Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

The overall goal of this procedure is to facilitate expeditious preclinical discovery of clinically relevant single nucleotide variants that may functionally drive resistance to various classes of therapies. This method can help answer key questions in the oncology field, such as which gene mutations may contribute to functional resistance to therapies in the clinic that may ultimately guide improvement in therapies and alternative clinical management strategies for drug resistant patients. The main advantage of this technique is that since we are facilitating the emergence of spontaneous mutations and applying an unbiased screening technology in resistant clones, there is a greater likelihood these variants are driving relapse targeted therapies in the clinic.

Demonstrating the procedure will be a very talented investigator from my laboratory To assess the GI 50 for cell lines. Prepare an assay plate by seeding cells in non-transparent clear. Bottom 96 well plates in 100 microliters of cell medium according to the map shown here.

Prepare a control plate in a similar manner, adding cells to the blue wells and only medium to the white wells. After incubating the plates at 37 degrees Celsius overnight, equilibrate the luminescent cell viability reagent to room temperature and mix gently by inverting. To obtain a homogeneous solution, add 80 microliters of reagent to the wells of the control plate and shake the contents for 30 minutes.

To induce cell lysis. Use a luminometer with an exposure of 0.1 to 1.0 seconds and a detection wavelength of 560 nanometers to record the luminescence. Next, to add the test compounds to the assay plates, make a series of one in four dilution of compounds in DMSO beginning with a 2000 micromolar concentration for a total of 10 dilution.

Then add the serially diluted compounds to medium to make a compound medium mix at a 10 x final concentration. Store the compound plate at minus 20 degrees Celsius for use on day three of the assay. Next, add 10 microliters of the diluted compounds to cells in triplicate such that the highest dose is 10 micromolar.

Incubate the plates at 37 degrees Celsius for three days. On day three, prepare a 400 microliter one X compound medium mix using the compound plates prepared three days prior. Invert the assay plates to remove the medium and pat dry on autoclave paper towels two to three times.

Add one x compound to well shaded in blue and add 100 microliters of medium to perimeter wells to prevent evaporation. On day six, assess the relative number of viable cells by taking luminescence reading as demonstrated earlier in the video. Then calculate the GI 50 values using software such as PRISM using the calculated GI 50 values set up cells for drug resistance assays.

If working with a cytostatic agent, seed cells at 30 to 40%confluence in 150 millimeter square culture dishes. If working with a toxic agent, see the cells at 70 to 80%confluence. After determining the initial compound concentrations according to the text protocol, treat the cells with the test compounds for highly toxic compounds, which cause cell death in a six day viability assay.

Start with the GI 50 and incrementally increase the concentration every two to three weeks until robust resistance is observed. To isolate single cell clones, use phase contrast microscopy at 40 x magnification to examine the culture dish and identify viable clusters of cells on the bottom of the dish. Use a pen to mark clones that are of average size and well isolated from other colonies to pick clones using a P 200 pipetter.

Remove the growth medium from the dish and use one XPBS to rinse away any floating cells. With the pipette tip, lift clones and transfer into the wells of a 48 well plate containing 50 microliters of 0.25%Trypsin incubate at 37 degrees Celsius for one to two minutes. Then add 200 microliters of fresh medium with half the concentration of compound to allow optimal recovery of cells.

To isolate the clones using three millimeter cloning discs, place the discs into a 10 centimeter tissue culture dish containing five milliliters of 0.25%tripsin EDTA for two minutes. Next, aspirate the medium from the dish containing the resistant clones and overlay the clones with the trips and soaked cloning discs. Incubate at 37 degrees Celsius for one to two minutes depending how easily the clones lift off the plates.

Then using sterile forceps, pick up the cloning discs and transfer into 48 well plates with 200 microliters of fresh medium, and have the concentration of compound gently pipette up and down to dislodge the cells from the cloning discs and incubate at 37 degrees overnight the following day, remove the cloning discs, incubate the cells, then assess for drug resistance. According to the text protocol, as shown here, HCT one 16 cells treated for a prolonged period with cytotoxic. Compound number one led to the spontaneous emergence of resistant clones that continued to grow during treatment.

The clones were picked using a pipette man. In this figure resistant clones one through three all demonstrated significant resistance to compound number one, and its close analog compound number two, whereas all clones showed sensitivity to an unrelated cytotoxic compound.Velcade. Following confirmation of phenotypic resistance, GDNA was isolated and submitted for whole exome sequencing analysis.

Structural variants that were recurrent and had the potential for functional impact were confirmed by an independent sequencing tool such as Sanger sequencing. An example shown here carried a heterozygous missense mutation, and the parental cell line was subsequently engineered to express the mutated CDNA. Whereas overexpression of the wild type CDNA failed to confer drug resistance.

The mutant CD NA significantly conferred phenotypic resistance to compound number one, confirming the functional role of the structural variation as the driver of resistance. After watching this video, you should have a good understanding of how to develop spontaneous resistant growth to screen for genes that are responsible for the drug resistance in cancer cells after its development. This technique takes the way for researchers in the field of oncology to explore drug resistance mechanisms in unbiased way in both cancer cell lines and animal models.