gms | German Medical Science

Objective: Patient-derived tumor organoids (TOs) are mini-tumors generated from tumor tissues within a few days while preserving their genotype and phenotype and maintaining the cellular heterogeneity and key components of the tumor microenvironment. This exciting new technology provides representative avatars of the patient tumor in large numbers and thus can be exploited for drug discovery and drug screening purposes more efficiently than conventional cell lines or xenografted mouse models. In our study, we established standardized TOs from glioblastoma (GBM) and implemented them in an automated large-scale drug screening.

Methods: Fresh tumor tissue was dissociated into a single cell suspension. Cells were seeded into anti-adhesive 384-well plates to allow the formation of standardized TOs. GBM TOs were assessed by light microscopy, immunostainings, and RNA-seq. Automated high-throughput drug screening (aHTS) on TOs was performed using the industry-leading liquid handler Hamilton MicroLAB STAR. The drug library consisted of 166 FDA-approved antineoplastic drugs. Cell viability was assessed by CellTiter-Glo3D.

Results: Over one thousand standardized TO were generated per patient with a success rate of over 90%. Morphologically, TOs fully compacted after two days and the size maintained stable over 10 days, with a slow outgrowth in some patient cases. TOs proliferated over time as seen as a steady increase of the ATP signal. Stainings for tumor cells (GFAP), the tumor microenvironment (myeloid cells: CD68, T cells: CD3), and extracellular matrix (Tensascin C) confirmed a strong resemblance of TOs to their corresponding parental tumor, which was further substantiated by RNA-seq. Next, aHTS on TOs from 11 GBM patients was performed demonstrating strong heterogeneity of drug responses. The top ten best-performing drugs among 11 GBM patients consisted of 35 compounds targeting 14 different modes of action. Proteasome (bortezomib, ixazomib, carfilzomib, and marizomib; average viability 25%) and HDAC-inhibitors (romidepsin and panobinostat; average viability 28%) demonstrated the highest efficacy.

Conclusion: We established a protocol to form standardized TO from GBMs resembling the parental tumors and demonstrating a high formation success rate. Furthermore, by utilizing the automated high-throughput drug screening platform, we identified proteasome inhibitors and HDAC-inhibitors as highly efficacious drugs with large inter-patient-individual drug responses.