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>

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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 border="0" cellpadding="0" cellspacing="0" style="width:835px;" width="834">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:352px;">10 cm dish</td>
			<td style="width:209px;">Corning</td>
			<td style="width:115px;">430293</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well dish</td>
			<td>CytoOne</td>
			<td>CC7682-7524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4T1</td>
			<td>ATCC</td>
			<td>CRL-2539</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 mm diameter Biopsy punch</td>
			<td>Integra Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate</td>
			<td>CytoOne</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A8960-5g</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Belzer UW cold storage solcution (UW solution)</td>
			<td>Bridge to Life</td>
			<td>500 mL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning Matrigel Membrane Mix</td>
			<td>Fisher scientific</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Corning</td>
			<td>MT-25950CQC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Edge Stainless Steel Cutting Blades</td>
			<td>TED PELLA</td>
			<td>121-6</td>
			<td>For slicing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Doxorubicin (hydrochloride)</td>
			<td>Cayman Chemical</td>
			<td>15007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine tip forceps</td>
			<td>ROBOZ</td>
			<td>RS-4974</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine tip forceps</td>
			<td>ROBOZ</td>
			<td>RS-4976</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767-500g</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS without Calcium, Magnesium or Phenol Red</td>
			<td>Gibco</td>
			<td>14-175-103</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCI010</td>
			<td colspan="2">Dr. Alana Welm, University of Utah</td>
			<td>Breast cancer PDX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffer Solution</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ITS + Premix</td>
			<td>Fisher Scientific</td>
			<td>354352</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IVIS Spectrum</td>
			<td>PerkinElmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica VT1200S Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Gibco</td>
			<td>25030164</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 &#181;m</td>
			<td>Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:352px;">Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 &#181;m</td>
			<td>Millipore</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636-500g</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PELCO Pro CA44 Tissue Adhesive</td>
			<td>TED PELLA</td>
			<td>10033</td>
			<td>Glue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140163</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RealTime-Glo MT Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9711</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Mouse EGF</td>
			<td>BioLegend</td>
			<td>585608</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI1640</td>
			<td>Gibco</td>
			<td>11875135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Single Edge Industrial Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td>For removing glued tissues</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Corning</td>
			<td>25-035-CI</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BW13115E</td>
			<td>For TSC medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Staurosporine</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-3510A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Synergy H4</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tooothed forceps</td>
			<td>ROBOZ</td>
			<td>RS-5155</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipettes</td>
			<td>Fisher scientific</td>
			<td>13-711-7M</td>
			<td>Wide tip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipettes</td>
			<td>Samco Scientific</td>
			<td>235</td>
			<td>Fine tip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">William&#39;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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Research

JoVE Journal

Cancer Research

11.1K 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

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

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 ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...