M. R., Owen, S. P., Heidelberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+organ+culture+of+human+tumors.">Studies on the organ culture of human tumors.</a> Cancer Research. 26 (4), 626-637 (1966).</li><li>Reinbold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Organotypic+differentiation+of+the+eye+of+the+chick+embryo+in+vitro].">[Organotypic differentiation of the eye of the chick embryo in vitro].</a> Comptes rendus des s&#233;ances de la Soci&#233;t&#233; de biologie et de ses filiales. 148 (15-18), 1493-1495 (1954).</li><li>Loffredo Sampaolo, C., Sampaolo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Organotypic+cultures+of+chick+embryo+lung;+some+histologic+and+histochemical+aspects].">[Organotypic cultures of chick embryo lung; some histologic and histochemical aspects].</a> Bollettino della Societa Italiano di Biologia Sperimentale. 32 (7-8), 797-801 (1956).</li><li>Bousquet, J., Meunier, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Organotypic+culture,+on+natural+and+artificial+media,+of+fragments+of+the+adult+rat+hypophysis].">[Organotypic culture, on natural and artificial media, of fragments of the adult rat hypophysis].</a> Comptes rendus des s&#233;ances de la Soci&#233;t&#233; de biologie et de ses filiales. 156, 65-67 (1962).</li><li>Merz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+of+human+glioblastoma+reveal+different+susceptibilities+to+treatments.">Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.</a> Neuro-Oncology. 15 (6), 670-681 (2013).</li><li>Gerlach, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+cultures+from+head+and+neck+squamous+cell+carcinoma:+a+novel+test+system+for+drug+susceptibility+and+mechanisms+of+resistance.">Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.</a> The British Journal of Cancer. 110 (2), 479-488 (2014).</li><li>Estes, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+anti-death+receptor+5+(DR5)+antibody+(TRA-8)+against+primary+human+ovarian+carcinoma+using+a+novel+ex+vivo+tissue+slice+model.">Efficacy of anti-death receptor 5 (DR5) antibody (TRA-8) against primary human ovarian carcinoma using a novel ex vivo tissue slice model.</a> Gynecologic Oncology. 105 (2), 291-298 (2007).</li><li>Kuip, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+term+culture+of+breast+cancer+tissues+to+study+the+activity+of+the+anticancer+drug+taxol+in+an+intact+tumor+environment.">Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment.</a> The British Journal of Cancer. 6, 86 (2006).</li><li>Vaira, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> Proceedings of the National Academy of Sciences. 107 (18), 8352-8356 (2010).</li><li>Zimmermann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+reproducibility+in+preparing+precision-cut+liver+tissue+slices.">Improved reproducibility in preparing precision-cut liver tissue slices.</a> Cytotechnology. 61 (3), 145-152 (2009).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+tissue+slices:+preparation,+handling,+and+successful+optical+mapping.">Cardiac tissue slices: preparation, handling, and successful optical mapping.</a> American journal of physiology. Heart and circulatory physiology. 308 (9), H1112-H1125 (2015).</li><li>Jaamaa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+recognition+via+activated+ATM+and+p53+pathway+in+nonproliferating+human+prostate+tissue.">DNA damage recognition via activated ATM and p53 pathway in nonproliferating human prostate tissue.</a> Cancer Research. 70 (21), 8630-8641 (2010).</li><li>Naipal, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+slice+culture+system+to+assess+drug+response+of+primary+breast+cancer.">Tumor slice culture system to assess drug response of primary breast cancer.</a> The British Journal of Cancer. 16, 78 (2016).</li><li>Gahwiler, B. H., Thompson, S. M., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+maintenance+of+organotypic+slice+cultures+of+CNS+tissue.">Preparation and maintenance of organotypic slice cultures of CNS tissue.</a> Current protocols in neuroscience. , (2001).</li><li>Alfaqeeh, S. A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+culture+method+for+following+development+of+tooth+germs+in+explant+culture.">The slice culture method for following development of tooth germs in explant culture.</a> Journal of Visualized Experiments. 10 (81), (2013).</li><li>Rak-Raszewska, A., Hauser, P. V., Vainio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+In+Vitro+Culture:+What+Have+We+Learned+about+Early+Kidney+Development?.">Organ In Vitro Culture: What Have We Learned about Early Kidney Development?.</a> Stem Cells International. , 959807 (2015).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental Cell Research. 16 (1), 118-147 (1959).</li><li>Gahwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures:+a+technique+has+come+of+age.">Organotypic slice cultures: a technique has come of age.</a> Trends in Neurosciences. 20 (10), 471-477 (1997).</li><li>Kiviharju-af Hallstrom, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+prostate+epithelium+lacks+Wee1A-mediated+DNA+damage-induced+checkpoint+enforcement.">Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement.</a> Proceedings of the National Academy of Science. 104 (17), 7211-7216 (2007).</li><li>Davies, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+complex+tumour+biology+in+vitro:+histological+and+molecular+characterisation+of+precision+cut+slices.">Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices.</a> Scientific Reports. 5, 17187 (2015).</li><li>Narhi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+aspects+of+oncogenic+signalling+determine+the+response+to+combination+therapy+in+slice+explants+from+Kras-driven+lung+tumours.">Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras-driven lung tumours.</a> The Journal of Pathology. 245 (1), 101-113 (2018).</li><li>Majumder, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+clinical+response+to+anticancer+drugs+using+an+ex+vivo+platform+that+captures+tumour+heterogeneity.">Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity.</a> Nature Communications. 6, 6169 (2015).</li><li>Nagaraj, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+of+Origin+Links+Histotype+Spectrum+to+Immune+Microenvironment+Diversity+in+Non-small-Cell+Lung+Cancer+Driven+by+Mutant+Kras+and+Loss+of+Lkb1.">Cell of Origin Links Histotype Spectrum to Immune Microenvironment Diversity in Non-small-Cell Lung Cancer Driven by Mutant Kras and Loss of Lkb1.</a> Cell Reports. 18 (3), 673-684 (2017).</li><li>Lv, D., Hu, Z., Lu, L., Lu, H., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+A+powerful+tool+in+tumor+research+and+drug+discovery.">Three-dimensional cell culture: A powerful tool in tumor research and drug discovery.</a> Oncology Letters. 14 (6), 6999-7010 (2017).</li><li>Weeber, F., Ooft, S. N., Dijkstra, K. K., Voest, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Organoids+as+a+Pre-clinical+Cancer+Model+for+Drug+Discovery.">Tumor Organoids as a Pre-clinical Cancer Model for Drug Discovery.</a> Cell Chemical Biology. 24 (9), 1092-1100 (2017).</li><li>Hoogt, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+and+characterization+data+for+2D,+3D,+and+slice-based+tumor+models+from+the+PREDECT+project.">Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project.</a> Scientific Data. 4, 170170 (2017).</li><li>Davidson, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+Impacts+the+Metabolic+Dependencies+of+Ras-Driven+Non-Small+Cell+Lung+Cancer.">Environment Impacts the Metabolic Dependencies of Ras-Driven Non-Small Cell Lung Cancer.</a> Cell Metabolism. 23 (3), 517-528 (2016).</li></ol>

Authors declare no conflict of interest.

Various complex in vitro tumor models, including 3D cultures and organoids, have been developed to recapitulate the architecture and oncogenic functions of in vivo tumor tissue29,30. However, the establishment of 3D cultures or organoids involves tissue dissociation and selective growth of a single cell type or co-culture of a select few cell types in an artificial environment. As a consequence, such models incompletely capture the intricacies of tumor heterogeneity and tumor-stroma interactions. Organotypic tumor slices, on the other hand, maintain the tissue architecture and biological complexities of the in situ tumor, without extensive manipulation. This ability of tissue slices to model tumor cells in their native microenvironment renders them particularly attractive for preclinical studies. We previously reported an optimized workflow for the establishment and analysis of precision-cut tumor slices, and showed that, compared to filter supports, a rotating incubation unit improve the viability of short-term murine NSCLC slice cultures25,31. However, cultivation on rotating units is technically challenging and requires constant monitoring. We here present a protocol for tumor tissue slice generation and practical use of a rotating incubation unit to culture them, as well as accompanying methods to monitor the ability of slices to capture in situ tumor biology, a prerequisite prior to drug response testing.
Several critical steps in the protocol ensure tissue integrity and viability of the tumor slices. If normal lung tissue surrounds the tumor, the vibratome can generate slices with inconsistent thickness or damage the slices, due to differences in texture and stiffness between normal and tumor tissues. It thus is important to remove the surrounding normal lung tissue prior to tumor slicing. Another critical step is the slicing thickness, which should be carefully optimized for each tissue type. Furthermore, once sliced, it is critical that the slice is placed approximately in the middle of the grid, so to ensure accurate intermittent dipping in culture medium and oxygen exposure. Finally, it is important to follow the position of a slice during its rotation period, as a slice can drop down in to the medium; if this happens, further actions can be taken as explained in step 3.6 of the protocol.
In addition to tissue handling to assure integrity, there are also critical steps in the IHC analysis to interpret how the slice resembles the native tissue. Our previous study showed that murine NSCLC tumors exhibit pronounced intra-tumor spatial heterogeneity in oncogenic signaling activities26. This means that the use of spatially distinct tissue slices for controls or drug-treated samples can affect reliable experimental data interpretation, and hence closely adjacent slices should be used as controls and test samples. We further showed that while proliferation or oncogenic phosphoprotein expression in freshly cut uncultured 0 h slices were similar to in situ tumors, cultured slices showed altered oncogenic phosphoprotein expression, specifically altered p4EBP1 and pSRC, as well as altered proliferation analyzed by Ki67 IHC. Altered p4EBP1 expression was similarly detected in 24 h human NSCLC and prostate cancer slice cultures (Narhi et al., Supplementary Figure S3B-S3C, S5 and S7)26. These findings endorse that comparison of cultured slices with their nearest 0 h uncultured slice is critical to assess the preservation of in situ tumor functions in cultured slices.
Despite improving the viability of organotypic slices25, there are limitations with the rotator system in terms of technicalities. Placing the tissue slices onto titanium grids is more challenging compared to filter inserts, and a rotating incubation unit may not be available. As an alternative, stagnant filter supports can be used, but in that case only the air-exposed side of the slices should be analyzed, as air-to-filter gradients in viability and hypoxia measured by HIF1&#945; expression are rapidly formed in filter-supported slice cultures (Davies et al., Figure 5 and Figure 7A-7B)25. We have further shown that tumor slice cultures can exhibit altered proliferation and oncogenic signaling activities compared to their native tumors26, possibly because of wound-healing responses or metabolic adaptation of the slices to ex vivo culture32. Although gross morphological features of the murine NSCLC tumors were maintained during 72 h cultivation, culture-induced proliferative changes may affect accurate grading of the cultivated slices. Thus, tissue slices should only be utilized for short-term functional studies.
Use of a rotating incubation unit at least partially rescues intra-slice viability or biomarker expression gradients, particularly during the first 24 h of culture. Once validated for integrity and function, this provides tissue material for functional studies, such as drug treatment studies. In addition to drug response profiling, altered target expression following drug treatment can also benefit antibody validation. This is particularly relevant for the detection of murine epitopes with mouse monoclonal antibodies, as these tend to give high staining background. In addition, well-validated antibodies are required to achieve reliable and reproducible data in diagnostic and clinical settings. Thus, modulation of the abundance or phosphorylation of relevant epitopes following drug treatment in tissues slices provides a handy practical application in antibody validation. A major advantage of tumor slice cultures is the ability to model spatially-distributed functions, including oncogenic signaling activities or drug response in tumor or stromal cells, which makes them an attractive ex vivo model. However, slices rotate during cultivation, and the process of cultivation can further damage the tissue particularly at the edges. It is therefore challenging to precisely overlay the biomarker-stained IHC images of 0 h slices with the necrotic regions detected in cultured slices, which compromises the ability to precisely link spatial biomarker activities to drug response. In addition, tumor-intrinsic, culture-induced and drug-induced necrotic responses are indistinguishable at least in murine NSCLC tissue slices, compromising accurate quantitation of spatial drug responses. Finally, the use of tumor slice cultures permits a researcher to the test multiple compounds on the same tumor, without a need to treat animals, thus refining, reducing, and replacing experiments on laboratory animals.
As a future application, the described protocol can be adopted to clinical solid tumor samples. Further tissue type-dependent modifications or optimizations are likely required, starting with adjustments to the vibratome settings including slice thickness and vibration speed to optimize these for tumor texture or stiffness. In addition, nutrient and growth factor requirement may vary for different tumor tissues. As an example, breast cancer slices have been cultured with insulin supplemented in the medium13,18. Given that limited tissue material is obtained during surgery or biopsy, optimization of patient-derived tumor slice cultures can be challenging due to difficulties in obtaining sufficient numbers of replicate samples. Furthermore, data reproducibility is also challenged by pronounced patient-to-patient sample heterogeneity, particularly in the percentage of tumor cells versus fibrotic regions or stromal infiltrates, as well as necrotic tissue components. Finally, application of tumor slices in diagnostic settings would require investigation of the extent to which drug responses in slice explants of pre-treatment biopsies matches to post-treatment in vivo responses.

Solid tumor tissues, including lung cancer, exhibit genetic and phenotypic heterogeneity, and harbor complex microenvironments1,2. The interplay between tumor cells and their surrounding microenvironment influence on drug sensitivity and resistance mechanisms3. This highlights the need for preclinical models that can accurately model biological complexities and functions acting in native tumors. Precision-cut slices immediately derived from fresh tumors provide a unique resource, as they have a principal capacity to represent in vivo biology, at least for a short window of time, including phenotypes spatially distributed in an individual tumor. Resected clinical tumors are one of the few personalized specimens that can be obtained from a cancer patient, and their diagnostic use deserves scrutiny.
The history of organotypic cultures dates back to the early 19th century, when human intracranial tumors were hand-cut into tissue pieces and cultured using the so-called hanging drop method. Tissue fragments were attached to a coverslip and allowed to dip into heparinized human plasma, after which the coverslips were inverted, sealed and cultured for several weeks4. Manually cut tumor pieces have since been cultured using a variety of other methods, such as on plasma clots5, in liquid media6, or on 0.45 &#181;m pore size filters6. The term &#34;organotypic&#34; was first used in 1954, in a study on retinal differentiation of the chick embryo eye7. This was followed by studies that used lung and heart tissue explants derived from chick embryos8, and brain explants from adult rats9.
Various slicing methods have been described, namely manual choppers10,11, the Krumdieck tissue slicer12,13, and vibratomes11,14,15,16. The Krumdieck tissue slicer generates cylindrical tissue cores, which are then sliced into circular tissue slices using a microtome. A vibratome, on the other hand, uses a vibrating blade microtome. In a study on liver slices, it was shown that the Leica vibratome generates more reproducible and consistent slices compared with the Krumdieck slicer15. Slice thicknesses ranging from 250 to 500 &#181;m have been used, and studies report maintenance of viability and morphological features even until 16 days14,10,17,18. However, tumors have variable metabolic profiles that can affect nutrient requirements, and parameters such as tissue stiffness and matrix composition can influence on aeration and nutrient flow. It is therefore likely that each tissue type requires optimization of slicing and culture conditions.
Different cultivation methods have been used to support slice cultures: i) stationary interphase culture, also called stagnant support culture, in which slices are placed on top of a semi-porous membrane insert immersed in the culture medium. In this, the top of the slices is exposed to the air, while the bottom is supplemented with nutrients via the porous insert14,19; ii) the Trowel method, originally developed to culture whole organs or embryonic tissue slices. In this, slices are placed on top of a cotton sheet or filter supported by a metal grid, and the filter is soaked in the culture medium. To keep the tissues moist, a thin layer of medium is added on top of the slices20,21,22. These first two are so called air-liquid interphase cultures; iii) roller tube cultures, in which slices are placed inside the flat side of a plastic tube containing medium, and slow tube rotation ensures that the tissue is covered with medium during the first part of a cycle, or aerated during the second23; iv) rotating incubation units, in which slices are intermittently exposed to medium with nutrients and aeration. Different from roller tubes, in this method slices are placed on top of porous titanium grids placed in 6-well plates with culture medium24.
Tissue slices derived from resected solid tumors logically present an attractive ex vivo model in which to test the treatment response of anti-cancer agents, as they permit the evaluation of viability, targeted pathway activity, and molecular profiles of a specific tumor in the presence of its native tumor microenvironment. However, to evaluate whether the drug responses measured in tumor slices are predictive of in situ responses, it is important to assess to what extent tissue slices preserve tumor-specific biological functions such as cell proliferation, histopathology-specific cell differentiation or oncogenic signaling activities. The impact of mechanical stress elicited during slice preparation, slice handling, or culture-induced adaptions on both the quality and biological functions of tissue slices are fundamental questions, tightly linked to the ability to implement tumor-derived slices for functional diagnostics.
Our IMI-funded consortium project PREDECT (http://www.predect.eu) set&#160;out to systematically address these fundamental questions, by studying slice explants from a variety of sources. Using slices derived from breast, prostate and lung cancer models, this joint effort utilized qualitative read-outs as well as quantitative hematoxylin and eosin (H&#38;E) - based readouts to demonstrate a requirement for atmospheric oxygen and stagnant filter supports to sustain the viability of cultured slices until 72 h. Furthermore, immunohistochemistry (IHC) analyses on cultured slices revealed intra-slice viability gradients, evidenced as necrosis gradients in slices derived from murine non-small cell lung cancer (NSCLC), estrogen receptor (ER), HIF1&#945; and &#947;H2AX gradients in breast cancer slices, or androgen receptor (AR) expression gradients in prostate cancer slices25. Interestingly, intra-slice viability gradients in 24 h cultures of murine NSCLC were rescued by cultivation in a rotating incubation unit, and our recent study showed that viability was extended to 72 h26. Particularly the top side remained most viable26, endorsing that drug response analyses on slices are best carried out on this side of the tissue.
Even though it remains a question as to how far tissue slices can recapitulate in situ tumor functions, they have been extensively used to test responses to anti-cancer agents, including targeted drugs, monoclonal antibodies and chemotherapy agents10,11,13,14,18,27. We recently showed that murine NSCLC slices show dynamic changes in proliferation and oncogenic signaling activities following cultivation when compared to freshly cut 0 h slices26. This indicates that it is important to investigate whether the targeted in situ biological functions are appropriately preserved during cultivation, prior to perturbation studies. Despite these findings, we showed that tumor slices can model spatial response to targeted therapies, midst drug treatments remain brief (24 h) and are initiated at the onset of culturing26. The following protocol describes important validation aspects relevant to the establishment and analysis of tumor slice cultures, prior to their application in pharmacological drug testing.

<table border="0" cellpadding="0" cellspacing="0" style="width:1091px;" width="1089">
	<tbody>
		<tr height="24">
			<td height="24" style="height:25px;width:486px;">Hank&#8217;s Balanced salt solution (HBSS)</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:176px;">H6648</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Penicillin Streptomycin solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Ham&#39;s F-12 medium</td>
			<td>Thermo Fischer Scientific</td>
			<td>21765-037</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Glucose</td>
			<td>VWR</td>
			<td>101174Y</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">FBS</td>
			<td>Thermo Fischer Scientific</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Glutamine 200mM</td>
			<td>Thermo Fischer Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Cyanoacrylate adhesive (GLUture)</td>
			<td>Abbott</td>
			<td>32046-90-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Leica VT1200 S vibrating blade microtome</td>
			<td>Leica Biosystems</td>
			<td>14048142066</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Slicing blade</td>
			<td>VWR</td>
			<td>PERS60-0138</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Titanium grids</td>
			<td>Albamma Research and Development</td>
			<td>MA0036</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Slice incubation unit</td>
			<td>Albamma Research and Development</td>
			<td>MD2500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">10 cm tissue culture plate</td>
			<td>Sarstedt</td>
			<td>83.1802</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">24-well plate</td>
			<td>Sarstedt</td>
			<td>83.1836</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">6-well plate</td>
			<td>Sarstedt</td>
			<td>83.1839</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Neolus Hypodermic Needles</td>
			<td>Terumo Neolus</td>
			<td>NN2525R</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">50 mL falcon tube</td>
			<td>Greiner&#160;</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">PBS</td>
			<td>Lonza</td>
			<td>BE17-517Q</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Formaldehyde</td>
			<td>Fisher</td>
			<td>F/1501/PB08</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Trifold histo cassette paper</td>
			<td>Cancer Diagnostics</td>
			<td>DX26280</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Histo cassettes</td>
			<td>VWR</td>
			<td>720-2199&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">KOS The microwave multifunctional tissue processor</td>
			<td>Milestone SRL</td>
			<td>CAT307EN-003</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Microtome</td>
			<td>Thermo Fischer Scientific</td>
			<td>HM355S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Superfrost Ultra plus slides</td>
			<td>VWR</td>
			<td>631-0099</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">NGS</td>
			<td>Thermo Fischer Scientific</td>
			<td>16210-064</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Citric acid</td>
			<td>Sigma</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">PT-Module</td>
			<td>Thermo Fischer Scientific</td>
			<td>A80400012</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Hematoxylin for H&#38;E staining</td>
			<td>Merck</td>
			<td>1.09249.0500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Hematoxylin for counter staining</td>
			<td>Dako</td>
			<td>S3309</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Eosin</td>
			<td>Sigma</td>
			<td>E4382</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">NKX2-1 antibody</td>
			<td>Abcam</td>
			<td>ab133638</td>
			<td>Lot Number: GR98031-12</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">pERK 1/2 antibody</td>
			<td>Cell Signaling Technologies</td>
			<td>CST 4370</td>
			<td>Lot Number: 17</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">p4EBP1 antibody</td>
			<td>Cell Signaling Technologies</td>
			<td>CST 2855</td>
			<td>Lot Number: 20</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">BrightVision poly-HRP Goat anti-rabbit secondary antibody</td>
			<td>ImmunoLogic</td>
			<td>VWRKDPVR-110HRP</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Mounting medicum pertex</td>
			<td>VWR</td>
			<td>MEDT41-4021-00</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">DAB&#160;</td>
			<td>ImmunoLogic</td>
			<td>VWRKBSO4-110</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Dactolisib (NVP BEZ-235)</td>
			<td>Selleckchem</td>
			<td>S1009</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Selumetinib (AZD2644)</td>
			<td>Selleckchem</td>
			<td>S1008</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Pannoramic 250 slide scaner</td>
			<td>3DHISTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td><a href="https://se.mathworks.com/products/matlab.html">https://se.mathworks.com/products/matlab.html</a></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">3DHISTECH PANNORAMIC VIEWER&#160;</td>
			<td>3DHISTECH</td>
			<td></td>
			<td>https://www.3dhistech.com/pannoramic_viewer</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Adobe Photoshop CS6</td>
			<td>Adobe</td>
			<td></td>
			<td>https://www.adobe.com/products/photoshop.html?sdid=KKQIN&#38;mv=search&#38;s_kwcid=AL!3085!3!247821564908!e!!g!!adobe%20photoshop&#38;ef_id=U8T1GwAABVTK3gDR:20180712103259:s</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">Fiji-ImageJ</td>
			<td>ImageJ</td>
			<td></td>
			<td>https://imagej.net/Fiji</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">CellProfiler</td>
			<td>CellProfiler cell image analysis software</td>
			<td></td>
			<td>http://cellprofiler.org/</td>
		</tr>
	</tbody>
</table>

establishment and analysis of tumor slice explants as a prerequisite for diagnostic testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

Figure 1 represents the workflow for the generation, cultivation and analysis of precision-cut tissue slices derived from murine NSCLC tumors. For this demonstration, we utilized tumors from a genetically engineered mouse model (GEMM) harboring conditional activation of KrasG12D together with the loss of Lkb1 (also known as Serine/Threonine Kinase 11), hereafter called KL. Mouse breeding and lung tumor initiation was performed as described in26,28. Figure 2A demonstrates the effect of tissue slice thickness on the viability of slices cultured for 24 h using a rotating incubation unit. The results show that 160 &#181;m thin slices contain large necrotic areas across the slice. In addition, 250 &#181;m thin slices show a necrosis gradient across the slice compared to 200 &#181;m thin slices. It is likely that the poor overall viability of the thinnest 160 &#181;m is caused by technical handling during positioning of the slices on top of the grids, as these are fragile and tend to curl. On the other hand, when slices are too thick, they can become prone to deficiencies in oxygen or nutrient diffusion across the slices, which in murine NSCLC explants is evidenced as necrotic death gradient25. However, it should be noted that slices with variable thicknesses can be generated from one tumor, despite use of identical vibratome settings. It is therefore recommended to analyze multiple replicates from different tumor samples. Importantly, each tissue type requires slice thickness optimization to achieve maximum viability, as the tissue texture and hardness can affect oxygenation and nutrient flow. Figure 2B-2C illustrates quantitative IHC analyses of NKX2-1 expression, a marker of well-differentiated lung adenocarcinoma (AC) in samples cultivated up to 72 h and matched 0 h slices. Results show that NKX2-1 expression is not significantly altered in cultured slices as compared to 0 h uncultured slices, suggesting that the process of cultivation does not overtly affect the differentiation status of AC tumor tissue. Figure 2D demonstrates the utility of tumor tissue slices for assessing the effectiveness of targeted drugs. We recently showed that Kras mutant murine ACs exhibit high expression of phosphorylated ERK1/2 (marking increased MAPK pathway activity) when compared to adenosquamous (ASC) tumors, while expression of phosphorylated 4EBP1 (marking mTOR activity) is similarly expressed in both AC and ASC tumors26. To test if these pathways can be effectively targeted on tissue slices, KL AC tissue slices were treated with DMSO or titrated amounts of compounds, namely 0.1 &#8211; 1 &#181;M dactolisib to target the mTOR pathway or 0.05&#8211;0.5 &#181;M selumetinib to target the MAPK pathway. Results show that 1 &#181;M dactolisib or 0.5 &#181;M of selumetinib are effective in inhibiting the phosphorylation of 4EBP1 or ERK1/2, respectively. Furthermore, dose-dependent inhibition of the targeted phosphoproteins indicates that tissue slices can also be utilized to validate phosphorylation-specific antibodies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58569/58569fig1.jpg" /> 
Figure 1: Schematic representation of the workflow for establishment and analysis of murine NSCLC tumor-derived slice explants. (A) Schematic describing the collection and preparation of tumor-bearing lungs for slicing. Lung lobes are harvested from a mouse and tumor tissue is dissected away from normal tissue. The black arrowhead and asterisk indicate approximately 4 mm and 1 mm tumors, respectively. The white arrowhead indicates lung tissue glued to the surface of the specimen holder. The red arrow points at an additional piece of normal lung support tissue to retain the tumor in an upright position. (B) Vibratome slicing and collection of tissue slices. White arrow indicates the slicing direction. Collection of sequential slices into a 24-well plate containing cold HBSS + P/S. The slices can either be cultured for different time points (here, 24&#8211;72 h) to assess tumor-specific marker expression during cultivation (top row), or can be used to perform drug treatments. C: vehicle control, T: drug treatment (bottom row). (C) Placing the tissue slice for cultivation using rotating incubation units. Tilt the 6-well plate so that some medium covers the top of the grid, place the tissue slice in the middle of the grid on top of the medium, and spread the slice using forceps. Ensure that the 6-well plates are weight balanced for a smooth rotation cycle. X: indicates incorrect, and <img alt="Equation" src="/files/ftp_upload/58569/58569eq1.jpg" />: indicates correct positioning of the slice. (D) Photograph of the FFPE block of a tumor slice. Black arrow points at paraffin-embedded tissue slice stained with hematoxylin. (E) Schematics showing the sectioning order of the slices in FFPE blocks; these sections can be processed to assess tissue viability and tumor-specific biomarker expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58569/58569fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58569/58569fig2.jpg" /> 
Figure 2: Assessment of viability and histotype-specific marker expression, and targeted drug treatment on NSCLC tissue slices. (A) Representative H&#38;E images of AC NSCLC slices of the indicated thicknesses cultured for 24 h. 200 &#181;m thin slices maintain better viability compared to 160 &#181;m or 250 &#181;m thin slices. Dark blue represents H&#38;E stained viable tissue, and pink indicates pseudocolored necrotic regions. Light blue indicates regions excluded from the analysis, either due to poor tissue quality or presence of fibrous stroma. T1 and T2 represent biological replicates derived from two different tumors. Scale bar = 500 &#181;m. (B) Representative IHC images of NKX2-1 expression in AC slices cultured for the indicated time points. Arrow indicates the area shown in higher magnification. Results show that NKX2-1 expression is not altered in the cultured slices compared to 0 h slices. Scale bar = 500 &#181;m and 50 &#181;m for low and high magnifications, respectively. (C) Quantification of the data shown in (B). (D) Representative IHC images of phosphorylated 4EBP1 or ERK1/2 expression in 0 h slices, or slices treated with DMSO or titrated amounts of dactolisib (dact, top row) or phosphorylated ERK1/2 expression in 0 h slice, or slices treated with DMSO or selumetinib (sel, bottom row). Black square boxes indicate areas shown in higher magnification. Scale bar = 1 mm or 50 &#181;m for low or high magnification, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58569/58569fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing at JoVE.com

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

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.

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

establishment-analysis-tumor-slice-explants-as-prerequisite-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.0K Views.  University of Helsinki. 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.

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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

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

Summary

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Chapters in this video

Introducing Shear Stress in the Study of Bacterial Adhesion

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Analysis of ¹⁸FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Zika Virus Specific Diagnostic Epitope Discovery

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster