RESUMEN
Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry.
Asunto(s)
Neoplasias Óseas/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Organoides/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Andrógenos/farmacología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Benzamidas/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Nitrilos/farmacología , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Virales/genética , Receptores Virales/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Trasplante Heterólogo , Internalización del VirusRESUMEN
Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient's bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.
