Medium-throughput Drug Screening of Patient-derived Organoids from Colorectal Peritoneal Metastases to Direct Personalized Therapy.

Patients with colorectal cancer with peritoneal metastases (CRPMs) have limited treatment options and the lowest colorectal cancer survival rates. We aimed to determine whether organoid testing could help guide precision treatment for patients with CRPMs, as the clinical utility of prospective, functional drug screening including nonstandard agents is unknown. CRPM organoids (peritonoids) isolated from patients underwent parallel next-generation sequencing and medium-throughput drug panel testing ex vivo to identify specific drug sensitivities for each patient. We measured the utility of such a service including: success of peritonoid generation, time to cultivate peritonoids, reproducibility of the medium-throughput drug testing, and documented changes to clinical therapy as a result of the testing. Peritonoids were successfully generated and validated from 68% (19/28) of patients undergoing standard care. Genomic and drug profiling was completed within 8 weeks and a formal report ranking drug sensitivities was provided to the medical oncology team upon failure of standard care treatment. This resulted in a treatment change for two patients, one of whom had a partial response despite previously progressing on multiple rounds of standard care chemotherapy. The barrier to implementing this technology in Australia is the need for drug access and funding for off-label indications. Our approach is feasible, reproducible, and can guide novel therapeutic choices in this poor prognosis cohort, where new treatment options are urgently needed. This platform is relevant to many solid organ malignancies.

Narasimhan V ，Wright JA ，Churchill M ，Wang T ，Rosati R ，Lannagan TRM ，Vrbanac L ，Richardson AB ，Kobayashi H ，Price T ，Tye GXY ，Marker J ，Hewett PJ ，Flood MP ，Pereira S ，Whitney GA ，Michael M ，Tie J ，Mukherjee S ，Grandori C ，Heriot AG ，Worthley DL ，Ramsay RG ，Woods SL ... -  《-》

Patient-Derived Organoids from Multiple Colorectal Cancer Liver Metastases Reveal Moderate Intra-patient Pharmacotranscriptomic Heterogeneity.

Molecular tumor heterogeneity may have important implications for the efficacy of targeted therapies in metastatic cancers. Inter-metastatic heterogeneity of sensitivity to anticancer agents has not been well explored in colorectal cancer. We established a platform for ex vivo pharmacogenomic profiling of patient-derived organoids (PDO) from resected colorectal cancer liver metastases. Drug sensitivity testing (n = 40 clinically relevant agents) and gene expression profiling were performed on 39 metastases from 22 patients. Three drug-response clusters were identified among the colorectal cancer metastases, based primarily on sensitivities to EGFR and/or MDM2 inhibition, and corresponding with RAS mutations and TP53 activity. Potentially effective therapies, including off-label use of drugs approved for other cancer types, could be nominated for eighteen patients (82%). Antimetabolites and targeted agents lacking a decisive genomic marker had stronger differential activity than most approved chemotherapies. We found limited intra-patient drug sensitivity heterogeneity between PDOs from multiple (2-5) liver metastases from each of ten patients. This was recapitulated at the gene expression level, with a highly proportional degree of transcriptomic and pharmacological variation. One PDO with a multi-drug resistance profile, including resistance to EGFR inhibition in a RAS-mutant background, showed sensitivity to MEK plus mTOR/AKT inhibition, corresponding with low-level PTEN expression. Intra-patient inter-metastatic pharmacological heterogeneity was not pronounced and ex vivo drug screening may identify novel treatment options for metastatic colorectal cancer. Variation in drug sensitivities was reflected at the transcriptomic level, suggesting potential to develop gene expression-based predictive signatures to guide experimental therapies.

Bruun J ，Kryeziu K ，Eide PW ，Moosavi SH ，Eilertsen IA ，Langerud J ，Røsok B ，Totland MZ ，Brunsell TH ，Pellinen T ，Saarela J ，Bergsland CH ，Palmer HG ，Brudvik KW ，Guren T ，Dienstmann R ，Guren MG ，Nesbakken A ，Bjørnbeth BA ，Sveen A ，Lothe RA ... -  《-》

Organoids from colorectal peritoneal metastases as a platform for improving hyperthermic intraperitoneal chemotherapy.

Patients with peritoneal metastases from colorectal cancer have a poor prognosis. If the intraperitoneal tumour load is limited, patients may be eligible for cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC). This treatment has improved overall survival, but recurrence rates are high. The aim of this study was to create a preclinical platform for the development of more effective intraperitoneal chemotherapy strategies. Using organoid technology, five tumour cultures were generated from malignant ascites and resected peritoneal metastases. These were used in an in vitro HIPEC model to assess sensitivity to mitomycin C (MMC) and oxaliplatin, the drugs used most commonly in HIPEC. The model was also used to test a rational combination treatment involving MMC and inhibitors of the checkpoint kinase ATR. MMC was more effective in eliminating peritoneal metastasis-derived organoids than oxaliplatin at clinically relevant concentrations. However, the drug concentrations required to eliminate 50 per cent of the tumour cells (IC50) were higher than the median clinical dose in two of five organoid lines for MMC, and all five lines for oxaliplatin, indicating a general resistance to monotherapy. ATR inhibition increased the sensitivity of all peritoneal metastasis-derived organoids to MMC, as the IC50 decreased 2·6-12·4-fold to well below concentrations commonly attained in clinical practice. Live-cell imaging and flow cytometric analysis showed that ATR inhibition did not release cells from MMC-induced cell cycle arrest, but caused increased replication stress and accelerated cell death. Peritoneal metastasis-derived organoids can be used to evaluate existing HIPEC regimens on an individual-patient level and for development of more effective treatment strategies. Surgical relevance Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has improved prognosis of patients with peritoneal metastases from colorectal cancer, but disease recurrence is common. More effective and personalized HIPEC is urgently needed. Organoid technology is frequently used for drug screens, as patient-derived organoids can accurately predict clinical therapeutic response in vitro. A panel of organoids was established from peritoneal metastases from colorectal cancer and used to develop a model for testing HIPEC regimens in vitro. Patient-derived organoids differed in sensitivity to commonly used chemotherapeutics, in line with variable clinical outcomes following cytoreductive surgery-HIPEC. Combining MMC with an ATR inhibitor improved the efficacy of MMC. Peritoneal metastasis-derived organoids can be used as a platform to test novel (combination) strategies that increase HIPEC efficacy. In the future, organoids could be used to select patent-tailored HIPEC regimens.

Ubink I ，Bolhaqueiro ACF ，Elias SG ，Raats DAE ，Constantinides A ，Peters NA ，Wassenaar ECE ，de Hingh IHJT ，Rovers KP ，van Grevenstein WMU ，Laclé MM ，Kops GJPL ，Borel Rinkes IHM ，Kranenburg O ... -  《-》

Appendiceal Cancer Patient-Specific Tumor Organoid Model for Predicting Chemotherapy Efficacy Prior to Initiation of Treatment: A Feasibility Study.

Votanopoulos KI ，Mazzocchi A ，Sivakumar H ，Forsythe S ，Aleman J ，Levine EA ，Skardal A ... -  《-》

Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids.

Drug sensitivity testing of patient-derived tumor organoids (PDTOs) is a promising tool for personalizing cancer treatment. Here, we present a protocol for generation of and high-throughput drug testing with PDTOs. We describe detailed steps for PDTO establishment from colorectal cancer tissues, preparation of PDTOs for high-throughput drug testing, and quantification of drug testing results using image analysis. This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies, along with exploring the activity of new agents, for translational research. For complete details on the use and execution of this protocol, please refer to Tan et al.1.

Tan T ，Mouradov D ，Gibbs P ，Sieber OM ... -  《-》