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Characterization of gastric tumor organoids with specific oncogenic pathway activations

ABSTRACT: Gastric cancer ranks the fifth most common malignancy and the third leading cause of cancer related death worldwide. Classical chemotherapy is still the standard treatment in advanced stages, with only two targeted therapies currently in clinical use. Several targeted trials have failed, likely due to missing relevant biomarkers that predict response. Patient derived cancer organoids (PDOs) constitute a three dimensional cell culture system showing self renewal, self organization and regularly unlimited proliferation while faithfully recapitulating many aspects of the tumor they are derived from. Nevertheless, the complex individual mutational landscape hampers the use of PDOs for basic and translational research. We therefore characterized three murine tumor organoid models with defined mutational patterns altering specific oncogenic pathways: a RAS activated (KrasG12D, Tp53R172H), a WNT activated (Apcfl/fl, Tp53R172H) and a diffuse (Cdh1fl/fl, Apcfl/fl) tumor model. The models were analyzed phenotypically, a proteome signature was established and a drug screen performed. The models were morphologically diverse, were characterized by individual protein expression signatures and showed differential drug sensitivities. The developed organoid models allow functional assays as well as pathway specific drug interference testing in a genetically defined setting.

INSTRUMENT(S): maXis, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Stomach

DISEASE(S): Stomach Carcinoma

SUBMITTER: Fernando Garcia

LAB HEAD: Javier Muñoz Peralta

PROVIDER: PXD022015 | Pride | 2022-08-22

REPOSITORIES: Pride

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			Action	DRS
	20170213_Organoids_F1_01_2-C1_01_9241.d.zip	Other
	20170213_Organoids_F2_01_2-C2_01_9242.d.zip	Other
	20170213_Organoids_F3_01_2-C3_01_9243.d.zip	Other
	20170213_Organoids_F4_01_2-C4_01_9244.d.zip	Other
	20170213_Organoids_F5_01_2-C5_01_9245.d.zip	Other

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Project description:The interaction of neoplastic cells with their tumor microenvironment (TME) is required for cancer progression. However, in vitro cancer models, including recent in vitro 3-dimensional (3D) organoid cultures of primary human tumors, are typically comprised exclusively of neoplastic epithelium, with stromal and/or immune interactions requiring artificial reconstitution. As relevant to cancer immunotherapy, the unified co-culture of primary tumor epithelia with their endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has remained particularly elusive. Here, we used a single 3D air-liquid interface (ALI) methodology to successfully propagate 3D Patient-Derived Tumor Organoids (PDOs) as primary tumor epithelia together with native immune and myofibroblast stromal compartments without reconstitution. Derived from >100 diverse human surgically resected tumor samples or from murine tumors in syngeneic immunocompetent mice, PDOs preserved cancer histologic subtypes and mutational spectrum with endogenous T, B, NK cells and macrophages integrally embedded amidst the tumor epithelium. PDO-based TILs accurately recapitulated the T cell receptor (TCR) spectrum of the original tumors, as determined by a robust droplet-based immune profiling solution that links gene expression and immune repertoire in single cells. Anti-PD-1 or anti-PD-L1 treatment of organoids from murine tumors from syngeneic immunocompetent hosts induced activation and cytolytic activity of tumor antigen-specific TILs, indicating successful PDO modeling of immune checkpoint blockade (ICB) and anti-tumor immunity. Crucially, the anti-PD-1 antibody nivolumab activated TILs in PDOs from human lung, renal and melanoma clinical tumor resections. The organoid-based propagation of primary tumor epithelium en bloc with its endogenous immune stroma should facilitate mechanistic investigation of TME-specific local tumor immunity, with applications for functional testing of individualized patient immunotherapy responses.

Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.

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Characterization of gastric tumor organoids with specific oncogenic pathway activations

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