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Circulating tumour cell (CTC)–derived organoids have the potential to provide a powerful tool for personalised cancer therapy but are restrained by low CTC numbers provided by blood samples. Here, we used diagnostic leukapheresis (DLA) to enrich CTCs from patients with metastatic prostate cancer (mPCa) and explored whether organoids provide a platform for ex vivo treatment modelling. We prospectively screened 102 patients with mPCa and performed DLA in 40 patients with ≥5 CTCs/7.5 mL blood. We enriched CTCs from DLA using white blood cell (WBC) depletion alone or combined with EpCAM selection. The enriched CTC samples were cultured in 3D to obtain organoids and used for downstream analyses. The DLA procedure resulted in a median yield of 5312 CTCs as compared with 22 CTCs in 7.5 mL of blood. Using WBC depletion, we recovered 46% of the CTCs, which reduced to 12% with subsequent EpCAM selection. From the isolated and enriched CTC samples, organoid expansion succeeded in 35%. Successful organoid cultures contained significantly higher CTC numbers at initiation. Moreover, we performed treatment modelling in one organoid cell line and identified substantial tumour heterogeneity in CTCs using single cell DNA sequencing. DLA is an efficient method to enrich CTCs, although the modest success rate of culturing CTCs precludes large scale clinical application. Our data do suggest that DLA and subsequent processing provides a rich source of viable tumour cells. Therefore, DLA offers a promising alternative to biopsy procedures to obtain sufficient number of tumour cells to study sequential samples in patients with mPCa. NL6019. • Leukapheresis offers 240-fold more circulating tumour cells (CTCs) than blood samples. • Using optimised methods, 46% of the CTCs were recovered. • Organoid expansion succeeded in 35% of the patient samples. • Substantial intra-patient heterogeneity was suggested by single cell DNA sequencing. • CTC derived organoid line reflected patient's treatment response. [ABSTRACT FROM AUTHOR]

• Steroid measurements in mechanically separated plasma and serum are comparable. • Levels in CellSave are slightly lower than in serum, but within the acceptable range. • Plasma from CellSave tubes can be used for multi-steroid profiling of CRPC patients. Steroid hormones are essential signalling molecules in prostate cancer (PC). However, many studies focusing on liquid biomarkers fail to take the hormonal status of these patients into account. Steroid measurements are sensitive to bias caused by matrix effects, thus assessing potential matrix effects is an important step in combining circulating tumour DNA (ctDNA) analysis with hormone status. We investigated the accuracy of multi-steroid hormone profiling in mechanically-separated plasma (MSP) samples and in plasma from CellSave Preservative (CS) tubes, that are typically used to obtain ctDNA, compared to measurements in serum. We performed multiplex steroid profiling by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in samples obtained from ten healthy controls and ten castration-resistant prostate cancer (CRPC) patients. Steroid measurements were comparable between MSP and serum. A small but consistent decrease of 8–21% compared to serum was observed when using CS plasma, which was considered to be within the acceptable margin. The minimal residual testosterone levels of CRPC patients could be sensitively quantified in both MSP and CS samples. We validated the use of MSP and CS samples for multi-steroid profiling by LC–MS/MS. The optimised use of these samples in clinical trials will allow us to gain further insight into the steroid metabolism in PC patients. [ABSTRACT FROM AUTHOR]