Human pancreatic cancer samples

Surgically resected specimens were obtained from pancreatic cancer patients at Kyoto University Hospital. Analyses for human subjects were approved by the Ethical Committee of Kyoto University Hospital. All experiments have been conducted in accordance with the Declaration of Helsinki as well as the guidelines and regulations of the Committee.

Organoid culture

Mouse pancreatic organoids (StemCell Technologies #70933) were cultured in PancreaCult Organoid Growth Medium (StemCell Technologies #06040) according to the manufacturer’s protocol.

Patient-derived pancreatic cancer organoids were established from fresh surgical specimens obtained from patients who underwent surgical resection at Kyoto University Hospital, approved by the Ethics Committees (R1281) and by the Ethical Committee of Sumitomo Pharma (2017–04). The pathological characteristics of the primary tumor are presented in Table 1. Primary tumor tissue samples were processed as previously reported, with some modifications [7, 8, 14]. Briefly, the cell aggregates were embedded in Matrigel (Corning, Cambridge, MA, USA) and covered by a medium composed of 50% L-WRN conditioned medium (ATCC) containing L-Wnt3A, R-spondin 3, and Noggin, consisting of Advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 5% FBS, 2 mmol/l L-Alanyl-L-Glutamine (Wako, Tokyo, Japan), 100 units/ml penicillin, 0.1 mg/ml streptomycin (Nacalai Tesque), 2.5 μg/ml Plasmocin prophylactic (Invitrogen), 10 μM Y-27632 (Tocris Bioscience), 1x B27 Supplement (Thermo Fisher Scientific, Waltham, MA, USA), 1 μM SB431542 (Tocris Bioscience), 100 ng/ml recombinant human fibroblast growth factor-basic (bFGF; Thermo Fisher Scientific), and 20 ng/ml recombinant human epidermal growth factor (EGF; Thermo Fisher Scientific). After confirming several passages of the PDOs, the organoids were also cultured with the following “complete medium” consisting of Advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 2 mM Glutamax-I (Wako, Tokyo, Japan), 10 mM HEPES (Thermo Fisher Scientific), 100 units/ml penicillin, 0.1 mg/ml streptomycin (Nacalai Tesque), 10 μM Y-27632 (Tocris Bioscience), 1x B27 Supplement (Thermo Fisher Scientific, Waltham, MA, USA), 1 μM inhibitor of transforming growth factor-β (TGF-β) type I receptor, SB431542 (Tocris Bioscience), 50 ng/ml Wnt3A(R&D systems), 500 ng/ml R-spondin-1 (Peprotech Inc), 100 ng/ml Noggin (R&D systems), 100 ng/ml bFGF (Peprotech Inc), and 50 ng/ml EGF (Peprotech Inc). For culture of SMAD4-mutants, Sph18–06 was cultured in the complete medium without SB431542 (Tocris Bioscience). The passage number of PDOs was as follows: for in vitro experiments, Sph18–02 (≥P19), Sph18–06 (≥P8), Sph18–14 (≥P23), Sph18–21 (≥P12), Sph18–25 (≥P12), Sph19–07 (≥P12), Sph19–14 (≥P10), Sph19–22 (≥P6); and for in vivo transplantation experiments, Sph18–02 (≥P25), Sph18–06 (≥P16), Sph18–14 (≥P31), Sph18–21 (≥P31), Sph18–25 (≥P28), Sph19–07 (≥P19), Sph19–14 (≥P15), Sph19–22 (≥P16). Cell proliferation of PDOs was examined by seeding the same number of cells in triplicate and counting the cell number at day 7 using a Countess II FL automated cell counter (Thermo Fisher Scientific). Bright field images of PDOs were taken on an inverted microscope system (Olympus, IX73, 10x or 20x objective lenses).

Table 1 Additional data that provide clinical information about the established PDOs

For evaluation of effects of kinase inhibitor compounds on PDOs, cells of PDOs, Sph18–06 and Sph18–14, were dissociated, and the same number of cells (1 × 103 cells/well) were plated in each of 384-well plates. After three days in culture, compounds from kinase inhibitor libraries (Selleck chemicals, L1200 and L2000) were added and further cultured for five days. Cell viability was examined by CellTiter-Glo 3D Reagent (Promega) according to the manufacturer’s instructions.

Genetic mutation analysis of organoid lines

Organoids were dissociated, and DNA was isolated using the QIAamp DNA Mini Kit (Qiagen). Genetic mutations of PDOs were determined by next generation sequencing analysis using the Ion AmpliSeq 50-gene Cancer Hotspot Panel v2 with additional genes (Thermo Fisher Scientific, sequencing, mapping alignment, and annotation was outsourced to Takara Bio, Kusatsu, Japan). The panel included mutation hotspots for the following cancer-related genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, JAK2, JAK3, IDH1, IDH2, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VH, ARID1A, ARID2, ATRX, BAP1, DAXX, MEN1, RNF43, and TGFBR2. To preserve the quality of mutation detection, mutation candidates with homopolymer regions with lengths of ≥5 base pairs and those with sequencing coverage of 250 or fewer base pairs were excluded from analysis.

Cell culture

The human pancreatic cancer cell lines, Panc-1 and BxPC-3 (ATCC), were cultured in DMEM or RPMI1640 supplemented with 10% FBS, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Nacalai Tesque) in a 5% CO2 incubator at 37 °C.

Histochemical analysis

For immunohistochemical analysis, 3D-organoids were embedded in iPGell (Geno Staff) and fixed overnight in 4% paraformaldehyde (Nacalai Tesque). Tumor specimens were isolated and fixed overnight in 4% paraformaldehyde (Nacalai Tesque), embedded in paraffin and sectioned at a thickness of 3 or 4 μm. Sections were then deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE). For immunohistochemical analyses, standard IHC procedures were performed in a BOND-RX automated immunostaining machine (Leica) according to the manufacturer’s instructions using anti-CD44 (1:600, Cell Signaling Technologies) and anti-CD133 (1:200, Abnova) antibodies. Images of the stained slides were captured and analyzed using an Aperio ImageScope (Leica, 20x objective lens) or inverted microscope systems (Olympus IX83 or Keyence BZ9000, 10x or 20x objective lenses) with the built-in software and ImageJ.

Western blot and ELISA analysis

Samples were extracted using ice-cold RIPA buffer (Pierce) and separated using SDS-PAGE in 10–20% acrylamide gel (Wako). Proteins were transferred onto PVDF membranes using the iBlot dry transfer system (Invitrogen), and blocked using 3% skim milk (Wako). Proteins were incubated with the primary antibodies overnight at 4 °C. The primary antibodies used in this study were as follows: anti-PROM1/CD133 (1:1000, Abnova), anti-SOX2 (1:1000, Cell Signaling Technologies), anti-CD24 (1:500, Sigma Aldrich), anti-CA19–9 (1:500, Gene Tex). Samples were then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 60 minutes at room temperature. HRP-conjugated anti-beta actin (1:2000, Cell Signaling Technologies) antibody was also used as a loading control. Immunoreactive protein bands were identified with chemiluminescent HRP substrate (SuperSignal West Pico Plus Luminol/Enhancer Solution). Chemiluminescence signals were captured and analyzed using an ImageQuant LAS 500 (Cytiva) and ImageJ. For measurement of CA19–9 in cultured medium, same number of PDO cells (1 × 105 cells / well) were embedded in Matrigel and cultured with 0.5 mL of the complete medium for 3 days, and the supernatant was collected and stored at − 80 °C until assay. The samples were analyzed using CA19–9 ELISA kit (RayBiotech) according to the manufacturer’s protocol.

PCR array analysis

Total RNA was purified and DNase-treated using the RNeasy Mini Kit (Qiagen). PCR array analysis was performed using RT2 Profiler PCR array (Human Cancer Stem Cells) (PAHS-176Z) (SABiosciences, Frederick, MD, USA) according to the manufacturer’s protocol. Synthesis of cDNA was performed using iScript Reverse Transcription Supermix (Biorad, #1708840). Real time PCR was conducted using CFX-384 (Biorad). Fold changes relative to the control sample were calculated on the Qiagen Data Analysis Webportal (https://dataanalysis.qiagen.com/pcr/arrayanalysis.php). All signals were normalized to the levels of GAPDH and ACTB probes. RT2 Profile PCR Array Human Cancer Stem Cells (PAHS-176Z) was purchased from Qiagen. The assays were performed according to the manufacturer’s instructions.

Flow cytometry

PDO samples were washed once with PBS (Nacalai Tesque), and then cells were dissociated with TrypLE Express (Thermo Fisher Scientific) and centrifuged. Single cell suspensions were washed once with Advanced DMEM/F12 (Thermo Fisher Scientific) containing 10% FBS. Cell pellets were resuspended in PBS containing 1% FBS and incubated for 30 min on ice with 10-fold dilution of the following antibodies: PE/Cy7 anti-CD44 (Bio-legend) and PE/Cy7 control IgG2b antibody (Bio-legend). Samples were passed through a 40 μm cell strainer (BD Biosciences) and resuspended in 500 μL incubation 1x PBS + 2% FBS to reach a final concentration of 106 cells per 100 μl. Flow cytometry was carried out using a MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec). Cell debris was excluded by forward scatter pulse width and side scatter pulse width. Dead cells were excluded by labeling with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). The data were analyzed using software FlowJo (Tree Star, Ashland, OR, USA).

Xenograft assay

All procedures for animal experiments were conducted in compliance with the ARRIVE guidelines and in accordance with the guidelines of the Animal Care and Use Committee at Sumitomo Pharma, Japan. Balb/c (Nude) mice were purchased from Charles River Laboratories Japan (Yokohama, Japan), and NOD/Shi-scid, IL-2RγKO Jic (NOG) mice were purchased from In-Vivo Science Inc. (Kawasaki, Japan). Mice were maintained in cages under standard conditions of ventilation, temperatures (20–26 °C), and lightning (Light/dark: 12 h / 12 h) and kept under observation for 1 week prior to experimentation. Drinking water and standard pellet diets were provided throughout the study. For subcutaneous grafts, 1 × 106 or 3 × 105 cell suspensions were resuspended in 50% Matrigel / 50% Hank’s balanced salt solution (HBSS) (Nacalai Tesque), and transplanted into the flanks of 6- to 8-week-old nude or NOG mice. Tumor size was measured with calipers once or twice a week after the injection. Volumes were calculated by applying the formula v = 0.5 × L × w × h, where v is volume, L is length, w is width and h is height. For the peritoneal dissemination model, PDOs were injected intraperitoneally with 1 or 3 × 106 cells in 100 μL HBSS. For evaluation of the in vivo efficiency of gemcitabine and CHK1 inhibitor, prexasertib, mice with established subcutaneous tumors were randomized by splitting size-matched tumors into two groups (vehicle / gemcitabine or prexasertib), and the mice were subcutaneously administered 10 mg/kg prexasertib twice per day, three times a week. Gemcitabine was administered intraperitoneally at a dose of 30 or 60 mg/kg, two times a week.

Statistics

All values are presented as mean ± SD unless otherwise stated. Statistical analysis was conducted using Prism v6 (GraphPad). Significant differences between groups were determined using a Student’s t-test. P-values < 0.05 were considered significant. Data distribution was assumed to be normal, but this was not formally tested.

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