This work was supported by the NIH/NCI [K22CA201229, P30CA015704], and Sidney Kimmel Foundation [Kimmel Scholar Award], Lung Cancer Discovery Award (LCD-505536). We would like to thank Dr. Alana Welm (University of Utah) for providing the breast cancer PDX tumor. We would also like to thank the staff at Comparative Medicine, Fred Hutchinson Cancer Research Center (FHCRC) for maintenance of the PDX model and members of the Gujral lab for helpful discussions. N.N.A is supported by the JSPS Overseas Research Fellowship and Interdisciplinary Training Grant from FHCRC. A.J.B. is supported by the Chromosome Metabolism and Cancer Training Grant from FHCRC.

All mouse experiments were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of animals in biomedical research.
1. Preparation of tumor tissue slices
<ol>
	<li>Prepare tissue slice culture (TSC) medium following the recipe in Table 1. Filter the medium with a 0.45 &#181;m vacuum filter unit to sterilize.</li>
	<li>Aliquot 250 &#181;L of TSC medium per well for a 24 well plate, or 1 mL of medium per well for a 6 well plate. Keep the plate in a 37 &#176;C, 5% CO2 humidified incubator until use. 
	NOTE: The medium and supplements can be optimized depending on tissue types and experimental goals. The amount of medium should be just enough to soak the porous membrane of a culture insert. Do not exceed the level of the membrane. Adjust the medium volume if the medium level is too low or too high. If researchers are testing immunomodulatory agents, we recommend supplementation of the medium with IL-210.</li>
	<li>If using tumor tissue derived from mice, euthanize mice with a recommended procedure, sanitize the exposed skin of mice by spraying 70% ethanol, and dissect tumors with aseptic techniques. Store the tumor tissue in a tube containing ice-cold Hank&#8217;s Balanced Salt Solution (HBSS).</li>
	<li>If using fresh patient tumor tissues, store in ice-cold HBSS for short term, or ice-cold Belzer University of Wisconsin (UW) solution for longer term storage (up to 24 h) to preserve tissue viability. 
	NOTE: Slices from a PDX tissue shipped overnight in ice-cold UW solution have been successfully prepared.</li>
	<li>Transfer the tumor tissue into a 10 cm culture plate containing ice-cold HBSS. Cut tumor into halves with a scalpel while submerged in ice-cold HBSS. Shape tumor tissues into cylinders using a 6 mm diameter biopsy punch and trim one side to create a flat surface. Store the tissues in ice-cold HBSS until use. 
	NOTE: Avoid including the necrotic area of the tissue, which is generally at the center and has an opaque and fragile appearance.</li>
	<li>Set up the vibratome. Disinfect all the equipment using 70% ethanol. Place the vibratome buffer tray covered with an acrylic glass lid at the center of the vibratome ice bath. Add ice to the ice bath and fill up the buffer tray with cooled HBSS to keep the tissue cool while slicing. Attach a razor blade to the blade holder. 
	NOTE: Although keeping vibratomes in a semisterile environment has not affected prior experiments, it is recommended to store the vibratome under a biosafety cabinet if available.</li>
	<li>Carefully lift up a biopsy-punched tumor tissue from the HBSS using toothed forceps and remove excess buffer by dabbing the tissue with a lint-free paper towel.</li>
	<li>Place a drop of medical grade cyanoacrylate glue on the specimen plate and place the tissue on this drop. Air-dry the glue for 2&#8211;3 min and place the plate into the buffer tray. Supplement with some HBSS until the tissue is fully immersed in the buffer. 
	NOTE: Mounting tissues in an upright, stable position is critical to generate uniformly cut slices. If mounted tissues are unstable, either add more glue to the surrounding tissue to retain an upright position or unload the tissue, flatten the bottom, and glue it again. Elastic tissues tend to get pushed and bent more easily during slicing, in which case shorter lengths (&#60;5 mm) and multiple runs may be appropriate. For extremely fragile tissues, paper towels can destroy the tissue. In this case, the tissue can be placed on a plastic surface to wick away some excess HBSS.</li>
	<li>Adjust the vibratome settings. The following settings are used: cutting thickness = 250 &#181;m, amplitude = 3.00 mm, slicing speed = 0.01&#8211;0.25 mm/s, and blade angle = 15&#176;&#8211;21&#176;. 
	NOTE: Optimize the slicing settings depending on the stiffness of the tissue. Softer tissues are more easily cut with a deeper blade angle at lower speed. A blade angle of 18&#176;, cutting speed between 0.10&#8211;0.18 mm/s, and an amplitude of 3.00 mm works well for mouse 4T1 and PDX HCI010 tumors. Stiffer tissue can be cut with a faster blade speed.</li>
	<li>Set the start and end locations of the blade and adjust the height of the stage. Run the instrument with continuous mode to cut slices for several cycles until the tissues are cut uniformly.</li>
	<li>Continue slicing the tissue. Transfer the slices along with a small amount of HBSS buffer onto the cell culture insert with medium with a wide-tip transfer pipette, using suction to lift the whole slice. Place one slice per insert on a 24 well plate. An insert for 6 well plates can accommodate up to four slices for a replicate assay. 
	NOTE: If the last edge of the tissue slice is still attached to the uncut tissue, stop the vibratome and separate the tissue slice with two pairs of fine tip forceps without touching or poking the tissue slices. A separate razor blade can be useful in removing hanging tissue.</li>
	<li>Remove any excess buffer with a fine tip transfer pipette.</li>
	<li>When the blade reaches close to the glued tissues, stop the instrument, lower the stage, remove the stiffened tissue with a separate blade, and mount a new tissue. Continue slicing until enough slices have been collected.</li>
	<li>Culture the plates in a humidified incubator set at 37 &#176;C, 5% CO2. The tissue slices are cultured at the air-liquid interphase on the cell culture insert. The tissue slices are ready for experiments 24&#8211;48 h after the initial slice preparation. For long-term cultivation, exchange the medium every 2&#8211;3 days.</li>
</ol>
2. Viability measurement
<ol>
	<li>For viability measurement of the tissue slice, exchange the medium with a luminescence-based viability measurement reagent containing both luciferase and prosubstrate at 1:1,000 dilution in TSC medium following the manufacturer&#8217;s instructions. The reagent measures the reduction activity of live cells of metabolized prosubstrate to luciferin11. Transfer 50 &#181;L of enzyme-substrate mixture on top of each tissue slice.</li>
	<li>Incubate with gentle agitation on an orbital shaker located inside a humidified incubator set at 37 &#176;C with 5% CO2 overnight.</li>
	<li>Luminescent signals can be measured using either a microplate reader or an in vivo imaging instrument for tissues cultured in a 24 well plate. To measure the individual viability of multiple tissues on a 6 well plate, an in vivo imaging system should be used.
	<ol>
		<li>When using a microplate reader, set the microplate without the lid. Any type of microplate reader capable of measuring luminescent intensity should be suitable for the experiment. We used the following settings: read time = 1 s, measurement from the top, gain = 200. 
		NOTE: For higher accuracy, use a white-wall microplate to set up the experiment. White-wall, clear-bottom 24 well plates have been tested, and in most cases, clear well plates do not compromise the results.</li>
		<li>When using an in vivo imaging system to measure the viability, place the plate at the center of the stage and remove the lid. Acquire normal photographic images followed by luminescent images with a field of stage setting at C, auto exposure time, f-stop = 1, objective setting as microplate with subject height = 0.0 cm.</li>
		<li>Quantify the luminescent intensity using the imaging software accompanied with the in vivo imaging system. Draw consistent regions of interest (ROI) around each tissue slice. This protocol uses a 1 cm diameter circle as an ROI (See Figure 1). Measure the total flux (p/s) of the area.</li>
	</ol>
	</li>
</ol>
3. Evaluation of drug effect on the tissue slices
<ol>
	<li>Measure baseline viability prior to treatment using the luminescence-based viability measurement reagent as explained in section 2. 
	NOTE: If several tissues have significantly lower viability compared to others, omit the tissues from the assay.</li>
	<li>Dissolve drugs in dimethyl sulfoxide (DMSO) to make stock solutions. Prepare a 10x drug solution with TSC or other culture medium (25 &#181;L/well for a 24 well plate containing 250 &#181;L of medium) at the desired concentrations. For vehicle control, prepare medium containing an equivalent volume of DMSO.</li>
	<li>Supplement the 10x drug solution to the culture medium at the bottom of the well. Mix by pipetting and transfer 50 &#181;L of the medium on top of the tissues. 
	NOTE: If several tissue slices are available, test duplicates or triplicates for each condition.</li>
	<li>Incubate overnight with gentle shaking on an orbital shaker in a 37 &#176;C, 5% CO2, humidified incubator.</li>
	<li>Remove the plate from the shaker and incubate statically in the 37 &#176;C, 5% CO2, humidified incubator.</li>
	<li>At desired timepoints, measure the luminescent intensity from the tissues using a microplate reader or an in vivo imaging system. Usually, drug effects become detectable after 1&#8211;6 days of treatment. 
	NOTE: The luminescence-based viability reagent is stable for at least 3 days under culture conditions. However, the substrate may be depleted during the assay depending on the metabolic activity of the tissue. If a significant signal drop is observed in tissues with previously high signals, supplement the substrate in all the wells.</li>
	<li>Calculate the remaining viability of the treated tumor tissues using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/61036/61036eq1v2.jpg" /> 
	The viability of the treated tissue is normalized by its baseline viability and viability shift of control tumor tissues.</li>
</ol>

<ol>
	<li>Klemm, F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+therapeutic+response+in+cancer.">Microenvironmental regulation of therapeutic response in cancer.</a> Trends in Cell Biology. 25 (4), 198-213 (2015).</li><li>Horvath, P., 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=Screening+out+irrelevant+cell-based+models+of+disease.">Screening out irrelevant cell-based models of disease.</a> Nature Reviews: Drug Discovery. 15 (11), 751-769 (2016).</li><li>Seruga, B., Ocana, A., Amir, E., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failures+in+Phase+III:+Causes+and+Consequences.">Failures in Phase III: Causes and Consequences.</a> Clinical Cancer Research. 21 (20), 4552-4560 (2015).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Nishida-Aoki, N., Gujral, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+approaches+to+study+cell-cell+interactions+in+tumor+microenvironment.">Emerging approaches to study cell-cell interactions in tumor microenvironment.</a> Oncotarget. 10 (7), 785-797 (2019).</li><li>van der 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> BMC Cancer. 6, 86 (2006).</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=Establishment+and+Analysis+of+Tumor+Slice+Explants+As+a+Prerequisite+for+Diagnostic+Testing.">Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing.</a> Journal of Visualized Experiments. (141), e58569 (2018).</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> BMC Cancer. 16, 78 (2016).</li><li>Jiang, X., 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=Long-lived+pancreatic+ductal+adenocarcinoma+slice+cultures+enable+precise+study+of+the+immune+microenvironment.">Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment.</a> Oncoimmunology. 6 (7), 1333210 (2017).</li><li>Sivakumar, 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=Organotypic+tumor+slice+cultures+provide+a+versatile+platform+for+immuno-oncology+and+drug+discovery.">Organotypic tumor slice cultures provide a versatile platform for immuno-oncology and drug discovery.</a> Oncoimmunology. 8 (12), 1670019 (2019).</li><li>Duellman, S. 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=Real-Time+Cell+Viability+Assay+and+Use+in+Inhibitor+Screening.">Real-Time Cell Viability Assay and Use in Inhibitor Screening.</a> Assay and Drug Development Technologies. 13 (8), 456-465 (2015).</li><li>DeRose, Y. 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=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nature Medicine. 17 (11), 1514-1520 (2011).</li><li>Lazaro, C. 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=Establishment,+characterization,+and+long-term+maintenance+of+cultures+of+human+fetal+hepatocytes.">Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes.</a> Hepatology. 38 (5), 1095-1106 (2003).</li><li>Chadwick, 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=A+Brain+Tumor/Organotypic+Slice+Co-culture+System+for+Studying+Tumor+Microenvironment+and+Targeted+Drug+Therapies.">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> Journal of Visualized Experiments. (105), e53304 (2015).</li><li>Parker, J. J., Lizarraga, M., Waziri, A., Foshay, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Human+Glioblastoma+Organotypic+Slice+Culture+Model+for+Study+of+Tumor+Cell+Migration+and+Patient-specific+Effects+of+Anti-Invasive+Drugs.">A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs.</a> Journal of Visualized Experiments. (105), e53304 (2017).</li></ol>

The authors have nothing to disclose.

In this protocol, we demonstrate a platform for quantitative, and real-time drug efficacy studies on organotypic tumor tissue slices. The tissue slice culture system provides distinct advantages over traditional cell-based in vitro methods by capturing cell heterogeneity and physiological characteristics of the native tumor microenvironment. This platform also enables higher throughput for drug efficacy testing, helping to bridge the gap between cell culture studies and in vivo experiments.
To...

Cancer cell interactions with the physical and biochemical properties of the surrounding stromal tissue forms the TME. TME can stimulate tumor growth, metastasis, and modulate tumor response to therapy1. In conventional preclinical drug development, drug candidates are typically first screened using cultured cancer cell lines, an assay platform that critically lacks the TME2. This lack of physiological TME in cell-based prescreening stages may limit the discovery of effective agents in tumor-bearing animal models and may contribute to the high attrition rate of many promising oncology drugs in later clinical stages of development3.
Despite the importance of TME in modulating tumor drug responses, experimental constraints limit the application of more physiologically relevant systems during the early stages of drug discovery and development. It is impractical to screen hundreds of therapeutic agents on tumors from animal models or patient tumor specimens. Indeed, surgical specimens are scarce resources with varied genetic backgrounds and screening thousands of candidate molecules in animal models is not feasible due to experimental scale, cost, and animal welfare.
Tumor tissue slice culture, where precisely cut, thin tumor sections are cultured ex vivo, can address the limitation of physiological TME in drug screening assays. Historically, the field of neuroscience pioneered and made extensive optimizations of slice culture for brain tissue4. Recently, many studies have demonstrated preparation of slices from various types of tumor tissue including cell line-derived tumors, spontaneous tumor models, patient-derived xenografts (PDX), and primary patient tumors. Ex vivo tissue slice culture integrates benefits from both in vivo and in vitro culture5. Tumor tissue slices retain intact tissue architecture and variegated cell composition, enabling the study of cancer cells within the TME context.
This protocol introduces an organotypic tumor tissue slice culture system combined with a real-time, highly sensitive viability assay to evaluate drug responses. Drug efficacy tests on organotypic tumor tissue slices in previously introduced protocols rely on measuring changes in cell viability by fluorescent dye incorporation, immunohistochemistry (IHC), or MTT ((3- [4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay6,7,8,9. However, all of these methods are end point assays and suffer from low sensitivity, long processing time, complex data analyses, narrow signal range, and high experimental error. Our luminescence-based live cell-compatible reagent improves these assays by providing a wide signal range and instantaneous (~5 min) measurement without prior processing and minimal post processing. This reagent is highly sensitive and can coexist in the cell culture media, allowing continuous and time-course measurement of cell viability. This assay system is applicable to high-throughput screening of drug candidates on tissue slices in preclinical drug development.

<table><tbody><tr><td>10 cm dish</td><td>Corning</td><td>430293</td><td></td></tr><tr><td>24-well dish</td><td>CytoOne</td><td>CC7682-7524</td><td></td></tr><tr><td>4T1</td><td>ATCC</td><td>CRL-2539</td><td></td></tr><tr><td>6 mm diameter Biopsy punch</td><td>Integra Miltex</td><td>33-36</td><td></td></tr><tr><td>6-well plate</td><td>CytoOne</td><td>CC7682-7506</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A8960-5g</td><td>For TSC medium</td></tr><tr><td>Belzer UW cold storage solcution (UW solution)</td><td>Bridge to Life</td><td>500 mL</td><td></td></tr><tr><td>Corning Matrigel Membrane Mix</td><td>Fisher scientific</td><td>356234</td><td></td></tr><tr><td>DMSO</td><td>Corning</td><td>MT-25950CQC</td><td></td></tr><tr><td>Double Edge Stainless Steel Cutting Blades</td><td>TED PELLA</td><td>121-6</td><td>For slicing</td></tr><tr><td>Doxorubicin (hydrochloride)</td><td>Cayman Chemical</td><td>15007</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>Fine tip forceps</td><td>ROBOZ</td><td>RS-4974</td><td></td></tr><tr><td>Fine tip forceps</td><td>ROBOZ</td><td>RS-4976</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5767-500g</td><td>For TSC medium</td></tr><tr><td>HBSS without Calcium, Magnesium or Phenol Red</td><td>Gibco</td><td>14-175-103</td><td></td></tr><tr><td>HCI010</td><td>Dr. Alana Welm, University of Utah</td><td>Breast cancer PDX</td><td></td></tr><tr><td>HEPES Buffer Solution</td><td>Gibco</td><td>15630080</td><td>For TSC medium</td></tr><tr><td>ITS + Premix</td><td>Fisher Scientific</td><td>354352</td><td>For TSC medium</td></tr><tr><td>IVIS Spectrum</td><td>PerkinElmer</td><td>124262</td><td></td></tr><tr><td>Leica VT1200S Vibratome</td><td>Leica</td><td>VT1200S</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030164</td><td>For TSC medium</td></tr><tr><td>Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 &amp;micro;m</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 &amp;micro;m</td><td>Millipore</td><td>PICM03050</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636-500g</td><td>For TSC medium</td></tr><tr><td>PELCO Pro CA44 Tissue Adhesive</td><td>TED PELLA</td><td>10033</td><td>Glue</td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Fisher Scientific</td><td>15140163</td><td>For TSC medium</td></tr><tr><td>RealTime-Glo MT Cell Viability Assay</td><td>Promega</td><td>G9711</td><td></td></tr><tr><td>Recombinant Mouse EGF</td><td>BioLegend</td><td>585608</td><td>For TSC medium</td></tr><tr><td>RPMI1640</td><td>Gibco</td><td>11875135</td><td></td></tr><tr><td>Single Edge Industrial Razor Blades</td><td>VWR</td><td>55411-050</td><td>For removing glued tissues</td></tr><tr><td>Sodium Bicarbonate</td><td>Corning</td><td>25-035-CI</td><td>For TSC medium</td></tr><tr><td>Sodium Pyruvate</td><td>Fisher Scientific</td><td>BW13115E</td><td>For TSC medium</td></tr><tr><td>Staurosporine</td><td>Santa Cruz Biotechnology</td><td>sc-3510A</td><td></td></tr><tr><td>Synergy H4</td><td>BioTek</td><td></td><td></td></tr><tr><td>Tooothed forceps</td><td>ROBOZ</td><td>RS-5155</td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisher scientific</td><td>13-711-7M</td><td>Wide tip</td></tr><tr><td>Transfer pipettes</td><td>Samco Scientific</td><td>235</td><td>Fine tip</td></tr><tr><td>William&#039;s medium E, no glutamine</td><td>ThermoFisher Scientific</td><td>12551032</td><td>For TSC medium</td></tr></tbody></table>

measuring real-time drug response in organotypic tumor tissue slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

Here, we demonstrate a time-course, multiple drug efficacy evaluation protocol for tumor tissue slices prepared from mouse 4T1 breast tumor tissues and a breast cancer PDX model, HCI01012. We successfully prepared tissue slices from several mouse-derived tumors, PDX, and fresh patient tumors10. The overall workflow of the tumor tissue preparation and drug efficacy test is described in Figure 1. In general, we could prepare 20&#8211;40 tissue sl...

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Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

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11.2K Views.  Fred Hutchinson Cancer Research Center. We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Video: Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicine

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

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

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

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

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

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.

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 ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

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Chapters in this video

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

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

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