Name: measuring real-time drug response in organotypic tumor tissue slices
Uploaded: 2020-05-02T14:00:00
Description: Watch this Scientific Journal Video about Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices at JoVE.com

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;...

<ol>
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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...

Watch this Scientific Journal Video about Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices at JoVE.com

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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