br Differentially In GO False br GO GO names
Differentially In GO False
GO GO names expressed category discovery rate
antigen via MHC class II
in a shaking incubator at 37 C. The tissue fragments were washed with Hank’s balanced salt solution twice, and pre-warmed incubation buffer (RPMI supplemented with colla-genase [1.5 mg/mL] and hyaluronidase [20 mg/mL]) was added to the fragments and incubated at 37 C for 30 minutes in a shaking incubator. The digest was diluted with 20 mL DPBS without Ca2þ and Mg2þ supplemented with antibiotics and filtered through a 70-mm filter. The cells were centrifuged at 1200 rpm for 5 minutes and embedded into Matrigel supplemented with gastric growth medium as described above.
Orthotopic Transplantation of Gastric Organoids
All mouse studies were approved by the University of Cincinnati Institutional Animal Care and Use Committee that maintains an American Association of Assessment and Accreditation of Laboratory Animal Care facility. Orthotopic transplantation of gastric organoids was performed in NOD scid gamma (NSG) mice according to a previously published protocol.29 Briefly, an acetic Actinomycin D injury was induced. After injury, approximately 500 organoids were resuspended in 1:1 Matrigel/PBS solution and injected within the submu-cosa. Stomach tissues were collected 14, 30, and 60 days after transplantation.
Mouse Xenograft Assay
Xenograft assays were performed by injecting approxi-mately 500 organoids subcutaneously in the right flank of NSG mice. Tumor dimensions were measured every 3–7 days.
Stomach tissues were collected and fixed in 4% para-formaldehyde for 16 hours, and longitudinal sections were paraffin-embedded and sectioned at 5 mm. Tissue slides were deparaffinized and boiled in antigen citrate buffer (Vector Laboratories, Burlingame, CA; H3300) for 10 minutes. Sections were then blocked with 20% donkey serum for 20 minutes and immunostained with primary
antibodies overnight at 4 C, followed by incubation with secondary antibodies for 1 hour. Whole mount staining of gastric organoids derived from fresh tissue was performed as previously described.28 Briefly, organoids were fixed in 3.7% formaldehyde for 15 minutes at room temperature. Organoids were permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. Organoids were incubated with primary antibody overnight and washed in PBS con-taining 0.01% Triton-X 100. Secondary antibody incubation was also performed overnight in gastric organoids and subsequently immunostained for cell nuclei using 10 mg/mL Hoechst. The following primary antibodies and dilutions were used: 1:100 human-specific rabbit anti histone (Abcam, Cambridge, United Kingdom; ab125027), 1:100 rabbit anti-HER2 (Novus Biologicals, Littleton, CO; NBP1-84584), and 1:400 goat anti-Ecad (R&D Systems, Minneap-olis, MN; AF648). For measurement of proliferation, EdU solution was added to the organoid medium of huTGOs or huFGOs for 1-hour uptake. EdU staining was performed by using the Click-iT Alexa Fluor 594 Imaging Kit, according to the manufacturer’s instructions (Life Technologies, Carls-bad, CA). Coverslips were mounted onto slides with Vecta-shield Mounting Medium (Vector Laboratories; H-1400), and slides and whole mount organoids were imaged on a Zeiss LSM710 LIVE (Carl Zeiss AG, Oberkochen, Germany) duo confocal microscope.
Stomach sections spanning both the fundic and antral regions collected from mice orthotopically transplanted with huTGOs were fixed for 16 hours in 4% para-formaldehyde, paraffin embedded, and sectioned at 5 mmol/L. Prepared slides were deparaffinized with antigen retrieval performed by submerging in boiling solution (1:100 dilution Antigen Unmasking Solution in dH2O; Vector Laboratories; H-3300) for 10 minutes, followed by 20 minutes at room temperature. Sections were then blocked and immuno-stained with 1:100 CK7 (Novus Biologicals; NBP2-44814), 1:100 CK20 (Novus Biologicals; NBP1-85599), or 1:400
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Ki67 (Thermo Fisher Scientific; RM-9106-SO). Slides were incubated with biotinylated anti-mouse or anti-rabbit sec-ondary antibodies for 30 minutes, followed by additional 30-minute incubation with ABC reagent (Vectastain ABC kit; Vector Laboratories). Color was developed with 3,30-diaminobenzidine (DAB) using the DAB Substrate Kit (Vec-tor Laboratories), and slides were then counterstained with hematoxylin (Fisher Scientific Company, Kalamazoo, MI). Immunohistochemical slides were dehydrated and mounted using Permount (Fisher Scientific), and images were viewed and captured under light microscopy (Olympus BX60 with Diagnostic Instruments “Spot” Camera; Tokyo, Japan).
Drug Assay in Tumor-Derived Organoids
Organoids were grown in 96-well plates and treated with epirubicin, oxaliplatin, or 5-FU (EOX) (Selleckchem, Houston, TX) at concentrations of 0, 0.5, 1, 5, 10, 50, 100, and 200 mmol/L for 48 hours. In a separate series of experiments, organoids were pretreated with HER2 inhibi-tor Mubritinib (Sigma-Aldrich) at concentrations of 0, 0.5, 1, 5, 10, 50, 100, and 200 mmol/L for 2 hours before epi-rubicin, oxaliplatin, or 5-FU treatment at the calculated IC50 for each drug for an additional 48 hours. After 48 hours, organoid proliferation was measured by using MTS Assay (Promega 93582; Madison, WI). Dose-response curves were calculated on the basis of the absorbance readings collected from the MTS assay relative to drug concentrations. Absorbance was normalized to the vehicle controls, and drug concentrations were converted to logarithms by using GraphPad Prism (GraphPad Software, San Diego, CA).