Table 3 Recommendations for CRC organoid drug screening

1. Medium composition

Resistance to oxaliplatin-based treatment increases with NAC in screening medium.

Remove NAC from screening medium in oxaliplatin-based drug screens.

2. Readouts

CellTiter-Glo measurements are in agreement with CyQUANT measurements.

Both readouts can be used. CyQUANT provides the advantage of performing drug screens with 5–10 PDOs per well.

3. DRC metrics

GR metrics showed better correlation with patient response than percentage viability metrics. GR_AUC is the most robust DRC metric and best reflects PDO and patient response.

Apply GR metrics to correct for confounders in organoid drug sensitivity, related to differences in natural cell division rate. Employ GR_AUC for comparison with patient response, or GR₅₀(2) if a clear lower curve plateau is present.

4. DRC fitting

PDOs exhibit a biphasic drug response to 5-FU & oxaliplatin.

Apply a biphasic model for DRC fitting instead of a log-logistic model.

5. Combination treatment set-up

SN-38 & 5-FU in a ratio combination screen did not reflect patient response to 5-FU & irinotecan. Oxaliplatin & 5-FU with a fixed oxaliplatin concentration did not reflect patient response to 5-FU & oxaliplatin.

Use a fixed concentration of SN-38 and increase the 5-FU concentration for 5-FU & SN-38 combination screens. Use a concentration ratio 5-FU:oxaliplatin for oxaliplatin-based combination screens.