Abstract 43: A living biobank of triple-negative breast cancer organoids for screening drug sensitivity
Authors: Hong Malin , Gao Lin , Hu Wenlong , Qiu Junying , Peng Zheng , Zhou Wenbin , Zou Chang 
Published: August 2019

Abstract
Introduction: Triple Negative Breast Cancer (TNBC) is the only type of breast cancer for which no specific targeted therapy, it presents drug resistance to targeted therapies of hormomal therapy, HER2-targeting therapy and chemical treatment. At present, due to patient of TNBC suffered with drug resistance, there are no beneficial human cancer models to screening anti-cancer drug in vivo. In this study, we performed TNBC tissues from patients were used for a long-term expansion by using self-proliferating organoids to test drugs in ex vivo.
Methods Triple-negative breast cancer (TNBC) tissues from patients were cut and digested with the human tumor dissociation kit. The digested pellet was resuspended in cold Matrigel and 50μl drops of Matrigel-Cell suspension were allowed to solidify on prewarmed 24-well culture plates at 37°C for 10 min. Upon completed solidification, cytokines and OAD12 medium was added advanced DMEM/F12 medium, the organoids were cultured at 37°C, 5% CO2 incubators. Medium was replaced every 1-2 weeks and TNBC organoids were passaged every 1-3 weeks. For immunofluorescence assay, TNBC organoids were fixed with 2% formaldehyde for 30 min, and then incubated with antibodies. For counting the percentage of tumoroids, four markers of CD44, CD24, CD133 and CD338 were used to identify tumoroids. IC50 of alazoparib (target PARP), ribociclib (CDK4/6) and SNX-2112 (Hsp90) were used for drug sensitivity screens through CCK-8 assay.
Results For establishing a living TNBCs tissues from patients, tissue samples were operated to isolate cells through mechanical and enzymatic digestion. Digested cells were resuspended with BME drops and seeded into organoid culture medium with various factors. The results showed that TNBC organoids were generated efficiently as well as term expansion over 10 passages. For testing whether TNBC organoids match the cutting TNBC tissues type, we performed a classical combined (CD44+; CD24-; CD133+; CD338-) markers in the tumoroids through immunofluorescence staining, the results showed that CD44 and CD133 were highly expressed in TNBC organoids and in the corresponding tissues. In addition, the flow cytometry assay was used to detect expression of positive, it showed that positive rates of tumoroids expressed up to 80%. In order to test TNBC tumoroids as functional in vitro cultured, talazoparib (target PARP), ribociclib (CDK4/6) and SNX-2112 (Hsp90) were used for drug sensitivity screens. The results indicated an effective response to 3 drugs yielding a single IC50. The tumoroids were sensitive to 3 drugs inhibiting PARP, CDK4/6 and Hsp90 signaling pathway, respectively.
Conclusions In this study, we reveal a novel culture system of living TNBC organoids. Described TNBC organoids are available for clinical treatment of drug sensitivity. It may provide a novel of drugs testing for anti-TNBC therapy in ex vivo.
Citation Format: Malin Hong, Lin Gao, Wenlong Hu, Junying Qiu, Zheng Peng, Wenbin Zhou, Chang Zou. A living biobank of triple-negative breast cancer organoids for screening drug sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 43.