br antibody specific for human histone
antibody specific for human histone protein (Figure 11H and J).
The expression of cytokeratin (CK) 7 and CK20 is often used for the diagnosis of gastric cancer. Immunohisto-chemical staining revealed high expression of CK7 within the lesions originating from the huTGO1 (Figure 12A) and
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huTGO2 (Figure 12B) organoid orthotopic transplantations. In contrast, CK20 was only detected in the stomachs of mice transplanted with huTGO2 (Figure 12D) compared with those animals transplanted with huTGO1 (Figure 12C). Lesions arising from huTGO1 and huTGO2 were highly proliferative (Figure 12E and F). Expression of E-cadherin (Figure 12G and H) was also detected within these lesions of mice transplanted with huTGO1 and 2 organoids. The negative control for the human-specific histone immuno-fluorescence is shown in Figure 13. Collectively, these data suggest that transplantation of patient-derived gastric can-cer organoids engraft within the gastric epithelium and mimic their parental histology.
Gastric Cancer Organoids Resemble the Patient’s Tumor Tissue From Which They Are Derived
HuTGO1-7 organoid lines were able to grow efficiently without organoid media and rapidly formed cell lines. To test the dependence of normal (huFGOs, organoids derived from normal human gastric tissue) and tumor-derived gastric organoids (huTGOs) on key growth factors sup-plied in the organoid growth medium, organoids were dissociated to single 61909-81-7 and re-suspended in organoid media with or without the key growth factors. HuTGOs grew in a growth factor–independent manner relative to control organoids (Figure 14A and B).
RNA sequencing followed by patient-matched statistical analysis identified 251 genes differentially expressed between samples derived from organoids and tissue sam-ples and samples derived from two-dimensional cultures (false discovery rate <0.1). Hierarchical clustering analysis of differentially expressed genes (Figure 15) and samples, including the samples from 2 commonly used gastric cancer cell lines (AGS and NCI-N87), revealed 3 major patterns of expression: genes down-regulated in two-dimensional cul-tures, genes up-regulated in two-dimensional cultures, and genes that were down-regulated in both TGO and two-dimensional cultures. Interestingly, genes down-regulated in both TGO and two-dimensional cultures were enriched by genes with several immune-response Gene Ontology categories (Table 2). This is consistent with the lack of the immune response within organoid and cell line cultures. Furthermore, gene expression profiles from AGS and NCI-N87 gastric cell lines were virtually identical to profiles of our two-dimensional cultures and different from TGO and cancer tissue samples, although these samples were not used in the selection of differentially expressed genes.
The genes that were highly expressed in gastric cancer tissue and organoids (TGOs) included GPD1, CXCR4, OLFM4, IL13Ra2, and carbonic anhydrase (CA9). Genes that were identified as being uniquely expressed in the cell lines
included KRT80, AMIGO2, CDKN2B, KRT23, and BAMBI. The expression of the genes among gastric cancer tissue 1, 2, 4, 5, and 7, TGO1, 2, 4, 5 and 7 lines, and cell lines was verified by quantitative real-time polymerase chain reaction (Figure 16). Collectively, these data suggest that gastric cancer organoids resemble the patient’s tumor tissue from which they were derived.
We demonstrate the proof of concept for the use of gastric cancer organoids as a preclinical model to potentially evaluate the efficacy of cancer therapeutics. The develop-ment of these organoid cultures represents the first step that is required to establish in vivo and in vitro patient-derived organoid-based platforms for personalized medi-cine. Cell lines have been the most frequently used models in cancer research, and their use has certainly advanced our understanding of cancer biology. As opposed to standard-of-care chemotherapeutic agents, targeted therapy is applied to the percentage of patients expressing a specific molecular abnormality. Thus, a large part of our ability to develop personalized medicine depends on cultures that capture this genetic heterogeneity. However, many studies report genomic differences between cancer cell lines and tissue samples from which they are derived.7–11 On the basis of RNA sequencing data and hierarchical clustering, we docu-ment a phenotypical similarity between the organoids and the patient’s tumor tissue. This is in stark contrast to a cell line derived from the gastric cancer organoids, which has a similar transcriptional program to that of the well-established gastric cancer cell lines AGS and NCI-N87 cells. Importantly, a limitation of the organoid and cell line cul-tures is the lack of the immune component that is found within the patient’s tumor environment. These findings are of significance because tumors can evade immune sur-veillance by expressing molecules such as programmed death-ligand 1 (PD-L1) that interacts with PD-1 and sub-sequently inhibiting CD8þ cytotoxic T-lymphocyte prolif-eration, survival, and effector function.13–15 On average, PD-L1 expression is detected in approximately 42.2% of gastric adenocarcinomas.16 Although anti-PD1 antibodies are already in clinical trials for gastric cancer treatment,17–19 there are currently no preclinical models that allow us to test the efficacy of therapy to predict patient treatment response and outcome. Refining the organoid culture system to include the patient’s own immune response would be beneficial to identifying the efficacy of immunotherapy in patients. Collectively, our data and pub-lished studies direct us toward developing in vitro models, such as the gastric cancer organoids, that would help to better predict the success or failure of chemotherapeutic agents and targeted therapies. Of importance, we have