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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

ANTECEDENTES: Los pacientes con metástasis peritoneales (peritoneal metastasis, PM) de cáncer colorrectal tienen un mal pronóstico. Si la carga tumoral intraperitoneal es reducida, los pacientes pueden ser candidatos a cirugía citorreductora seguida de quimioterapia intraperitoneal hipertérmica (hyperthermic intraperitoneal chemotherapy, HIPEC). Este tratamiento ha mejorado la supervivencia global, pero las tasas de recidiva son altas. El objetivo de este estudio fue crear una plataforma preclínica para el desarrollo de las estrategias de quimioterapia intraperitoneal más efectivas. MÉTODOS: Mediante la utilización de la tecnología de organoides, se generaron cinco cultivos tumorales a partir de ascitis maligna y PM resecadas. Se utilizó un modelo de HIPEC in vitro para evaluar la sensibilidad a la mitomicina C (mitomycin C, MMC) y al oxaliplatino, los fármacos más utilizados en la HIPEC. El modelo también se usó para probar un tratamiento combinado de MMC e inhibidores de control inmunitario de la quinasa ATR. RESULTADOS: A concentraciones clínicamente relevantes, la MMC fue más efectiva que el oxaliplatino para eliminar los organoides derivados de PM. Sin embargo, las concentraciones de fármaco necesarias para eliminar el 50% de las células tumorales (IC50) fueron más elevadas que la mediana de la dosis clínica en 2/5 (MMC) o 5/5 (oxaliplatino) de las líneas de organoides, lo que indica una resistencia general a la monoterapia. La inhibición de ATR aumentó la sensibilidad a MMC de todos los organoides derivados de PM, ya que la IC50 disminuyó (2,6-12,4 veces) a concentraciones muy por debajo de las que se alcanzan comúnmente en la práctica clínica. Los análisis de viabilidad celular y de citometría de flujo (FACS) mostraron que la inhibición de ATR no liberaba células tras la detención del ciclo celular inducida por la MMC, sino que causaba un aumento en el estrés replicativo y muerte celular acelerada. CONCLUSIÓN: Se pueden usar los organoides derivados de PM para evaluar los regímenes HIPEC existentes a nivel del paciente individual y para desarrollar estrategias terapéuticas más efectivas.

Oncogenic KRAS sensitises colorectal tumour cells to chemotherapy by p53-dependent induction of Noxa.

BACKGROUND: Oxaliplatin and 5-fluorouracil (5-FU) currently form the backbone of conservative treatment in patients with metastatic colorectal cancer. Tumour responses to these agents are highly variable, but the underlying mechanisms are poorly understood. Our previous results have indicated that oncogenic KRAS in colorectal tumour cells sensitises these cells to chemotherapy. METHODS: FACS analysis was used to determine cell-cycle distribution and the percentage of apoptotic and mitotic cells. A multiplexed RT-PCR assay was used to identify KRAS-controlled apoptosis regulators after exposure to 5-FU or oxaliplatin. Lentiviral expression of short-hairpin RNAs was used to suppress p53 or Noxa. RESULTS: Oncogenic KRAS sensitised colorectal tumour cells to oxaliplatin and 5-FU in a p53-dependent manner and promoted p53 phosphorylation at Ser37 and Ser392, without affecting p53 stabilisation, p21 induction, or cell-cycle arrest. Chemotherapy-induced expression of the p53 target gene Noxa was selectively enhanced by oncogenic KRAS. Suppression of Noxa did not affect p21 induction or cell-cycle arrest, but reduced KRAS/p53-dependent apoptosis after exposure to chemotherapy in vitro and in tumour xenografts. Noxa suppression did not affect tumour growth per se, but strongly reduced the response of these tumours to chemotherapy. CONCLUSION: Oncogenic KRAS determines the cellular response to p53 activation by oxaliplatin or 5-FU, by facilitating apoptosis induction through Noxa.