Summary

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Published: July 06, 2021
doi:

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, including macrophages, myeloid-derived suppressor cells (MDSCs), and lymphocytes (reviewed in 4,5). Interactions between cancer stem cells and the tumor microenvironment (TME) substantially impact tumor characteristics and the response of the patient to treatment. This approach strives to allow investigators to acquire knowledge for preclinical drug development and biomarker discovery for the personalized treatment of gastric cancer.

The method presented here uses human-derived autologous organoid/immune cell co-cultures generated from gastric cancer patients to understand the immunosuppressive role of the MDSCs. Presented is a preclinical model that may predict the efficacy of targeted therapies to improve the survival of patients. Cancer organoid co-cultures with immune cells have been extensively reported in the pancreatic cancer field6,7,8,9,10. However, such co-cultures have not been reported to study gastric cancer. Overall, this method demonstrates the co-culturing of autologous human-derived immune cells within the same matrix environment as the cancer organoids, thus allowing the immune cells to be in contact with the target organoids.

The study by Tiriac et al.10 reported that patient-derived pancreatic cancer organoids, which exhibited heterogeneous responses to standard-of-care chemotherapeutics, could be grouped into organoid-based gene expression signatures of chemosensitivity that could predict improved patient responses to chemotherapy. The investigators proposed that combined molecular and therapeutic profiling of pancreatic cancer organoids may predict clinical response10. Co-clinical trial data from Yao et al.11 also showed that rectal cancer-derived organoids represent similar pathophysiology and genetic changes similar to the patient tumor tissues in response to chemoradiation. Thus, it is fundamental for organoid cultures to be used in the context of the patient's immune cells and tumor immune phenotype when using these cultures as predictive models for therapy.

Tumors expressing PD-L1 that interact with PD-1 inhibit CD8+ cytotoxic T lymphocyte proliferation, survival, and effector function 12,13,14. While approximately 40% of gastric cancers express PD-L1, only 30% of these patients respond to immunotherapy15,16,17. Anti-PD1 antibodies are used in clinical trials for gastric cancer treatment18,19,20. However, there are currently no preclinical models that allow testing of therapeutic efficacy for each patient. Optimizing the organoid culture such that the patient's immune cells are included in the system would potentially allow for the individualized identification of the efficacy of immunotherapy.

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 …

Disclosures

The authors have nothing to disclose.

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. We sincerely thank the patients who consented to donate tissue and blood for the development of the gastric organoid/immune cell co-cultures. Without their willingness to participate in the study, this work would not be possible.

Materials

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

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Cite This Article
Chakrabarti, J., Koh, V., So, J. B. Y., Yong, W. P., Zavros, Y. A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies. J. Vis. Exp. (173), e61443, doi:10.3791/61443 (2021).

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