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A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies
* These authors contributed equally
In This Article
Summary
The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.
Abstract
Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.
Introduction
Gastric cancer is the fifth most common cancer worldwide 1. The effective diagnosis and treatment of Helicobacter pylori (H. pylori) have resulted in a low incidence of gastric cancer in the United States 2. However, the 5-year survival rate for patients diagnosed with this malignancy is only 29%, making gastric cancer an important medical challenge3. The purpose of the methods presented here is to develop an approach to predict immunotherapy responses in individual patients accurately. Solid tumors consist of cancer cells and various types of stromal, endothelial, and hematopoietic cells....
Protocol
Approval was obtained for the collection of human-biopsied tissues from patient tumors (1912208231R001, University of Arizona Human Subjects Protection Program; IRB protocol number: 1099985869R001 , University of Arizona Human Subjects Protection Program TARGHETS).
1. Establishing patient-derived gastric organoids from biopsies
- Collect 1-2 mm of human biopsied tissues from the tumor region of gastic cancer patients undergoing esophageal gastro-duodenoscopy in 1x phosphate-buffered .......
Representative Results
When completed, gastric organoids appear as spheres within the well, typically within 2-4 days post embedding (Figure 1). Figure 1A demonstrates a thriving gastric organoid culture that exhibits a regular membrane. Tumor organoids will often exhibit a divergent morphology that is unique to the patient sample. Unsuccessful cultures will appear dense or not exhibit any growth from the initial digestion of tissue (Figure 1B). Cultures .......
Discussion
We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture that may be used as a preclinical model to predict the efficacy of targeted therapies to ultimately improve treatment outcome and patient prognosis. Although cancer organoid co-cultures with immune cells have been reported, this is the first report of such a co-culture system for the study of gastric cancer. Numerous other organoid-based patient profiling efforts are well-developed at multiple institutions, including co-culture .......
Acknowledgements
This work was supported by NIH (NIAID) 5U19AI11649105 (PIs: Weiss and Wells, Project Leader 1: Zavros) and NIH (NIDDK) 2 R01 DK083402-06A1 (PI: Zavros) grant. This project was supported in part by PHS Grant P30 DK078392 (Integrative Morphology Core) of the Digestive Diseases Research Core Center in Cincinnati and 5P30CA023074 UNIVERSITY OF ARIZONA CANCER CENTER – CANCER CENTER SUPPORT GRANT (PI: Sweasy). We would like to acknowledge the assistance of Chet Closson (Live Microscopy Core, University of Cincinnati) and past members of the Zavros laboratory, Drs. Nina Steele and Loryn Holokai, for their contribution to the development of the organoid culture system. ....
Materials
| Name | Company | Catalog Number | Comments |
| 12 well plate | Midwest Scientific | 92012 | |
| 15 mL Falcon tube | Fisher scientific | 12-565-269 | |
| 24 well plate | Midwest Scientific | 92024 | |
| 30 μm filters | Miltenyi Biotec | 130-041-407 | |
| 40 μm filters (Fisher Scientific) | Fisher scientific | 352340 | |
| 5 mL round bottom polystyrene tubes | Fisher scientific | 14956-3C | |
| 50 mL Falcon tube | Fisher scientific | 12-565-271 | |
| Advanced DMEM/F12 | Thermo Fisher Scientific | 12634010 | |
| AIMV | Thermo Fisher Scientific | 12055091 | Basal medium for PBMCs and DCs |
| Amphotericin B/ Gentamicin | Thermo Fisher Scientific | R-01510 | |
| B-27 supplement | Thermo Fisher Scientific | 12587010 | |
| β-mercaptoethanol | Thermo Fisher Scientific | 800-120 | |
| Bone morphogenetic protein inhibitor (Noggin) | Peprotech | 250-38 | |
| Bovine Serum Albumin (BSA) | Sigma Aldrich | A7906 | |
| Cabozantinib | Selleckchem | S1119 | |
| Carboxyfluorescein diacetate succinimidyl ester (CFSE) | Biolegend | 423801 | |
| Collagenase A | Sigma Aldrich | C9891 | |
| Dulbecco’s Phosphate Buffered Saline (DPBS) | Fisher scientific | 14190-144 | cell separation buffer |
| EasySep Buffer | Stem Cell Technologies | 20144 | Contains Enrichment Cocktail and Magnetic Particles used in CTL culture |
| EasySep Human CD8+ T Cell Enrichment Kit | Stem Cell Technologies | 19053 | cell separation magnet |
| EasySep Magnet | Stem Cell Technologies | SN12580 | |
| EDTA | Sigma Aldrich | E6758 | |
| Epidermal Growth Factor (EGF) | Peprotech | 315-09 | |
| Farma Series 3 Water Jacketed Incubator | Thermo Fisher Scientific | 4120 | |
| Fetal Calf Serum (FCS) | Atlanta Biologicals | SI2450H | |
| Fibroblast growth factor 10 (FGF-10) | Peprotech | 100-26 | density gradient medium |
| Ficoll-Paque | GE Healthcare | 171440-02 | |
| Gastrin 1 | Tocris | 30061 | |
| Gelatin | Cell Biologics | 6950 | |
| GM-CSF | Thermo Fisher Scientific | PHC6025 | |
| Hank's Balanced Salt Solution (HBSS) | Thermo Fisher Scientific | 14175095 | |
| HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) | Fisher scientific | BP299-100 | |
| Human Epithelial Cell Basal Medium | Cell Biologics | H6621 | |
| human serum AB | Gemini Bioscience | 21985023 | |
| Hyaluronidase Type IV-S | Sigma Aldrich | H3884 | |
| Insulin-Transferrin-Selenium | Thermo Fisher Scientific | 41400045 | |
| Interleukin 1β (IL-1β) | Thermo Fisher Scientific | RIL1BI | |
| Interleukin 6 (IL-6) | Thermo Fisher Scientific | RIL6I | |
| Interleukin 7 (IL-7) | Thermo Fisher Scientific | RP-8645 | |
| Kanamycin | Thermo Fisher Scientific | 11815024 | |
| L-glutamine | Fisher scientific | 350-50-061 | basement membrane matrix |
| Matrigel (Corning Life Sciences, Corning, NY) | Fisher scientific | CB40230C | |
| N-2 supplement | Thermo Fisher Scientific | 17502048 | |
| N-acetyl-L-cysteine | Sigma Aldrich | A7250 | |
| Nicotinamide (Nicotinamide) | Sigma Aldrich | N0636 | |
| PD-L1 inhibitor | Selleckchem | A2002 | |
| Penicillin/Streptomycin | Thermo Fisher Scientific | SV3000 | |
| Petridish | Fisher scientific | 07-202-030 | |
| Potassium chloride (KCl) | Fisher scientific | 18-605-517 | |
| Potassium dihydrogenphosphate (KH2PO4) | Fisher scientific | NC0229895 | |
| prostaglandin E2 (PGE2) | Sigma Aldrich | P0409 | |
| RPMI 1640 | Thermo Fisher Scientific | 11875119 | |
| Sodium chloride (NaCl) | Fisher scientific | 18-606-419 | |
| Sodium hydrogen phosphate (Na2HPO4) | Fisher scientific | NC0229893 | cell dissociation reagent |
| StemPro Accutase solution | Thermo Fisher Scientific | A1110501 | |
| Transforming growth factor beta 1 (TGF-β1) | Thermo Fisher Scientific | 7754-BH-005/CF | |
| Tumor necrosis factor α (TNF-α) | Thermo Fisher Scientific | PHC3015 | |
| Vascular endothelial growth factor (VEGF) | Thermo Fisher Scientific | RVGEFI | |
| Y-27632 ROCK inhibitor | Sigma Aldrich | Y0350 |
References
- Ferlay, J., et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer. 136 (5), 359-386 (2015).
- Piazuelo, M. B., Epplein, M., Correa, P. Gastri....
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