Drug Sensitivity Assays of Human Cancer Organoid Cultures
Hayley E. Francies, Andrew Barthorpe, Anne McLaren-Douglas, William J. Barendt, Mathew J. Garnett
Abstract
Drug sensitivity testing utilizing preclinical disease models such as cancer cell lines is an important and widely used tool for drug development. Importantly, when combined with molecular data such as gene copy number variation or somatic coding mutations, associations between drug sensitivity and molecular data can be used to develop markers to guide patient therapies. The use of organoids as a preclinical cancer model has become possible following recent work demonstrating that organoid cultures can be derived from patient tumors with a high rate of success. A genetic analysis of colon cancer organoids found that these models encompassed the majority of the somatic variants present within the tumor from which it was derived, and capture much of the genetic diversity of colon cancer observed in patients. Importantly, the systematic sensitivity testing of organoid cultures to anticancer drugs identified clinical gene–drug interactions, suggestive of their potential as preclinical models for testing anticancer drug sensitivity. In this chapter, we describe how to perform medium/high-throughput drug sensitivity screens using 3D organoid cell cultures.
Steps
3.1 Preparation of Drug Stocks and Drug Plates
Design the layout of your drug plate ( see Note 4 and Fig. 2).
Drugs are reconstituted in 100% volume and stored frozen at -80°C or ideally in StoragePods®(Roylan Developments) kept at Room temperature, providing a moisture-free, low-oxygen environment and protection from UV damage ( see Note 5 ).
Master stocks of drugs are reconstituted at 1000–10,000× of the final desired maximum screening concentration.
Following the reconstitution of the drug(s) in DMSO, the master stock drugs are cherry-picked to a 384-well polypropylene plate using a liquid-handling robot Biomek FX (Beckman Coulter). This plate is called the stock plate. If the master stock is at 1000× of the final screening concentration, a direct transfer to the stock plate can be performed. If at greater than 1000× concentration, a dilution in DMSO is performed during the transfer to the stock plate, resulting in all drugs on the stock plate being at 1000×.
Using the stock plate we generate a source plate (384-well low dead volume (LDV) plate). The source plate contains all the drugs to be screened along with the dilution series of the drugs. Half-log dilutions are performed in DMSO and all wells are 1000× of the final desired concentration, typically containing 10 μL per well. A single source plate can be used for drugging multiple organoid screening plates and over multiple dates ( see Notes 6 and 7 ).
The generation of the source plate is performed using an Echo 555 acoustic dispenser.
For storage, seal plates using adhesive plate foil seals.
Both the stock and source drug plates can be stored in compound StoragePods®and used multiple times. If plates are stored at -80°C we recommended you limit the number of freeze–thaw cycles.
3.2 Dispensing the Layer of BME-2 to 384-well Plates
Program a FluidX XRD-384 reagent dispenser to dispense 8µL. Flush the XRD-384 with ethanol, followed by sterile water and lastly organoid media.
Prime the XRD-384 with 7.5mg/mL until all tubing is loaded. Once primed, dispense 8µL in to all wells of a 384-well flat clear bottom polypropylene plate. Ensure the BME-2 is kept chilled while dispensing.
Centrifuge the plates at 182x g to ensure the BME-2 covers the bottom of each well.
Place the plates in a 37°C incubator for at least 0h 20m 0s allowing the BME-2 to polymerize, forming a polymerized layer of BME-2 across the bottom of all wells ( see Note 8 ).
3.3 Preparation of Organoid Cultures
Organoids are routinely cultured embedded within 10–15 μL droplets of 7.5 mg/mL BME-2. We seed 15 droplets per well of a 6-well cell culture plate together with 2mL.
To harvest organoids from a 6-well plate, use a cell scraper to disrupt the 7.5 mg/mL BME-2 droplets containing organoids from the bottom of the well.
Using a 1 mL pipette, mix the disrupted organoids along with the media to initiate release of the organoids from the BME-2. This should physically disrupt the organoids but avoid breaking them down to single cells, the goal being to plate formed organoids, not single cells.
Transfer the organoid suspension to a 15 mL centrifuge tube and centrifuge at 364x g.
Aspirate the supernatant from the 15 mL falcon tube and resuspend the organoid pellet in 5mL.
As organoids are too large to be counted by an automated cell counter or a hemocytometer, dispense three 10µL of the organoid suspension on to a microscope slide and count the number of organoids in each 10 μL droplet ( see Note 9 ). Multiply the average number of organoids per droplet by 100 to calculate the number of organoids per mL ( see Note 10 ).
A suspension of 15,000–20,000 organoids per mL is required for drug screening. To dispense two 384-well plates requires 35 mL of organoids prepared in drug screening media at 15,000 organoids per mL. Approximately three confluent 6-well plates are sufficient to generate the 525,000 organoids necessary.
Program the XRD-384 to dispense 32 μL. Flush the XRD-384 as previously described in Section 3.2 "Dispensing the Layer of BME-2 to 384-well Plates" with ethanol, sterile water and organoid media:
Ensure the XRD-384 is primed, then dispense 32µL into all wells of column 1 (media only, no organoids).
Once completed, dispense 32µL to all wells with the exception of column 1 ( see Note 11 ). This gives a total well volume of 40µL.
Once completed, place the plates in a 37°C incubator until the following day allowing the organoids to settle ( see Note 12 ).
3.4 Drugging of Screening Plates
Following overnight incubation, organoid plates are manually inspected using a phase-contrast microscope to check cell health prior to the addition of drugs.
Using an Echo 555 acoustic dispenser, 40nL of compound previously prepared at 1000× are dispensed from a source plate (described in Section 3.1 "Preparation of Drug Stocks and Drug Plates") into the cell plate, to a final concentration of 1× compound and 0.1 % DMSO.
Return plates to a 37°C incubator.
3.5 Cell Viability Assay
Organoid plates can be incubated with compound/drug for up to 6 days before ending the assay. After more than 6 days, the plates are liable to be effected by evaporation and a reduction in data quality may be observed.
Following incubation with drug, the plates are manually checked under the microscope to ensure the positive control drug(s) have effectively killed organoids, and organoids present in the negative control wells are healthy.
To quantitate the effect of drugs on cell viability, CellTiter-Glo®2.0 Luminescent Cell Viability Assay (Promega) can be used. CellTiter-Glo®2.0 determines the number of viable cells in a well based on the quantification of ATP present, an indicator of metabolically viable cells. Addition of CellTiter-Glo®2.0 leads to cell lysis and generation of a luminescent signal proportional to the amount of ATP present, and thus indirectly measures cell number.
For each 384-well plate approximately 4 mL of CellTiter-Glo®2.0 is required, add 10mL to the total volume required to account for dead volume of the XRD-384.
Prior to the addition of CellTiter-Glo®2.0, remove plates from the incubator and allow them to equilibrate to Room temperature for 0h 10m 0s.
Program the XRD-384 to dispense 10µL. Following the flushing of the machine as described in Section 3.2 "Dispensing the Layer of BME-2 to 384-well Plates", prime the machine with CellTiter-Glo®2.0 and dispense 10µL to all wells of the 384-well plate(s) ( see Note 13 ).
Incubate the plates at Room temperature for 0h 20m 0s before reading plate luminescence using a Paradigm plate reader (Molecular Devices) ( see Note 14 ).
3.6 Data Quality Checks and Downstream Analysis
Quality control metrics are calculated for each plate prior to further analysis:
Coefficient of variation (CV): The CV of the negative control wells determines the variation in the luminescence of the negative control wells on each plate. It is calculated by dividing the standard deviation of the luminescence of the negative controls wells by their mean luminescence. For an organoid drug screen, CV values of less than 0.22 are acceptable.
Z-factor: The Z-factor (also known as Z-prime) is commonly used in high-throughput screens and measures the assay dynamic range and data variation using both the positive and negative controls [17]. The calculation determines if the effect observed between the positive and negative controls is large enough to make comparisons with drugs of unknown effect. The Z-factor can be calculated using each of the positive control drugs present on the drug plate ( see Note 4d ). Organoid plates with a Z-factor of 0.4 or greater are generally of good quality. This threshold is slightly lower than we would typically use for a cancer cell line (Z-factor >0.5) due to the increased technical complexity of screening 3D organoid cultures.
DMSO effect: The ratio between the negative control wells containing DMSO and those without DMSO is calculated. This is to ensure that the concentration of DMSO present in the drugged wells (0.1 % DMSO) does not have a significant effect on cell viability. A ratio of between 0.8 and 1.2 is required.
Following completion of the above quality metrics, the data can be normalized or the raw intensity data can be used for curve-fitting.
Data normalization.Normalization is completed using the following calculation:
For drugs where the concentrations selected have generated a dose–response curve, measurements such as the IC50(half-maximal inhibitory concentrations) and AUC (area under the curve) can be calculated to assess and compare sensitivity.
Curve-fitting.Commercial software packages such as GraphPad Prism and Microsoft Office Excel can also be used to analyze the data generated from the drug screen assay. Curve-fitting algorithms for modeling drug response are also available [18].
