TITLE: Drug Sensitivity Assays of Human Cancer Organoid Cultures
AUTHORS: Hayley E. Francies, Andrew Barthorpe, Anne McLaren-Douglas, William J. Barendt, Mathew J. Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [3.1 Preparation of Drug Stocks and Drug Plates]
Design the layout of your drug plate (see Note 4 and Fig. 2).
?. [3.1 Preparation of Drug Stocks and Drug Plates]
Drugs are reconstituted in  and stored frozen at  or ideally in StoragePods® (Roylan Developments) kept at , providing a moisture-free, low-oxygen environment and protection from UV damage (see Note 5).
[DMSO]
-80 °C
0 Room temperature
?. [3.1 Preparation of Drug Stocks and Drug Plates]
Master stocks of drugs are reconstituted at 1000–10,000× of the final desired maximum screening concentration.
?. [3.1 Preparation of Drug Stocks and Drug Plates]
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×.
?. [3.1 Preparation of Drug Stocks and Drug Plates]
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).
?. [3.1 Preparation of Drug Stocks and Drug Plates]
For storage, seal plates using adhesive plate foil seals.
?. [3.1 Preparation of Drug Stocks and Drug Plates]
Both the stock and source drug plates can be stored in compound StoragePods®and used multiple times. If plates are stored at  we recommended you limit the number of freeze–thaw cycles.
-80 °C
?. [3.2 Dispensing the Layer of BME-2 to 384-well Plates]
Program a FluidX XRD-384 reagent dispenser to dispense . Flush the XRD-384 with ethanol, followed by sterile water and lastly organoid media.
8 µl
?. [3.2 Dispensing the Layer of BME-2 to 384-well Plates]
Prime the XRD-384 with  until all tubing is loaded. Once primed, dispense  in to all wells of a 384-well flat clear bottom polypropylene plate. Ensure the BME-2 is kept chilled while dispensing.
[BME-2]
[7.5 mg/mL BME-2 media]
?. [3.2 Dispensing the Layer of BME-2 to 384-well Plates]
Centrifuge the plates at  to ensure the BME-2 covers the bottom of each well.
Centrifuge: 182 34, 1 min
?. [3.2 Dispensing the Layer of BME-2 to 384-well Plates]
Place the plates in a  incubator for at least  allowing the BME-2 to polymerize, forming a polymerized layer of BME-2 across the bottom of all wells (see Note 8).
[,  5 % CO2]
?. [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 .
[organoid media]
?. [3.3 Preparation of Organoid Cultures]
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.
?. [3.3 Preparation of Organoid Cultures]
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.
?. [3.3 Preparation of Organoid Cultures]
Transfer the organoid suspension to a 15 mL centrifuge tube and centrifuge at .
Centrifuge: 364 34, 2 min
?. [3.3 Preparation of Organoid Cultures]
Aspirate the supernatant from the 15 mL falcon tube and resuspend the organoid pellet in .
[drug screening media]
?. [3.3 Preparation of Organoid Cultures]
As organoids are too large to be counted by an automated cell counter or a hemocytometer, dispense three  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).
[droplets]
?. [3.3 Preparation of Organoid Cultures]
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.
?. [3.3 Preparation of Organoid Cultures]
Ensure the XRD-384 is primed, then dispense  into all wells of column 1 (media only, no organoids).
[drug screening media]
?. [3.3 Preparation of Organoid Cultures]
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:
?. [3.3 Preparation of Organoid Cultures]
Once completed, dispense  to all wells with the exception of column 1 (see Note 11). This gives a total well volume of .
[organoid suspension]
40 µl
?. [3.3 Preparation of Organoid Cultures]
Once completed, place the plates in a  incubator until the following day allowing the organoids to settle (see Note 12).
[, 5 % CO2]
?. [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.
?. [3.4 Drugging of Screening Plates]
Using an Echo 555 acoustic dispenser,  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.
40
?. [3.4 Drugging of Screening Plates]
Return plates to a  incubator.
[, 5 % CO2]
?. [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.
?. [3.5 Cell Viability Assay]
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.
?. [3.5 Cell Viability Assay]
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.
?. [3.5 Cell Viability Assay]
For each 384-well plate approximately 4 mL of CellTiter-Glo® 2.0 is required, add  to the total volume required to account for dead volume of the XRD-384.
10 mL
?. [3.5 Cell Viability Assay]
Prior to the addition of CellTiter-Glo® 2.0, remove plates from the incubator and allow them to equilibrate to  for .
0 Room temperature
?. [3.5 Cell Viability Assay]
Program the XRD-384 to dispense . 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  to all wells of the 384-well plate(s) (see Note 13).
10 µl
10 µl
?. [3.5 Cell Viability Assay]
Incubate the plates at  for  before reading plate luminescence using a Paradigm plate reader (Molecular Devices) (see Note 14).
0 Room temperature
?. [3.6 Data Quality Checks and Downstream Analysis]
Quality control metrics are calculated for each plate prior to further analysis:
?. [3.6 Data Quality Checks and Downstream 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.
?. [3.6 Data Quality Checks and Downstream Analysis]
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.
?. [3.6 Data Quality Checks and Downstream Analysis]
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.
?. [3.6 Data Quality Checks and Downstream Analysis]
Following completion of the above quality metrics, the data can be normalized or the raw intensity data can be used for curve-fitting.
?. [3.6 Data Quality Checks and Downstream Analysis]
Data normalization.Normalization is completed using the following calculation:(Raw\  intensity\ signal−Mean\ of\ positive\ control)/(Mean\ of\ negative\ control−Mean\ positive\ control)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.
?. [3.1 Preparation of Drug Stocks and Drug Plates]
The generation of the source plate is performed using an Echo 555 acoustic dispenser.
?. [3.6 Data Quality Checks and Downstream Analysis]
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].