1. Volume 27, Issue 4, Pages 1265-1276.e4 (April 2019)
Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma 
Yoshimasa Saito, Toshihide Muramatsu, Yae Kanai, Hidenori Ojima, Aoi Sukeda, Nobuyoshi Hiraoka, Eri Arai, Yuko Sugiyama, Juntaro Matsuzaki, Ryoei Uchida, Nao Yoshikawa, Ryo Furukawa, Hidetsugu Saito 
Cell Reports 
Volume 27, Issue 4, Pages e4 (April 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 1265-1276.e4DOI: (10.1016/j.celrep.2019.03.088)
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3. Figure 1 Establishment of Organoids Derived from IHCC
(A) Macroscopic appearance of the surgically resected IHCC tissue specimen 9T. The tumor tissue specimens were cut into small pieces and subsequently subjected to organoid culture.
(B) Microscopic appearance of the primary IHCC tissue specimens 1T, 9T, and 24T, and organoids derived from them. Scale bars: 100 μm (H&E staining) and 1,000 μm (bright-field image). M/D, moderately differentiated; P-M/D, poorly to moderately differentiated.
(C) Similarity between the primary IHCC tissues and established organoids. H&E and PAS staining and immunohistochemistry for CK7 and MUC1 in the primary tissue of IHCC (1T) and organoids established using this specimen. Scale bars: 100 μm.
See also Figures S1 and S5 and Table S1.
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4. Figure 2
Establishment of Organoids Derived from PDA, GBC, and NEC of the Ampulla of Vater
(A) Macroscopic appearance of the surgically resected PDA tissue specimen 3T. The region of tumor tissue used for the establishment of organoids is shown in the square.
(B) Bright-field images of organoids derived from PDA tissue specimen 3T. Scale bars: 1,000 μm.
(C) Similarity between the primary PDA tissues and established organoids. H&E staining and immunohistochemistry for CK7 in the primary tissue of PDA specimen 3T and organoids established from it. Scale bars: 100 μm.
(D) Macroscopic appearance of the surgically resected GBC tissue specimen 19T. Tumor tissue specimens were cut into small pieces and subsequently subjected to organoid culture.
(E) Bright-field images of organoids derived from GBC tissue specimen 19T. Scale bars: 1,000 μm.
(F) Similarity between the primary GBC tissues and established organoids. H&E and PAS staining and immunohistochemistry for CK7 in the primary tissue of GBC specimen 19T and organoids established from it. Scale bars: 100 μm.
(G) Macroscopic appearance of the surgically resected NEC of the ampulla of Vater, specimen 36T. Tumor tissue specimens were cut into small pieces and subsequently subjected to organoid culture.
(H) Bright-field images of organoids derived from NEC of the ampulla of Vater specimen 36T. Scale bars: 1,000 μm.
(I) Similarity between the primary NEC tissues and established organoids. HE staining and immunohistochemistry for synaptophysin and chromogranin A in the primary tissue of NEC of the ampulla of Vater specimen 36T and organoids established from it. Scale bars: 100 μm.
See also Figures S1 and S5 and Table S1.
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5. Figure 3
Culture Courses of Organoids Derived from BTCs and Non-cancer Organoids
(A) Passage numbers and time in culture of 6 cancer organoid lines, including specimens 1T (IHCC), 3T (PDA), 9T (IHCC), 19T (GBC), 24T (IHCC), and 36T (NEC of the ampulla of Vater). These cancer organoid lines were cultured stably for >1 year.
(B) Passage numbers and time in culture of organoids derived from non-cancerous GB tissues (19N and 26N) and non-cancerous BD tissues (30N and 32N). Non-cancer organoids showed robust proliferation at an early stage, but they ceased proliferation at approximately passage 15.
(C) Passage numbers and time in culture of organoids established using BDC (25T) and PDA (28T) tissues for which long-term culture failed.
See also Figure S2 and Tables S1 and S2.
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6. Figure 4
Gene Expression Profiles of Organoids Derived from BTCs and Non-cancer Organoids
(A) PCA map for gene expression profiles of organoids derived from BTCs and non-cancer organoids. Cancer organoid lines with different passages (1T P7, 1T P32, 1T P58, 1T P177, 3T, 9T, 19T, 24T, and 36T) and the NCC-CC1 cell line were clearly segregated from non-cancer organoids (26N, 29L, 30N, 32N, 33N, 34N, 35L, and 37L) and organoids for which long-term culture failed (22T, 25T, 26T, 28T, 30T, and 32T). This map also demonstrated that xenograft and primary tumor tissue samples (1T Xenograft and 9T Tissue) were segregated from cancer and non-cancer organoids.
(B) Heatmap showing the gene expression profiles associated with cholangiocarcinoma (CC) and hepatocellular carcinoma (HCC) in cancer organoid lines with different numbers of passages (1T P7, 1T P32, 1T P58, 1T P177, 3T, 9T, 19T, 24T, and 36T), xenograft and tumor tissues (1T-Xe and 9T-Ti), and the NCC-CC1 cell line (CC1).
(C) Heatmap showing genes differentially expressed between cancer and non-cancer organoids (fold change >4 and Bonferroni-corrected p < 0.01).
(D) Heatmap showing miRNAs differentially expressed between cancer and non-cancer organoids (fold change >2 and Bonferroni-corrected p < 0.05).
(E) Survival plots for groups with high and low expressions of SOX2 in the TCGA-CHOL cohort. The best cutoff levels were identified by receiver operating characteristic (ROC) analysis, and the significance of differences between the 2 groups was assessed by log-rank test (p = ).
See also Figures S3 and S4 and Tables S1 and S2.
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7. Figure 5
Genetic Alterations of Organoids Derived from BTCs and Their Primary Tumor Tissues
(A) Concordance rate of exonic variants between the primary tumor tissues we obtained from BTC patients and the corresponding organoids (O) at early and late passages.
(B) Overall distribution of base substitutions detected in all of the samples of both cancer organoids and the corresponding BTC tissues.
(C) Proportion of exonic variations detected in each cancer organoid line at early and late passages and the corresponding BTC tissues.
(D) Overview of the mutations detected in cancer organoid lines (O) at early and late passages and the corresponding BTC tissues, as well as organoids established from tumor tissues (2T and 25T) for which long-term culture failed.
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8. Figure 6
Drug Sensitivity Associated with Gene Mutations and Gene Expression Profiles
(A) Dose-response curves after 6 days of treatment of organoids derived from BTCs with nutlin-3a in association with TP53 mutation status. All of the experiments were carried out in triplicate, and data are represented as means ± SDs.
(B) Dose-response curves after 6 days of treatment of organoids derived from BTCs with erlotinib in association with KRAS mutation status. All of the experiments were carried out in triplicate, and data are represented as means ± SDs.
(C) Heatmap for genes that were differentially expressed between cancer organoids showing high and low sensitivity to erlotinib (fold change >16).
(D) Survival plots for groups with high and low expressions of KLK6 and CPB2 in the TCGA-CHOL cohort. The best cutoff levels were identified by ROC analysis, and the significance of differences between the 2 groups was assessed by log-rank test (p = and 0.046).
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9. Figure 7
Drug Screening Using a Compound Library of Clinically Used Drugs
(A) Protocol for the treatment of organoids with various compounds. Cells from organoid line 1 were plated (1.2 × 103/well) and cultured for 4 days, and compounds were added to the culture media at a final concentration of 0.1 μM. After 6 days of treatment with the compounds, cell viability was examined by WST assay.
(B) A representative example of organoid-based drug screening using a compound library. When the ratio of the average level of cell viability in the presence of the compounds (n = 3) compared to the control (0.1% DMSO, n = 3) was <0.5, the suppressive effect was considered to be significant. In this case, compounds (d), (e), and (f) were considered to have an ability to suppress organoids. Data are represented as means ± SDs. Scale bars: 1,000 μm.
(C) Use of organoids for drug screening using a compound library. From a library of 339 medicines already in clinical use, we successfully screened 22 compounds that were able to significantly suppress organoids. Among them, amorolfine, fenticonazole, cerivastatin, and talipexole were identified, as well as already established anticancer agents.
(D) Dose-response curves after 6 days of treatment of organoids derived from BTCs with amorolfine, cerivastatin, and fenticonazole. These drugs exhibited a significant suppressive effect against organoids derived from BTCs at 0.1 or 1.0 μM. Bright-field images of IHCC organoids (1T) after treatment are shown. All of the experiments were carried out in triplicate, and data are represented as means ± SDs. Scale bars: 1,000 μm.
(E) Effects of amorolfine, cerivastatin, and fenticonazole on the growth of normal biliary epithelial cells (HIBEpiC). All of the experiments were carried out in triplicate, and data are represented as means ± SDs.
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