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  • PKC412 br Our data suggest that gastric cancer organoids

    2020-08-12


    Our data suggest that gastric cancer organoids may be used to help predict patient response to targeted therapies such as HER2 inhibition. Expression of HER2 within orga-noid cultures (huTGO1 and huTGO2) that were 2 of the resistant lines to chemotherapy treatment was sensitized to these chemotherapeutic agents with HER2 inhibitor pretreatment. HER2 overexpression is becoming recog-nized as a frequent molecular abnormality in gastric can-cer.26 Amplification of the HER2 gene was first discovered in breast cancer and is significantly associated with worse prognosis.27 With the recent introduction of HER2 molec-ular targeted therapy for patients with metastatic gastric cancer, determination of HER2 status is crucial to select patients who may benefit from this treatment. However, HER2 testing in gastric cancer differs from testing in breast cancer because of inherent differences in tumor biology, tumor heterogeneity of HER2 expression, and incomplete membrane staining that are commonly observed in gastric cancers.6 The organoid culture system may provide a reliable method for identifying HER2-positive patients because the culture is designed to select for the cancer 
    stem cells. Treating organoids alongside the patients from whom the cultures were derived will ultimately test their usefulness to predict individual therapy response and patient outcome.
    Materials and Methods
    Generating Human Fundic Gastric Organoids
    Human fundus was collected from sleeve gastrectomies (IRB protocol number: 2015-5537, University of Cincinnati and 2014-0427, Cincinnati Children’s Hospital Medical Center), and gastric glands were generated as previously described.28,29 Briefly, PKC412 was separated from the muscle layer, cut into small fragments, and washed in Dulbecco phosphate-buffered saline (DPBS) without Ca2þ/Mg2þ. Tissue fragments were placed in a buffer containing collagenase (1 mg/mL) from Clostridium histolyticum and bovine serum albumin (2 mg/mL) for 30 minutes at 37 C. Gastric glands were suspended in 50 mL Matrigel (Thermo Fisher Scientific, Waltham, MA) and cultured in freshly generated human gastric organoid media (DMEM/F12 (Thermo Fisher Scientific), HEPES (10 mmol/L), 1X L-glutamine (Thermo Fisher Scientific), 1X Pen/Strep, 1X N2 (Thermo Fisher Scientific), 1X B27 (Thermo Fisher Scientific), N-acetylcysteine (1 mmol/L; Sigma-Aldrich, St Louis, MO), nicotinamide (10 mmol/L; Sigma-Aldrich), epidermal growth factor (50 ng/mL; Pepro-Tech, Rocky Hill, NJ), noggin (100 ng/mL; PeproTech), R-spondin conditioned media, wnt conditioned media, FGF10 (200 ng/mL; PeproTech), gastrin (1 nmol/L; Tocris Biosci-ence, Bristol, United Kingdom), Y-27632 (10 mmol/L; Sigma-Aldrich), 1X amphotericin B/gentamicin, 1X kanamycin). Organoids were harvested after 4–7 days of growth.
    178 Steele et al Cellular and Molecular Gastroenterology and Hepatology Vol. 7, No. 1
    2019 Human-Derived Gastric Cancer Organoids 179
    sequencing analysis of gastric cancer patient tissue (GC), organoid (TGOs), and cell lines (AGS, NCI, or TGOC). RNA sequencing analysis of gastric cancer cell lines (NCI, AGS), organoid-derived gastric cancer cell lines (TGO1C, TGO2C, TGO5C), patient tissue (GC1, GC2, GC5), and
    patient-derived gastric cancer organoids (TGO1, TGO2, TGO5).
    Organoids Derived From Gastric Cancer Tissue
    Tumor tissue was obtained from patients undergoing surgical resection for gastric cancer (IRB protocol number: 2015-5537, University of Cincinnati and H-35094, Baylor College of Medicine). Tumor organoids were generated as previously described.30 Briefly, tumor tissue was washed well in DPBS without Ca2þ and Mg2þ supplemented with
    antibiotics and minced into small pieces. Tumor fragments were placed in pre-warmed stripping buffer: Hank’s balanced salt solution, ethylenediamine tetraacetic acid (5 mmol/L), HEPES (25 mmol/L), 10% fetal calf serum. Fragments were incubated for 10 minutes in a shaking incubator at 37 C. The fragments were supplied fresh stripping buffer and incubated for an additional 5 minutes
    Figure 14. (See previous page). Growth factor independent growth of gastric cancer organoids. Light micrographs of organoids derived from (A) normal (huFGOs) or (B) gastric cancer (huTGO1) organoid line in various growth factor conditions. Quantification of number of organoids per 4 field is shown for both huFGOs and huTGO1. *P < .05 compared with complete media condition, n ¼ 3 individual organoid lines.
    180 Steele et al Cellular and Molecular Gastroenterology and Hepatology Vol. 7, No. 1
    Table 2.Immune Response GO Categories Identified in the Enrichment Analysis of the Genes in the Immune Responses
    Cluster in Figure 15