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  • Cellular and Molecular Gastroenterology and Hepatology Vol N


     Cellular and Molecular Gastroenterology and Hepatology Vol. 7, No. 1
    treatment, cancer staging, and tumor response and recur-rence were recorded when available (Table 1). The morphology of each patient-derived organoid line (huTGO) was unique (Figure 1A and B). Specifically, we observed that whereas huTGO1 and 2 appeared as spherical nests with a central lumen lined by multiple layers of cells, huTGO4 exhibited a cribriform glandular morphology with Solutol HS-15 forming multiple lumens of varying sizes (Figure 1A and B). HuTGO5 formed large spheres that consisted of a single epithelial layer by H&E staining (Figure 1A and B). All huTGO lines were passaged and re-formed organoids efficiently except for the huTGO3 line that lasted for only 4 passages before the line no longer persisted. Thus, we were unable to study the huTGO3 in the drug response assays. The proliferative response of each huTGO was measured by 5-ethynyl-2-deoxyuridine (EdU) uptake. This analysis revealed a divergent and significantly different proliferative rate among the different organoid lines (Figure 1C and D). In contrast to the huTGOs, normal human-derived normal fundic gastric organoid (huFGO) lines displayed similar morphologies both in culture (Figure 2A) and by H&E staining (Figure 2B). In addition, the proliferative rates of the huFGOs were not statistically different among the different organoid lines (Figure 2C and D).
    To investigate whether huTGOs are a potential in vitro platform to study the efficacy of standard-of-care chemo-therapeutic agents, organoids were treated with drugs that gastric cancer patients are typically treated with (epirubicin, oxaliplatin, 5-fluorouracil [5-FU]) (Figure 3A–C). As a com-parison, organoids generated from normal gastric tissue (huFGOs) were treated with the same drugs (Figure 3D–F). In the huFGO lines it was observed that the half maximal inhibitory concentration (IC50) values, as documented by an MTS cell viability assay, were similar among the organoid lines for each drug that was tested (Figure 3D–F). Statistical analysis revealed an overlapping 95% confidence interval between each huFGO line (Figure 4D–F), thus demon-strating that the IC50 concentrations were not statistically different among these organoids. However, cell viability assays documented divergent responses and varying IC50 values to drug treatments among the huTGO lines (Figure 3A–C, Figure 4A–C). Note that a shift of the curve to the right indicates a higher IC50 (ie, more resistant to that particular drug). Cell viability assays were normalized to vehicle-treated controls to ensure that toxicity was specific to the drug effects.
    Abbreviations used in this paper: CK, cytokeratin; DPBS, Dulbecco phosphate-buffered saline; EdU, 5-ethynyl-20-deoxyuridine; 5-FU, 5-fluorouracil; HER2, human epidermal growth factor receptor 2; huFGO, human-derived normal fundic gastric organoid; huTGO, human-derived tumor gastric organoid; IC50, half maximal inhibitory concentration; PD-L1, programmed death-ligand 1.
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    © 2019 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license ( 2352-345X
    Human-Derived Gastric Cancer Organoids 163
    Table 1.Histologic Classification, Tumor Response, Number of Cases, and Organoid Lines Derived From Patients With
    Gastric Cancer
    Histologic classification No. of cases
    Organoid line Patient treatment and tumor response
    Diffuse/intestinal 1 1: huTGO1
    EOX, T3N1M1, no evaluation of tumor response
    2: No cell growth
    No chemo, T2N3aM1, no evaluation of tumor response
    No chemo, T1bN0M0, no evaluation of tumor response
    Signet-ring cell
    1: No cell growth
    2: No cell growth
    Poorly differentiated adenocarcinoma
    with diffuse and signet-ring patterns
    We wanted to next correlate the drug response of each huTGO line to the corresponding patient’s tumor response from whom the cultures were derived. Lines huTGO1, huTGO2, and huTGO6 were among the more resistant to Solutol HS-15 chemotherapeutic drug treatment. Resistance was docu-mented on the basis of decreased percentage of dead cells in organoid response to combination treatment with epirubicin, oxaliplatin, and 5-FU through the use of a fluorescence-based live/dead cell viability assay (Figure 5A and C). Unfortunately, evaluation of tumor response in these patients was not performed (Table 1). This is because (1) the patient from whom huTGO1 orga-noids were derived exhibited metastatic gastric cancer and the tumor was not resected, and (2) the patients from whom huTGO2 and huTGO6 organoids were derived did not receive chemotherapy and therefore tumor response was not evaluated (Table 1). The huTGO4 line displayed decreased resistance in response to chemotherapeutic drug treatment; however, this particular patient did not respond to chemotherapy (Table 1). Also, compared with huTGO1, 2, and 6, huTGO4 responded partially to the combination in vitro treatment of the organoids as docu-mented by the significant increase in the percent of dead cells within the organoid cultures within 48 hours of treatment (Figure 5A). However, huTGO7 was highly responsive to drug treatment, and similarly the patient’s tumor exhibited a near complete response to the same chemotherapy combination therapy (Table 1, Figure 5A and D). Importantly, whereas the huTGO lines exhibited differences in the response to drug treatment, huFGOs showed similar response to the combination drug treat-ment (Figure 5B). We were unable to perform a similar analysis on huTGO5 because this culture did not persist. Our studies suggest that each organoid line may be useful to help determine an active chemotherapeutic drug(s) for patient treatment. However, just as important is our ability to define drugs for which a patient has a resistance and