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  • br Figure See previous page Organoid responses to combined t

    2020-08-12


    Figure 5. (See previous page). Organoid responses to combined treatment with epirubicin, oxaliplatin, and 5-FU. Quantification of flow cytometric cell viability analysis at 0, 24, 48, 72, and 96 hours after treatment with combined epirubicin, oxaliplatin, and 5-FU of (A) huTGOs and (B) huFGOs. Representative flow cytometry dot plots at 0, 24, 48, 72, and 96 hours after treatment with combined epirubicin, oxaliplatin, and 5-FU in (C) huTGO2 and (D) huTGO7. *P < .05 compared with t ¼ 0 hours. Each assay was run in triplicate for each individual organoid line.
    170 Steele et al Cellular and Molecular Gastroenterology and Hepatology Vol. 7, No. 1
    2019 Human-Derived Gastric Cancer Organoids 171
    Figure 7. IC50 values for huTGO1 dose-response curves. IC50 values for
    tumor-derived gastric organoids (huTGO) treated with (A) epirubicin with or without HER2I, (B) oxali-platin with or without HER2I, or (C) 5-FU with or without HER2I. CI, confi-dence interval.
    optimized these cultures such that organoids can be directly grown in a 96-well plate format and used for drug testing within 3 days of the patient’s surgery. Thus, it is feasible to inform the clinician of possible treatment options within 5–6 days of surgery.
    Investigating the impact of the endogenous environment of the stomach on tumor growth is an important direction of
    our future research plans. An orthotopic transplantation model using patient-derived gastric cancer organoids was developed. Organoids that were transplanted into the submucosa below the gastric epithelium of NSG mouse stomachs developed into adenocarcinoma, and this was confirmed by a board-certified pathologist. The lesions were originated from human organoids as
    Figure 6. (See previous page). HER2 CL316 , 243 and responsiveness to HER2 inhibition in patient-derived gastric cancer organoids. (A) Immunofluorescence of HER2 expression (red) in huTGOs. Dose-response curves generated by using (B) huTGO1, (C) huTGO2, (D) huTGO4, and (E) huTGO6 organoid lines in response to epirubicin, oxaliplatin, or 5-FU with or without HER2 inhibitor (HER2I). Each assay was run in triplicate for each individual organoid line.
    172 Steele et al Cellular and Molecular Gastroenterology and Hepatology Vol. 7, No. 1
    documented by the positive immunostaining using anti-human histone antibody. Organoid-derived lesions were highly proliferative, and huTGO1 and huTGO2 differentially expressed CK7 and CK20. The expressions of CK7 and CK20 are often used as prognostic markers for gastric cancer.20 Cytokeratin (CK), an intermediate filament that is expressed in epithelial cells, plays an important role as a cytoskeletal component in the maintenance of cell morphology. The CK gene has 20 subtypes, and the expression of CK depends primarily on the epithelial cell type and the degree of differentiation.21 Early studies report that the expression of CK7 and/or CK20 showed a tendency 
    Figure 8. IC50 values for huTGO2 dose-response curves. IC50 values for
    tumor-derived gastric organoids (huTGO) treated with (A) epirubicin with or without HER2I, (B) oxali-platin with or without HER2I, or (C) 5-FU with or without HER2I. CI, confi-dence interval.
    toward a high positive rate in the differentiated type of gastric cancer.20 Our data showed that whereas CK7 was highly expressed in lesions arising from the transplant of TGO1 and 2 organoid lines, CK20 was not expressed in TGO1-derived transplants. This is significant because TGO1 was an organoid line derived from a patient with a mixed, poorly differentiated, and intestinal-type gastric cancer. In support of our studies, a recent study demonstrated the development of an orthotopic mouse model whereby gastric cancer cell lines tagged with luciferase and injected into the subserosa of the stomach allows for monitoring of primary tumor growth and metastasis in real-time.22 Our RNA
    2019 Human-Derived Gastric Cancer Organoids 173
    Figure 9. IC50 values for huTGO4 dose-response curves. IC50 values for
    tumor-derived gastric organoids (huTGO) treated with (A) epirubicin with or without HER2I, (B) oxali-platin with or without HER2I, or (C) 5-FU with or without HER2I. CI, confi-dence interval.
    sequencing data suggest that the use of gastric cancer cell lines may be a limitation because the patient-derived organoids express a transcriptional profile more similar to the patient’s primary tumor tissue than the profile of gastric cancer cell lines. Thus, we advance these recent studies by demonstrating that orthotopic transplantation of cancer-derived organoid lines is clinically relevant and may recapitulate tumor growth and metastasis in vivo.