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.

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Our protocol is compatible with other molecular analysis methods and allows the assessment of how drugs affect the viability of in-tact tissue slices and the performance of medium throughput screening. The main advantage of this technique is that we can measure the viability of a whole tissue slice in real time with very little pre-or post-processing. This technique accelerate pre-clinical drug development and oncology by testing drug candidates or tissues slices acquired directly from patient tumor tissue samples.

Our real-time tissue viability assessment method is quite versatile and can be applied to cancer biology, neuroscience, and developmental biology studies. Preparing in-tact viable tissue slices is the key to the success of the procedure. The Vibratome settings in medium composition should be adjusted according to the tissue type and firmness.

On the day of the tumor tissue slice acquisition, aliquot 250 microliters of tissue slice culture medium per well in a 24 well plate and place one tissue culture insert into each well. Then place the plate in a 37 degrees Celsius humidified incubator with five percent carbon dioxide until use. To acquire tumor tissue fragments, submerge the tumor tissue in ice cold HBSS in a 10 centimeter culture plate and use a scalpel to cut the tissue in half.

Use a six millimeter biopsy punch to acquire cylinders of the tumor tissue and trim one end of each cylinder to create a flat surface. Place the tissue pieces in fresh, ice cold HBSS and turn on a Vibratome. Disinfect all of the equipment with seventy percent ethanol and place the Vibratome buffer tray, covered with an acrylic glass lid, at the center of the Vibratome ice bath.

Add ice to the bath, fill the buffer tray with cold HBSS. Load a razor blade into the blade holder and use toothed forceps to carefully select a biopsy punched tumor sample. Add a drop of medical grade cyanoacrylate glue onto the specimen plate.

Dab the tissue onto a piece of lint-free paper towel to remove any excess buffer and mount the cylinder of tissue on the drop in an upright stable position. After two to three minutes of air drying, place the plate into the buffer tray and add additional HBSS to the tray until the tissue is fully immersed in the buffer. Then assemble the Vibratome by placing the ice tray and buffer tray assembly onto the Vibratome and locking the arm.

Set the Vibratome cutting thickness to 250 micrometers, the amplitude to three millimeters, the slicing speed to 0.01 to 0.25 millimeters per second, and the blade angle to 15 to 21 degrees. Set the start and end locations of the blade and adjust the height of the stage. Run the instrument in continuous mode to cut slices for several cycles until the tissues are cut uniformly.

Use a wide tip transfer biped to transfer the slices and a small amount of HBSS into individual cell culture inserts in each well of the previously prepared 24 well plate and use a fine tip transfer biped to remove any excess buffer from the wells. When the end of the sample has been reached, mount a new piece of tissue and obtain additional slices until enough samples have been acquired. Then culture the tissue slices in the cell culture incubator for 24 to 48 hours.

To measure the viability of the tissue slices, prepare a luminescence-based viability measurement reagent containing both luciferase and prosubstrate at one to one thousand dilution in fresh tissue slice culture medium according to the manufacturer's instructions and replace the medium in each well of the 24 well plate with the mixture. Add 50 microliters of enzyme substrate mixture onto each tissue slice and place the plate onto an orbital shaker inside a humidified incubator with gentle agitation at 37 degrees Celsius and five percent carbon dioxide overnight. The next morning, measure the luminescence signal in each well on a microplate reader using the appropriate settings.

After determining the baseline viability of the tissue slices, dissolve the drugs of interest in tissue slice culture medium to obtain 10x stock solutions and add each 10x drug solution to the culture medium at the bottom of each tissue slice well. Transfer 50 microliters of the drug supplemented medium from the bottom of each well onto each tissue slice and return the slices to the orbital shaker overnight. The next morning, transfer the culture plate to the subculture incubator without shaking and measure the luminescent intensity in each tissue sample on the microplate reader at the appropriate experimental time points.

The viability of the treated tumor tissues at each time point can then be calculated using the equation as indicated. The viability of the tissue slices prepared from a mouse breast cancer tumor sample can be maintained for at least 21 days with occasional medium exchanges. Treatment with the multikinase inhibitor for four days reduces the luminescence signal intensity by 100 times compared to control tumor tissues treated with dimethyl sulfoxide.

Treatment with half the concentration of the same inhibitor decreases the luminescence as early as day one and reaches the lowest level at day four, remaining low for each subsequent measured time point. In contrast, the luminescent intensity of the controlled tumor tissue group remains stable throughout the six day culture. In addition, the treatment of mouse breast cancer tumor slices with serial doses of the inhibitor for four days illustrates the dose-dependent changes in the slice viability in the half maximal effective concentration of the drug.

Patient derived xenografts treated with doxorubicin, a standard chemotherapeutic drug, also exhibit dose-dependent changes in viability. Further, the testing of seventeen pre-clinical and clinically approved drugs in triplicate on tissue slices prepared from a single bulk patient-derived xenograft provides proof of concept that medium throughput drug screening can be performed on tumor slices with the native tumor microenvironment. When preparing the tumor tissue samples, take care to minimize contact with the tissue slices to avoid damaging the tissue.

Our method can be coupled with other approaches such as IHC, flow cytometry, or single cell sequencing, to obtain spatial and single-cell information from the tissues. Our technique allows testing of the effects of multiple drugs of interest at various doses under physiological conditions over time. When preparing tissue slices from tumors with an unknown pathogen status, be sure to perform all of the procedures in a biosafety cabinet.

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Journal (Cancer Research)

Journal (Cancer Research)

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

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