Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Summary

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.

Abstract

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. 

Introduction

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 de....

Protocol

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

1. Prepare tissue slice culture (TSC) medium following the recipe in Table 1. Filter the medium with a 0.45 µm vacuum filter unit to sterilize.
2. Aliquot 250 µL 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 °C, 5% CO₂ humidified incubator u....

Results

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 20–40 tissue slices f.......

Discussion

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.......

Materials

List of materials used in this article

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