Table 3.

Recommendations for CRC organoid drug screening

Topic	Evidence	Recommendations
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. GRAUC 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 GRAUC 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.

5-FU 5 Fluorouracil, CTG CellTiter-Glo, CQ CyQUANT, DRC Drug response curve, GR_AUC area under the growth rate inhibition curve, NAC N-acetylcysteine, PDO Patient-derived organoid, SN-38 active metabolite of irinotecan