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

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 field^6,7,8,9,10. 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 immunotherapy^15,16,17. Anti-PD1 antibodies are used in clinical trials for gastric cancer treatment^18,19,20. 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

1. Collect 1-2 mm of human biopsied tissues from the tumor region of gastic cancer patients undergoing esophageal gastro-duodenoscopy in 1x phosphate-buffered saline (PBS) (Table 1).
   NOTE: All procedures henceforth should be conducted in an aseptic environment using sterile materials and reagents.
2. Mince the biopsied tissues using scalpel blades on a Petri dish. Transfer the minced tissues to a 15 ml conical tube and add 5-10 mL of 1x PBS.
3. Centrifuge at 300 × g for 5 min at room temperature. Discard the supernatant. Add 1-2 mL of prewarmed digestion buffer (Table 1) to the pelleted tissues.
4. Incubate at 37 °C for 15-30 min, depending on the size of the minced tissues. Monitor the status of digestion by looking under the microscope for small cell clusters every 5-10 min.
5. Stop the digestion by diluting 5-fold with cold Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 medium (Advanced DMEM/F-12). If clumps of undigested material are visible, filter the tissues through 30 µm filters. Collect the flow-through.
6. Centrifuge the flow-through at 300 × g for 5 min at room temperature to pellet the cells. Discard the supernatant.
7. Resuspend the pelleted cells (10,000-100,000) in an appropriate volume (2-4 mL) of thawed basement membrane matrix (see the Table of Materials) on ice. Seed as 30-50 µL cell-basement membrane matrix drops in 24-well culture plates.
8. Incubate the plates at 37 °C for 15 min to allow the cell-basement membrane matrix drops to solidify. Overlay the cell-basement membrane matrix drops with prewarmed gastric organoid culture medium (Table 1).
9. Maintain organoid cultures at 37 °C in 5% CO₂. Replace the culture medium with fresh medium every 3-4 days, depending on the organoid growth. Passage the organoids once every 7-10 days in a 1:3 ratio.

2. Establishing patient-derived gastric organoids from surgical specimens

1. Collect human gastric cancer tissues from resected specimens of gastric cancer patients during surgical procedures in 1x Dulbecco's phosphate-buffered saline (DPBS) supplemented with antibiotics (Table 1).
   NOTE: All procedures hereafter should be processed in a biosafety cabinet, maintaining an aseptic environment and using sterile materials and reagents.
2. Mince the resected tissues using surgical scalpel blades on a Petri dish. Wash the minced tissues with 5-10 mL of 1x DPBS containing antibiotics.
3. Transfer the tissue to a 50 mL conical tube. Add 10 mL of prewarmed ethylenediamine tetraacetic acid (EDTA) stripping buffer to the minced tissues. Monitor the status of digestion every 5-10 min. Incubate the tube in a 37 °C shaker for 10 min.
4. Let the tissues settle at the bottom of the tube, remove the EDTA buffer without disturbing the tissues, and add 10 mL of fresh EDTA buffer. Incubate the tube in a 37 °C shaker for 5 min.
5. Let the tissues settle at the bottom of the tube. Remove the EDTA buffer and wash the tissues twice (follow step 2.4) with 10 mL of advanced DMEM/F-12 supplemented with antibiotics (no centrifuge) (Table 1).
6. Add 5-10 mL of prewarmed digestion buffer (Table 1) to the tissues, depending on the tissue size. Incubate the tube at 37 °C for 15-30 min with mild agitation, depending on the size and texture of the tissues. Check for the appearance of cell clusters under a microscope every 10 min.
7. Stop the digestion by diluting two-fold with cold Advanced DMEM/F-12 plus antibiotics. Filter the undigested tissues through 40 µm filters and collect the flow-through.
8. Centrifuge the flow-through at 400 × g for 5 min at 4 °C to pellet the cells. Discard the supernatant, and wash the cells with cold 1x DPBS plus antibiotics at 400 × g for 5 min at 4 °C. Carefully discard the supernatant, and store the cells on ice.
9. Resuspend the pelleted cells in an appropriate volume of the basement membrane matrix on ice. Seed 30-50 µL cell-basement membrane matrix drops in 24- or 12-well cell culture-treated plates.
10. Incubate the plates at 37 °C for 15 min to allow the cell-basement membrane matrix drops to solidify as a dome. Overlay the cell-basement membrane matrix dome with prewarmed gastric organoid culture medium (Table 1).
11. Maintain the organoid cultures at 37 °C in 5% CO₂. Replace the culture medium with fresh medium every 3-4 days, depending on the organoid growth. Passage the organoids once every 7-10 days in a 1:2 or 1:3 ratio, based on the organoid density.

3. Maintenance and expansion of organoid cultures

NOTE : All procedures should be conducted in an aseptic environment using sterile materials and reagents.

1. Maintenance and expansion
   1. Maintain the organoid cultures for 7-10 days until they are 70-80% confluent. Harvest the organoids in ice-cold DMEM. Transfer the organoids to 5 mL round-bottom polystyrene tubes.
   2. Centrifuge the organoids at 400 × g for 5 min at 4 °C to pellet cells. Carefully remove the supernatant and resuspend the pellet in 1 mL of prewarmed cell dissociation reagent solution. Incubate the organoids at 37 °C for 6 min.
   3. Gently pass the organoids through a 26 G needle 4 times. Add 2 mL of DMEM to stop the action of the cell dissociation reagent.
   4. Centrifuge the organoids at 400 × g for 5 min at 4 °C to pellet the cells. Carefully discard the supernatant and store the cells on ice.
   5. Resuspend the pelleted cells in an appropriate volume of basement membrane matrix on ice. Seed 30-50 µL of the cell-basement membrane matrix drops in 24- or 12-well cell culture treated plates.
   6. Incubate the plates at 37 °C for 15 min to allow the cell-basement membrane matrix drops to solidify as a dome. Overlay the cell- cell-basement membrane matrix dome with prewarmed gastric organoid culture medium (see Table 1).
   7. Maintain the organoid cultures at 37 °C in 5% CO₂. Replace the culture medium with fresh medium every 3-4 days, depending on the organoid growth. Repeat passaging of the organoids once every 7-10 days in a 1:2 or 1:3 ratio, based on the organoid density.
2. Expansion of organoid-derived cell lines
   1. Harvest the organoids when they are 70-80% confluent. Feed the cells adherent to the plastic plates with gastric organoid culture medium and maintain the culture by feeding them every 3-4 days depending on the cell growth.
   2. Passage the cells when they reach 80-90% confluence.
      1. Remove the culture medium and gently wash the plate with prewarmed DPBS. Add 1 mL of prewarmed cell dissociation reagent.
      2. Incubate the cells for 5 min at 37 °C. Harvest the cells in 2 mL of DMEM.
      3. Centrifuge the cells at 400 × g for 5 min at 4 °C. Remove the supernatant and resuspend the pellet in gastric organoid culture medium or human gastric epithelial cell culture medium (Table 1).
   3. Plate the cells from 3.2.2.3 either in basement membrane matrix-coated or gelatin-coated plates. Maintain the organoid cultures at 37 °C in 5% CO₂. Replace the medium with fresh medium every alternate day depending on the cell growth. Repeat the passaging of the cells once every 7-10 days in a 1:2 or 1:3 ratio, based on the cell density.
      1. To coat the plates with basement membrane matrix, dilute the matrix in ice-cold cell culture grade water in a 1:10 ratio. Uniformly coat the plate with 1 mL of ice-cold diluted matrix solution using a cell spreader, and incubate the coated plate at 37 °C for 2 h. Remove the remaining coating solution and let the plate dry without the lid inside the biosafety hood for 30 min, right before plating the cells.
      2. To coat the plates with gelatin, uniformly coat the plate with 1 mL of an ice-cold gelatin-based coating solution. Incubate the coated plate at room temperature for 5 min. Remove the remaining gelatin solution and plate the resuspended cells.

4. Culturing immune cells from peripheral blood mononuclear cells (PBMCs)

NOTE: All procedures should be conducted in an aseptic environment using sterile materials and reagents.

1. PBMC isolation
   1. Dilute whole blood with an equal volume of 1x PBS.
   2. Depending on the total volume of the diluted blood sample, dispense an appropriate volume of density gradient medium (see the Table of Materials) into a 15 mL tube.
      NOTE: The recommended ratio is 3 mL of the density gradient medium to 4 mL of the diluted blood sample.
   3. Carefully layer the diluted blood sample onto the density gradient medium. Centrifuge at 400 × g for 30 min-1 h without brake.
   4. After centrifugation, carefully transfer the mononuclear cells at the interface into a new 15 mL conical tube. Dilute the mononuclear cells with 3 volumes of 1x PBS.
   5. Centrifuge at 400 × g for 10 min. Discard the supernatant. Repeat once.
   6. Resuspend the pelleted cells in an appropriate medium for downstream applications or cryopreserve as frozen stocks.
   7. If needed, determine the yield and cell viability of PBMCs using the trypan blue dye exclusion assay.
2. Culture of human dendritic cells
   1. Resuspend the isolated PBMCs in PBMC medium (see the Table of Materials and Table 1) and plate them in a 24-well tissue culture plate for 1-2 h at 37 °C in 5% CO₂.
   2. Firmly tap the culture plate and discard the spent culture medium containing non-adherent cells.
   3. Add the DC culture medium (Table 1) to the adherent cells. Maintain the cultures for 3 days at 37 °C in 5% CO₂. Replace the supernatant medium with fresh culture medium on alternate days.
   4. On day 3, replace the exhausted medium with fresh DC maturation medium (Table 1). Maintain the cultures for 24 h at 37 °C in 5% CO₂.
3. Culture of human cytotoxic T lymphocytes (CTLs)
   1. Transfer 1 mL of PBMCs to a 5 mL polystyrene round-bottom tube.
   2. Add 50 µL of Enrichment Cocktail (see the Table of Materials) to the PBMCs. Incubate for 10 min at room temperature.
   3. Add 150 µL of Magnetic Particles (Table of Materials) to the sample. Incubate for 5 min at room temperature.
   4. Increase the sample volume to 2.5 mL with the cell separation buffer (Table of Materials). Place the polystyrene round-bottom tube into a cell separation magnet (Table of Materials) for 5 min to enable cell separation.
   5. Pour the enriched cell suspension into a new 15 mL conical tube. Centrifuge at 300 × g for 5 min. Discard the supernatant.
   6. Resuspend the pelleted cells and culture in CTL culture medium (Table 1) for 24 h at 37 °C in 5% CO₂.
4. Culture of human myeloid-derived suppressor cells (MDSCs)
   1. Culture PBMCs in MDSC culture medium (Table 1) for 7 days at 37 °C in 5% CO₂ to enrich for MDSCs. Replace the supernatant medium with fresh culture medium on alternate days.
5. Co-culture of DCs and CTLs
   1. Collect conditioned medium from the organoid cultures.
   2. Gently remove 50% of the medium from the matured DC culture and replace it with organoid-derived conditioned medium.
   3. Incubate the DCs with the organoid-derived conditioned medium for 2 h at 37 °C in 5% CO₂.
   4. Harvest the loosely adherent DCs and centrifuge them at 300 × g for 5 min at 4 °C. Remove the supernatant, resuspend the pellet in RPMI co-culture medium, and add this cell suspension back into the same well.
   5. Harvest the CTLs from the same patient line and transfer them to the matured DCs. Continue DC-CTL co-culture (Table 1) for 72 h at 37 °C in 5% CO2.

5. Establishing organoid/immune cell co-cultures

1. Isolate CTLs from co-cultures of DCs and CTLs using a kit (see the Table of Materials) as described previously in section 4.3.
2. Incubate the CTLs with 5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) at 37 °C for 20 min.
   NOTE: CFSE is a blue laser excitable dye used for flow cytometric monitoring of cell divisions.
3. Harvest the organoids and mix them with CFSE-labeled CTLs. Keeping the CTL/organoid ratio at 50,000 CTLs per 200 organoids, resuspend the organoids in an appropriate volume of thawed basement membrane matrix on ice. Seed as 25 µL cell-basement membrane matrix drops in 24-well tissue culture plates.
   NOTE: For experimental conditions requiring MDSCs, mix the MDSCs with the organoids and CFSE-labeled CTLs before seeding. The MDSC/CTL ratio should be 4:1.
4. Incubate the plates at 37 °C for 15 min to allow the cell-basement membrane matrix drops to solidify.
5. Overlay the cell-basement membrane matrix drops with prewarmed organoid culture medium.
6. Maintain the organoid/immune cell co-cultures at 37 °C in 5% CO₂ until analysis.

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 ...

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