Schoonbeek, M.;Valova, S.;Swaak, S.;Looze, E.;Watzeels, M.;Brink, L.;Roman, M.;Velzen, J.;ODuibhir, E.;Langenberg, K.;Wienke, J.;Hooff, S.;Boogaard, M.;Eising, S.;Molenaar, J.

High-risk neuroblastoma patients face poor survival despite intensive treatment. Drug testing using patient-derived models can support therapy prioritization for precision medicine and drug development. Models incorporating tumour microenvironmental components, such as co-cultures and short-term cultured patient samples containing substantial non-malignant cell fractions, could better recapitulate microenvironment-dependent drug responses. However, conventional viability assays measure the combined signal from all viable cells in a well and therefore cannot determine tumour-specific drug responses. Here, we establish a microscopy-based readout to quantify cell-type-specific viability in two complementary settings: neuroblastoma-PBMC co-cultures and freshly dissociated patient tumour samples. In the co-cultures, PBMCs were pre-labelled with a cell-tracking dye, and Calcein staining was used to independently quantify the viability of tumour cells and PBMCs in the same well. The Calcein-based viability readout correlated strongly with conventional CellTiter-Glo measurements and was compatible with automated high-throughput drug screening. The imaging workflow enabled identification of compounds with differential efficacy in co-culture versus monoculture and distinguished tumour-specific effects from PBMC toxicity. The microscopy-based viability readout was further adapted to short-term cultured patient samples. Neuroblastoma tumour cells were distinguished from the non-malignant cells using a combination of tumour-specific surface markers NCAM, L1CAM and B7H3. This enabled determination of tumour fractions and measurement of tumour-specific drug responses. Tumour fractions varied substantially between patient samples, highlighting the importance of tumour-specific viability measurements. Together, the microscopy-based viability readout for co-cultures and patient samples enables scalable assessment of tumour-specific drug responses.