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Organoid and Immune cell Co-Culture. Organoid/immune cell co-cultures of (a) mGO alone, (b) mGOs co-cultured with DCs and cytotoxic CD8+ T cells (CTLs), (c) mTGO alone, and (d) mTGOs co-cultured with DCs and CTLs
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The interaction between the receptor, programmed cell death protein 1 (PD-1) and ligand, programmed cell death 1 (PD-L1) is known to inhibit CD8+ cytotoxic T lymphocyte proliferation, survival, and effector function. The result of this interaction leads to evasion of immune surveillance by tumors and subsequently cancer cell proliferation. Immunoth...
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Context 1
... Add 1 mL DPBS/antibiotics each well and collect the DCs from individual wells. (Fig. 3a, c), or mGO/mTGO plus matured DCs and CTLs (Fig. 3b, d 12. Store the fixed slides in DPBS (w/o calcium and magnesium) at 4 °C until performing TUNEL ...
Citations
... Despite being heterogeneous, most patient-derived organoids (PDOs) are devoid of nearby stromal cells in the culture, which fails to re-establish the tissue microenvironment (TME). However, co-culturing fibroblasts, endothelial cells, and immune cells could provide the desired TME in vitro [14]. In organoid cultures, the basement matrix i.e., Matrigel, could affect functional/biochemical experiments and impede the collection of cells and sub-culturing in comparison to 2D cell culture. ...
Organoid models have recently been utilized to study 3D human-derived tissue systems to uncover tissue architecture and adult stem cell biology. Patient-derived organoids unambiguously provide the most suitable in vitro system to study disease biology with the actual genetic background. With the advent of much improved and innovative approaches, patient-derived organoids can potentially be used in regenerative medicine. Various human tissues were explored to develop organoids due to their multifold advantage over the conventional in vitro cell line culture approach and in vivo models. Gastrointestinal (GI) tissues have been widely studied to establish organoids and organ-on-chip for screening drugs, nutraceuticals, and other small molecules having therapeutic potential. The function of channel proteins, transporters, and transmembrane proteins was also explained. The successful application of genome editing in organoids using the CRISPR-Cas approach has been reported recently. GI diseases such as Celiac disease (CeD), Inflammatory bowel disease (IBD), and common GI cancers have been investigated using several patient-derived organoid models. Recent advancements on organoid bio-banking and 3D bio-printing contributed significantly in personalized disease management and therapeutics. This article reviews the available literature on investigations and translational applications of patient-derived GI organoid models, notably on elucidating gut-microbial interaction and epigenetic modifications.
Graphical Abstract
... Researchers have also established a triple co-culture scheme of tumor organoids with T-cells and other immune cells to assess toxicity. Chakrabarti and colleagues [36,37] isolated dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) from mouse bone marrow and thymus, respectively, and cultured them with tumor organoids. They found that Hedgehog signaling induced expression of programmed cell death ligand 1 (PD-L1) and proliferation of tumor cells in gastric cancer. ...
In recent years, the three-dimensional (3D) culture system has emerged as a promising preclinical model for tumor research owing to its ability to replicate the tissue structure and molecular characteristics of solid tumors in vivo. This system offers several advantages, including high throughput, efficiency, and retention of tumor heterogeneity. Traditional Matrigel-submerged organoid cultures primarily support the long-term proliferation of epithelial cells. One solution for the exploration of the tumor microenvironment is a reconstitution approach involving the introduction of exogenous cell types, either in dual, triple or even multiple combinations. Another solution is a holistic approach including patient-derived tumor fragments, air-liquid interface, suspension 3D culture, and microfluidic tumor-on-chip models. Organoid co-culture models have also gained popularity for studying the tumor microenvironment, evaluating tumor immunotherapy, identifying predictive biomarkers, Cancer Innovation. 2023;1-25. wileyonlinelibrary.com/journal/cai2 | 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
... When co-cultured with pancreatic ductal adenocarcinoma organoids, patient-matched fibroblasts and peripheral blood lymphocyte showed myofibroblast-like fibroblast activation, lymphocyte infiltration into Matrigel, and subsequent migration toward tumor organoids [50]. The co-culture of dendritic cells (DCs) primed by tumor antigens secreted from mouse gastric tumor organoids and cytotoxic T lymphocytes (CTLs) was a more sophisticated strategy [51]. Activated CTLs destroyed tumor organoids in the presence of anti-programmed cell death-ligand 1 (PD-L1); this indicated that the coculture of various immune cells would enable predicting the efficacy of immunotherapy in individual patients. ...
Recent advances in tumor microenvironment (TME) modeling as well as its applications to cancer therapy has brought various dramatical changes in multiple malignancies management. Understanding the mechanisms of response and resistance to cancer therapy requires a clear elucidation of the intricate interactions between TME cells, the surrounding stroma, and distant affected tissues or organs. To address this demand, various 3D cell culture techniques have been developed in order to recapitulate and understand cancer biology over the past decade. This review summarizes some saliant progresses in in vitro 3D TME modeling, including the cell-based, matrix-based, and vessel-based dynamic 3D modeling techniques and their applications in investigating tumor-stroma interactions and responses to cancer therapies. The review also discusses the limitations of current TME modelling approaches and proposes some new thoughts on the construction of more clinically relevant models.
... The major application of cancer organoid co-culture models is the co-culture of cancer organoids with immune cells, including cytotoxic T lymphocytes and dendritic cells (45), NK cells (55), macrophages (49,54), and lymphocytes (46, 47) ( Table 2). James et al. co-cultured PDAC organoids with CAFs and CD3 + T lymphocytes to develop a specific TME for PDAC (46). ...
Three-dimensional cancer organoids derived from self-organizing cancer stems are ex vivo miniatures of tumors that faithfully recapitulate their structure, distinctive cancer features, and genetic signatures. As novel tools, current cancer organoids have been well established and rapidly applied in drug testing, genome editing, and transplantation, with the ultimate aim of entering clinical practice for guiding personalized therapy. However, given that the lack of a tumor microenvironment, including immune cells and fibrous cells, is a major limitation of this emerging methodology, co-culture models inspire high hope for further application of this technology in cancer research. Co-culture of cancer organoids and immune cells or fibroblasts is available to investigate the tumor microenvironment, molecular interactions, and chimeric antigen receptor-engineered lymphocytes in cancer treatment. In light of the recent progress in cancer organoid co-culture models, it is only possible to recognize the advantages and drawbacks of this novel model to exploit its full potential. In this review, we summarize the recent advances in the application of cancer organoids and co-culture models and how they could be improved in the future to benefit cancer research, especially precision medicine.
... Coculture systems are essential in all studies of cellcell interactions and have long been used to investigate interactions between cell populations [30]. To study PD-L1/PD-1 interactions in the tumor microenvironment in vitro, mouse-derived gastric cancer organoids and autologous immune cells were cocultured [31]. After activating the androgen and Wnt/β-catenin signaling pathways, HFSC differentiation was assessed in a coculture model with DPC or culturing with DPC-conditioned media [32]. ...
Both hair follicle stem cells (HFSC) and dermal papilla cells (DPC) are essential for hair follicle growth and proliferation. In this study, HFSCs and DPCs that made signature proteins like KRT14, KRT15, KRT19, α-SMA, and Versican were obtained. Cell coculture systems between HFSCs and DPCs were used to measure the increased PCNA protein content in HFSCs. Additionally, exosomes from dermal papilla cells (DPC-Exos), the overexpression and silencing of Wnt3a, could regulate the Wnt/β-catenin signaling pathway downstream genes. After collecting DPC-ExosOE-Wnt3a, the treatment of HFSC with DPC-ExosOE-Wnt3a showed that DPC-ExosOE-Wnt3a could upregulate the mRNA expression of downstream genes in the Wnt/β-catenin signaling pathway and that DPC-ExosOE-Wnt3a enhanced the proliferation of HFSCs while inhibiting their apoptosis. These findings suggest that DPC-Exos could regulate HFSC cell proliferation via the Wnt3a/β-catenin signaling pathway. This research offers novel concepts for the molecular breeding and efficient production of Angora rabbits, as well as for the treatment of human hair problems.
... The methodology of co-cultures of murine GC organoids with immune cells has been described previously [85]. Mouse-derived normal and/or tumour GC organoids were cultured with mouse-derived dendritic cells (DCs) and cytotoxic T-cells. ...
Cancers affecting the gastrointestinal system are highly prevalent and their incidence is still increasing. Among them, gastric and pancreatic cancers have a dismal prognosis (survival of 5–20%) and are defined as difficult-to-treat cancers. This reflects the urge for novel therapeutic targets and aims for personalised therapies. As a prerequisite for identifying targets and test therapeutic interventions, the development of well-established, translational and reliable preclinical research models is instrumental. This review discusses the development, advantages and limitations of both patient-derived organoids (PDO) and patient-derived xenografts (PDX) for gastric and pancreatic ductal adenocarcinoma (PDAC). First and next generation multicellular PDO/PDX models are believed to faithfully generate a patient-specific avatar in a preclinical setting, opening novel therapeutic directions for these difficult-to-treat cancers. Excitingly, future opportunities such as PDO co-cultures with immune or stromal cells, organoid-on-a-chip models and humanised PDXs are the basis of a completely new area, offering close-to-human models. These tools can be exploited to understand cancer heterogeneity, which is indispensable to pave the way towards more tumour-specific therapies and, with that, better survival for patients.
... Autologous organoid/immune cell co-cultures were established as described previously [30,33]. Dendritic cells were initially pulsed with huTGO CM , followed by co-culturing them with CTLs for 24 h. ...
(1) Background: The expression of programmed death-ligand 1 (PD-L1), which interacts with programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTLs), enables tumors to escape immunosurveillance. The PD-1/PD-L1 interaction results in the inhibition of CTL proliferation, and effector function, thus promoting tumor cell evasion from immunosurveillance and cancer persistence. Despite 40% of gastric cancer patients exhibiting PD-L1 expression, only a small subset of patients responds to immunotherapy. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several solid tumors, including metastatic gastric cancer. Although half of PD-L1-positive gastric tumors co-express HER2, crosstalk between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined. (2) Methods: Human gastric cancer organoids (huTGOs) were generated from biopsied or resected tissues and co-cultured with CTLs and myeloid-derived suppressor cells (MDSCs). Digital Spatial Profiling (DSP) was performed on FFPE tissue microarrays of numerous gastric cancer patients to examine the protein expression of immune markers. (3) Results: Knockdown of HER2 in PD-L1/HER2-positive huTGOs led to a concomitant decrease in PD-L1 expression. Similarly, in huTGOs/immune cell co-cultures, PD-L1 expression decreased in huTGOs and was correlated with an increase in CTL proliferation which enhanced huTGO death. Treatment with Nivolumab exhibited similar effects. However, a combinatorial treatment with Mubritinib and Nivolumab was unable to inhibit HER2 expression in co-cultures containing MDSCs. (4) Conclusions: Our study suggested that co-expression of HER2 and PD-L1 may contribute to tumor cell immune evasion. In addition, autologous organoid/immune cell co-cultures can be exploited to effectively screen responses to a combination of anti-HER2 and immunotherapy to tailor treatment for gastric cancer patients.
... Human gastric organoid models have been reported derived from both primary human gastric tissue [80,[190][191][192][193][194][195][196][197] or hIPSCs [198]. Many studies have also used murine primary gastric organoids [192,196,199,200] including co-culture with autologous immune cells [200] and immortalized stomach mesenchymal cells [201], in addition to murine pluripotent stem-cell-derived gastroids [202,203]. ...
... Human gastric organoid models have been reported derived from both primary human gastric tissue [80,[190][191][192][193][194][195][196][197] or hIPSCs [198]. Many studies have also used murine primary gastric organoids [192,196,199,200] including co-culture with autologous immune cells [200] and immortalized stomach mesenchymal cells [201], in addition to murine pluripotent stem-cell-derived gastroids [202,203]. ...
... Mainly, these models have been used to investigate H. Pylori infection [190-192, 194, 195, 197, 198], cancer [80,193,196,199,200] and pharmaceuticals [201]. H2D human fundic organoid layers have been used to study H. Pylori infection [197]. ...
Orally ingestible medical devices offer significant opportunity in the diagnosis and treatment of gastrointestinal conditions. Their development necessitates the use of models that simulate the gastrointestinal environment on both a macro and micro scale. An evolution in scientific technology has enabled a wide range of in vitro, ex vivo and in vivo models to be developed that replicate the gastrointestinal tract. This review describes the landscape of the existing range of in vitro tools that are available to characterize ingestible devices. Models are presented with details on their benefits and limitations with regards to the evaluation of ingestible devices and examples of their use in the evaluation of such devices is presented where available. The multitude of models available provides a suite of tools that can be used in the evaluation of ingestible devices that should be selected on the functionality of the device and the mechanism of its function.
... This has been best exemplified in the context of intestinal cancers. Gastric organoids derived from murine and human tissue and co-cultured with CD8+ CTLs and DCs have been employed to elucidate important mechanistic insights into PD-L1 regulation in gastric tumor progression [134][135][136]. Such a system also allowed for convenient introduction of microbes such as Helicobacter pylori (H. ...
Immunosuppression in cancer has emerged as a major hurdle to immunotherapy efforts. Immunosuppression can arise from oncogene-induced signaling within the tumor as well as from tumor-associated immune cells. Understanding various mechanisms by which the tumor can undermine and evade therapy is critical in improving current cancer immunotherapies. While mouse models have allowed for the characterization of key immune cell types and their role in tumor development, extrapolating these mechanisms to patients has been challenging. There is need for better models to unravel the effects of genetic alterations inherent in tumor cells and immune cells isolated from tumors on tumor growth and to investigate the feasibility of immunotherapy. Three-dimensional (3D) organoid model systems have developed rapidly over the past few years and allow for incorporation of components of the tumor microenvironment such as immune cells and the stroma. This bears great promise for derivation of patient-specific models in a dish for understanding and determining the impact on personalized immunotherapy. In this review, we will highlight the significance of current experimental models employed in the study of tumor immunosuppression and evaluate current tumor organoid-immune cell co-culture systems and their potential impact in shedding light on cancer immunosuppression.
... Murine monocytes were isolated and cultured from bone marrow according to a published protocol [42,43]. Dendritic cells were cultured from bone marrow-derived monocytes according to [43]. ...
... Murine monocytes were isolated and cultured from bone marrow according to a published protocol [42,43]. Dendritic cells were cultured from bone marrow-derived monocytes according to [43]. CTLs were extracted from splenocytes using the EasySep TM Mouse CD8+ T cell Isolation kit according to the manufacturer's protocol (Stemcell Technologies, 19853) and cultured according to previously published studies [43]. ...
... Dendritic cells were cultured from bone marrow-derived monocytes according to [43]. CTLs were extracted from splenocytes using the EasySep TM Mouse CD8+ T cell Isolation kit according to the manufacturer's protocol (Stemcell Technologies, 19853) and cultured according to previously published studies [43]. ...
Simple Summary
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with an approximate 10% five-year survival rate despite therapy. A plausible reason for this observation may be that other, redundant, immune-suppressive mechanisms are at play. Thus, effective treatment of PDAC is a medical challenge and warrants the development of a pre-clinical model whereby the patient’s tumor immune phenotype is characterized and the immune response within the tumor microenvironment tested prior to therapy. These studies present a pre-clinical organoid model that may be used to test the efficacy of combinatorial therapies and targeted therapies, based on modulating the tumor microenvironment, to improve cancer patient response and survival.
Abstract
Purpose: Pancreatic ductal adenocarcinoma (PDAC) has the lowest five-year survival rate of all cancers in the United States. Programmed death 1 receptor (PD-1)-programmed death ligand 1 (PD-L1) immune checkpoint inhibition has been unsuccessful in clinical trials. Myeloid-derived suppressor cells (MDSCs) are known to block anti-tumor CD8+ T cell immune responses in various cancers including pancreas. This has led us to our objective that was to develop a clinically relevant in vitro organoid model to specifically target mechanisms that deplete MDSCs as a therapeutic strategy for PDAC. Method: Murine and human pancreatic ductal adenocarcinoma (PDAC) autologous organoid/immune cell co-cultures were used to test whether PDAC can be effectively treated with combinatorial therapy involving PD-1 inhibition and MDSC depletion. Results: Murine in vivo orthotopic and in vitro organoid/immune cell co-culture models demonstrated that polymorphonuclear (PMN)-MDSCs promoted tumor growth and suppressed cytotoxic T lymphocyte (CTL) proliferation, leading to diminished efficacy of checkpoint inhibition. Mouse- and human-derived organoid/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of arginase 1-expressing PMN-MDSCs within these co-cultures rendered the organoids susceptible to anti-PD-1/PD-L1-induced cancer cell death. Conclusions: Here we use mouse- and human-derived autologous pancreatic cancer organoid/immune cell co-cultures to demonstrate that elevated infiltration of polymorphonuclear (PMN)-MDSCs within the PDAC tumor microenvironment inhibit T cell effector function, regardless of PD-1/PD-L1 inhibition. We present a pre-clinical model that may predict the efficacy of targeted therapies to improve the outcome of patients with this aggressive and otherwise unpredictable malignancy.
