Human gastric cancer modelling using organoids

Abstract

Objective: Gastric cancer is the second leading cause of cancer-related deaths and the fifth most common malignancy worldwide. In this study, human and mouse gastric cancer organoids were generated to model the disease and perform drug testing to delineate treatment strategies.

Design: Human gastric cancer organoid cultures were established, samples classified according to their molecular profile and their response to conventional chemotherapeutics tested. Targeted treatment was performed according to specific druggable mutations. Mouse gastric cancer organoid cultures were generated carrying molecular subtype-specific alterations.

Results: Twenty human gastric cancer organoid cultures were established and four selected for a comprehensive in-depth analysis. Organoids demonstrated divergent growth characteristics and morphologies. Immunohistochemistry showed similar characteristics to the corresponding primary tissue. A divergent response to 5-fluoruracil, oxaliplatin, irinotecan, epirubicin and docetaxel treatment was observed. Whole genome sequencing revealed a mutational spectrum that corresponded to the previously identified microsatellite instable, genomic stable and chromosomal instable subtypes of gastric cancer. The mutational landscape allowed targeted therapy with trastuzumab for ERBB2 alterations and palbociclib for CDKN2A loss. Mouse cancer organoids carrying Kras and Tp53 or Apc and Cdh1 mutations were characterised and serve as model system to study the signalling of induced pathways.

Conclusion: We generated human and mouse gastric cancer organoids modelling typical characteristics and altered pathways of human gastric cancer. Successful interference with activated pathways demonstrates their potential usefulness as living biomarkers for therapy response testing.

Keywords: gastric cancer.

Figure 1

Human gastric cancer organoid cultures expand in vitro. (A) Scheme of cancer organoid culture establishment and performed assays. (B) Representative pictures of four different cancer organoid lines and one normal line (scale bar 100 µm). (C) Proliferation rate of gastric organoids assessed by an EdU proliferation assay. (D) Media component depletion. Each annotated component was omitted from the complete medium and organoids followed over 5 weeks. (E) Growth curve of organoid xenografts (n=3 for each organoid line).

Figure 2

Human gastric cancer organoids maintain primary tumour characteristics. Immunohistochemistry of (A) DD107, (B) DD109, (C) DD191 and (D) DD282 using HE, carcinoembryonal antigen (CEA), cytokeratin 7 (CK7), cadherin 17 (Cadh17) and periodic acid Schiff (PAS) reaction of primary cancers, organoids and organoid-derived xenograft tumours (scale bar 50 µm).

Figure 3

Human gastric cancer organoids show divergent therapy response to conventional chemotherapeutics. (A–E) Cell viability assay after treatment with 5-fluoruracil (5-FU), oxaliplatin, irinotecan, epirubicin and docetaxel in varying concentrations. Analysis for oxaliplatin, irinotecan, epirubicin and docetaxel treat was performed after 24 hours. Organoids treated with 5-FU were analysed 72 hours post-treatment. Values were normalised to untreated control organoids of the same patient. (F) Apoptosis assay using annexin V/propidium iodide (PI) flow cytometry (one example of three independent experiments is shown).

Figure 4

Molecular features of gastric cancer organoids in comparison to the The Cancer Genome Atlas (TCGA) cohort. Cancer organoid lines were assigned to the microsatellite instable (MSI), genomic stable (GS) and chromosomal instable (CIN) subtypes. Represented are somatic copy number aberrations (SCNA), microsatellite instability (MSI) status, Epstein-Barr virus (EBV) status, prominent genes and mutational features. Statuses are indicated as high (red), low (orange) and stable (light grey) and presence or absence of features in black or white respectively. *DD282 carries an MSH6 mutation conveying microsatellite instability.

Figure 5

Circos plots of overall genetic and chromosomal characteristics of gastric cancer organoids. Represented are DD107 (A), DD191 (B), DD109 (C) and DD282 (D). From the outside to the inside: known oncogenes (green: missense, black: non-sense, red: amplifications, blue: deletions), chromosomes, coding variants represented as small squares (green: missense, black: stop or splice site, red: frameshift), copy number variations (CNV) as a scatterplot and as gained (red) or lost (blue) regions as well as interchromosomal (red) and intrachromosomal (blue) rearrangements bigger than 1 Mb. CIN, chromosomal instable; GS, genomically stable; MSI, microsatellite instable.

Figure 6

Targeting of mutated pathways in human gastric cancer organoids. (A) Immunohistochemistry for ERBB2 confirming the genomic amplification in DD109 (scale bar 50 µm). (B) Cell viability measurement after 72 hours of 0.1 µM trastuzumab. Student’s t-test treated versus untreated; **<0.01; ***<0.001. Western blot (C) and densitometric analysis (D) for ERK1/2 and phospho-ERK1/2 after 72 hours of 0.1 µM trastuzumab. (E) Western blot for phospho-c-KIT 24 hours after 100 µM imatinib treatment on DD109 (p.Glu142Asp c-KIT mutation). (F) Targeting of CDK4/6 in DD109 (biallelic loss of CDKN2A) and DD191 (no CDKN2A alteration) as well as DD320N (normal control) using 5 µM palbociclib. Proliferation was analysed after 24 hours of treatment using an EdU assay. (G) Long-term treatment (11 days, two splits) using 10 µM palbociclib led to the loss of the DD191 and DD320N culture (scale bar 100 µm).

Figure 7

Tumour modelling using corpus mouse organoids simulating the chromosomal instable (CIN) and genomic stable (GS) subtype of stomach cancer. (A–H) Established tumour organoids were analysed concerning mutated pathways and compared with normal organoids. (A–B, E–F) Representative pictures of organoids showed differences in morphology. CIN tumours showed smooth layered epithelium compared with normal organoids. GS-WNT tumour organoids had a grape-like structure. (C, G) Immunohistochemistry of normal versus tumour organoids. CIN organoids showed a Tp53 accumulation in the nucleus. GS-WNT organoids showed β-catenin accumulation in the nucleus whereas normal organoids showed membranous expression. (D) Kras pathway of CIN organoids was quantified by Western blot. Tumour organoids were increased in Erk1/2 phosphorylation compared with normal organoids. (H) Quantitative RT-PCR experiments of GS-Wnt organoids. Tumours showed an increased Wnt signalling as measured by Axin2 and Cyclin D1 expression levels compared with normal organoids. Treatment with 5 mM calphostin C decreased pathway activity (scale bar A–G 100 µm, Student’s t-test treated vs non-treated *<0.05; ** < 0.01). (I) Media component release. Each indicated medium component was omitted from the whole medium one by one and organoids passaged two times per week in a 1:2 ratio. (J) Single cell plating of organoids. One hundred single cells were plated per well and originating organoids counted after 7 days. Student’s t-test tumour versus control (*<0.05).