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 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) assay^6,7,8,9. 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.

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 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.
3. 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’s Balanced Salt Solution (HBSS).
4. 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.
5. 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.
6. 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.
7. 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.
8. Place a drop of medical grade cyanoacrylate glue on the specimen plate and place the tissue on this drop. Air-dry the glue for 2–3 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 (<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.
9. Adjust the vibratome settings. The following settings are used: cutting thickness = 250 µm, amplitude = 3.00 mm, slicing speed = 0.01–0.25 mm/s, and blade angle = 15°–21°.
   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°, cutting speed between 0.10–0.18 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.
10. 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.
11. 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.
12. Remove any excess buffer with a fine tip transfer pipette.
13. 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.
14. Culture the plates in a humidified incubator set at 37 °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 24–48 h after the initial slice preparation. For long-term cultivation, exchange the medium every 2–3 days.

2. Viability measurement

1. 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’s instructions. The reagent measures the reduction activity of live cells of metabolized prosubstrate to luciferin¹¹. Transfer 50 µL of enzyme-substrate mixture on top of each tissue slice.
2. Incubate with gentle agitation on an orbital shaker located inside a humidified incubator set at 37 °C with 5% CO₂ overnight.
3. 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.
   1. 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.
   2. 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.
   3. 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.

3. Evaluation of drug effect on the tissue slices

1. 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.
2. Dissolve drugs in dimethyl sulfoxide (DMSO) to make stock solutions. Prepare a 10x drug solution with TSC or other culture medium (25 µL/well for a 24 well plate containing 250 µL of medium) at the desired concentrations. For vehicle control, prepare medium containing an equivalent volume of DMSO.
3. Supplement the 10x drug solution to the culture medium at the bottom of the well. Mix by pipetting and transfer 50 µL of the medium on top of the tissues.
   NOTE: If several tissue slices are available, test duplicates or triplicates for each condition.
4. Incubate overnight with gentle shaking on an orbital shaker in a 37 °C, 5% CO₂, humidified incubator.
5. Remove the plate from the shaker and incubate statically in the 37 °C, 5% CO₂, humidified incubator.
6. 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 1–6 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.
7. Calculate the remaining viability of the treated tumor tissues using the following equation:
   
   The viability of the treated tissue is normalized by its baseline viability and viability shift of control tumor tissues.

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