Potent and selective binders of the E3 ubiquitin ligase ZNRF3 stimulate Wnt signaling and intestinal organoid growth.

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small- or large-molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3.

Human Gastrointestinal Organoid Models for Studying Microbial Disease and Cancer.

One of the major discoveries in stem cell research in the past decade embraces the development of "organs in a dish," also known as "organoids." Organoids are three-dimensional cellular structures derived from primary stem cells of different organ-specific cell types which are capable of self-renewal and maintenance of the parental lineages. Researchers have developed in vitro organoid models to mimic in vivo host-microbial interactions and disease. In this review, we focus on the use of gastrointestinal organoids as models of microbial disease and cancer.

Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma.

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.

Increased Programmed Death-Ligand 1 is an Early Epithelial Cell Response to Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) is the major risk factor for the development of gastric cancer. Our laboratory has reported that the Sonic Hedgehog (Shh) signaling pathway is an early response to infection that is fundamental to the initiation of H. pylori-induced gastritis. H. pylori also induces programmed death ligand 1 (PD-L1) expression on gastric epithelial cells, yet the mechanism is unknown. We hypothesize that H. pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Shh signaling pathway during infection. To identify the role of Shh signaling as a mediator of H. pylori-induced PD-L1 expression, human gastric organoids generated from either induced pluripotent stem cells (HGOs) or tissue (huFGOs) were microinjected with bacteria and treated with Hedgehog/Gli inhibitor GANT61. Gastric epithelial monolayers generated from the huFGOs were also infected with H. pylori and treated with GANT61 to study the role of Hedgehog signaling as a mediator of induced PD-1 expression. A patient-derived organoid/autologous immune cell co-culture system infected with H. pylori and treated with PD-1 inhibitor (PD-1Inh) was developed to study the protective mechanism of PD-L1 in response to bacterial infection. H. pylori significantly increased PD-L1 expression in organoid cultures 48 hours post-infection when compared to uninfected controls. The mechanism was cytotoxic associated gene A (CagA) dependent. This response was blocked by pretreatment with GANT61. Anti-PD-L1 treatment of H. pylori infected huFGOs, co-cultured with autologous patient cytotoxic T lymphocytes and dendritic cells, induced organoid death. H. pylori-induced PD-L1 expression is mediated by the Shh signaling pathway within the gastric epithelium. Cells infected with H. pylori that express PD-L1 may be protected from the immune response, creating premalignant lesions progressing to gastric cancer.

An Organoid-Based Preclinical Model of Human Gastric Cancer.

BACKGROUND & AIMS: Our goal was to develop an initial study for the proof of concept whereby gastric cancer organoids are used as an approach to predict the tumor response in individual patients. METHODS: Organoids were derived from resected gastric cancer tumors (huTGOs) or normal stomach tissue collected from sleeve gastrectomies (huFGOs). Organoid cultures were treated with standard-of-care chemotherapeutic drugs corresponding to patient treatment: epirubicin, oxaliplatin, and 5-fluorouracil. Organoid response to chemotherapeutic treatment was correlated with the tumor response in each patient from whom the huTGOs were derived. HuTGOs were orthotopically transplanted into the gastric mucosa of NOD scid gamma mice. RESULTS: Whereas huFGOs exhibited a half maximal inhibitory concentration that was similar among organoid lines, divergent responses and varying half maximal inhibitory concentration values among the huTGO lines were observed in response to chemotherapeutic drugs. HuTGOs that were sensitive to treatment were derived from a patient with a near complete tumor response to chemotherapy. However, organoids resistant to treatment were derived from patients who exhibited no response to chemotherapy. Orthotropic transplantation of organoids resulted in the engraftment and development of human adenocarcinoma. RNA sequencing revealed that huTGOs closely resembled the patient's native tumor tissue and not commonly used gastric cancer cell lines and cell lines derived from the organoid cultures. CONCLUSIONS: The treatment of patient-derived organoids alongside patients from whom cultures were derived will ultimately test their usefulness to predict individual therapy response and patient outcome.

Establishment of Human- and Mouse-Derived Gastric Primary Epithelial Cell Monolayers from Organoids.

Organoid cultures generated from gastrointestinal tissues have been an invaluable advancement for in vitro studies of physiological function and disease. Here we present a comprehensive protocol for the establishment and culture of human- and mouse-derived 3-dimensional gastric organoids transferred to 2-dimensional gastric epithelial cell monolayers. We introduce two methods that include the establishment of monolayers from: (1) intact organoids, and (2) single cells dissociated from intact organoids.

Mouse-Derived Gastric Organoid and Immune Cell Co-culture for the Study of the Tumor Microenvironment.

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. Immunotherapy via PD-L1 blockade is used for a variety of malignancies, yet the prognostic value of immune checkpoint inhibition for the treatment of gastric cancer remains controversial. Thus, preclinical models that would predict the efficacy of such therapy in a subgroup of gastric cancer patients would be an advancement in the personalized treatment of this disease. Three-dimensional organoid cultures have not only been used to investigate the mechanisms regulating development and disease, but have also been used for high-throughput drug screening for targeted personalized therapy. Here we present the methodology for the co-culture of mouse-derived gastric cancer organoids with autologous immune cells specifically for the study of PD-L1/PD-1 interactions within the tumor microenvironment in vitro.

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Hedgehog signaling induces PD-L1 expression and tumor cell proliferation in gastric cancer.

Tumor cells expressing programmed cell death ligand 1 (PD-L1) interact with PD-1 on CD8+ cytotoxic T lymphocytes (CTLs) to inhibit CTL effector function. In gastric cancer, the mechanism regulating PD-L1 is unclear. The Hedgehog (Hh) signaling pathway is reactivated in various cancers including gastric. Here we tested the hypothesis that Hh-induced PD-L1 inactivates effector T cell function and allows gastric cancer cell proliferation. Mouse organoids were generated from tumors of a triple-transgenic mouse model engineered to express an activated GLI2 allele, GLI2A, in Lgr5-expressing stem cells, (mTGOs) or normal mouse stomachs (mGOs). Bone marrow-derived dendritic cells (DCs) were pulsed with conditioned media collected from normal (mGOCM) or cancer (mTGOCM) organoids. Pulsed DCs and CTLs were then co-cultured with either mGOs or mTGOs in the presence of PD-L1 neutralizing antibody (PD-L1Ab). Human-derived gastric cancer organoids (huTGOs) were used in drug and xenograft assays. Hh/Gli inhibitor, GANT-61 significantly reduced the expression of PD-L1 and tumor cell proliferation both in vivo and in vitro. PD-L1Ab treatment induced tumor cell apoptosis in mTGO/immune cell co-cultures. GANT-61 treatment sensitized huTGOs to standard-of-care chemotherapeutic drugs both in vivo and in vitro. Thus, Hh signaling mediates PD-L1 expression in gastric cancer cells and subsequently promotes tumor proliferation.