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OBJECTIVE: Tumors evade immune surveillance by expressing Programmed Death-Ligand 1 (PD-L1), subsequently inhibiting CD8+ cytotoxic T lymphocyte function. Response of gastric cancer to immunotherapy is relatively low. Our laboratory has reported that Helicobacter pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Hedgehog (Hh) signaling pathway. The PI3K/AKT/mTOR pathway is activated in gastric cancer and may have immunomodulatory potential. We hypothesize that Hh signaling mediates mTOR-induced PD-L1 expression. DESIGN: Patient-derived organoids (PDOs) were generated from gastric biopsies and resected tumor tissues. Autologous organoid/immune cell co-cultures were used to study the immunosuppressive function of MDSCs. NanoString Digital Spatial Profiling (DSP) of immune-related protein markers using FFPE slide-mounted tissues from gastric cancer patients was performed. RESULTS: DSP analysis showed infiltration of immunosuppressive MDSCs expressing Arg1, CD66b, VISTA and IDO1 within cancer tissues. Orthotopic transplantation of patient derived organoids (PDOs) resulted in the engraftment of organoids and the development of histology similar to that observed in the patient’s tumor tissue. PDO/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of PMN-MDSCs within these co-cultures sensitized the organoids to anti-PD-1/PD-L1-induced cancer cell death. Rapamycin decreased phosphorylated S6K, Gli2 and PD-L1 expression in PDO/immune cell co-cultures. Transcriptional regulation of PD-L1 by GLI1 and GLI2 was blocked by rapamycin. CONCLUSIONS: The PDO/immune cell co-cultures may be used to to study immunosuppressive MDSC function within the gastric tumor microenvironment. The mTOR signaling pathway mediates GLI-induced PD-L1 expression in gastric cancer.

Cushing’s disease (CD) is a serious endocrine disorder attributed to an ACTH-secreting pituitary neuroendocrine tumor (PitNET) that subsequently causes chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSCUSP48 and iPSCUSP8 cultures, mifepristone induced the predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD patients.HIGHLIGHTSCushing disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that leads to chronic hypercortisolemiaMifepristone (Korlym®), a non-selective glucocorticoid receptor (GR) antagonist, is an approved treatment for patients with Cushing disease, and competes with the binding of cortisol to the GR as well as the binding of progesterone to the progesterone receptor.Relacorilant is an investigational selective GR modulator in development for the treatment of Cushing syndrome that, unlike mifepristone, does not bind to the other hormone receptors.Unlike mifepristone, relacorilant does not significantly raise systemic cortisol levels, and cases of PitNET regression with relacorilant have been reported. However, the mechanisms behind these clinical differences remained unknown.PitNET organoids were generated from: 1) CRISPR-Cas9 gene editing of patient iPSCs, and 2) CD patient corticotroph PitNETs (hPITOs) and used to compare the diverse effects of mifepristone and relacorilant in a human-relevant model that recapitulates the PitNET microenvironment in vitro.Mifepristone and relacorilant have different effects on the induction of somatostatin receptor (SSTR) SSTR2 and SSTR5 expression, ACTH secretion and PitNET organoid proliferation and apoptosis.BRIEF COMMENTARYBackgroundCushing’s disease (CD), a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) leads to chronic hypercortisolemia. Approved for the treatment for CD, Mifepristone (Korlym®) is a non-selective glucocorticoid receptor (GR) antagonist with additional competitive binding with progesterone for the progesterone receptor. Relacorilant, an investigational selective GR modulator in development for the treatment of CD, does not bind to the other hormone receptors.Translational SignificancePatient-derived PitNET organoids recapitulate the tumor microenvironment in vitro. PitNET organoids revealed the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD.

Background Cushing's disease (CD) is an endocrine disorder caused by an ACTH-secreting pituitary adenoma that is associated with increased morbidity and mortality. Because somatostatin inhibits pituitary ACTH secretion, the somatostatin receptor subtypes (SSTR) 2 and 5 have been targets of medical therapies for CD. The SSTR5 is most consistently and strongly expressed in corticotroph pituitary tumors but the SSTR dynamics in CD during treatment and responses to cortisol level changes are far from being understood. Studies in adult stomach demonstrated that the Hedgehog (Hh) signaling pathway is critical for the regulation of somatostatin and SSTR signaling. While Hh signaling is known to be essential during the embryonic development of the pituitary and in the adult gland, the mechanism by which Hh signaling regulates SSTR expression in CD is unknown. Hypothesis Activation of glucocorticoid receptor (NR3C1, GR) results in the inhibition of Hh transcription factor GLI1 leading to reduced SSTR expression. Methods We developed a human pituitary adenoma organoid model (hPITOiPSC) from induced pluripotent stem cells treated with the glucocorticoid receptor (GR) antagonist mifepristone, and co-treated them with or without SSTR agonists pasireotide or octreotide. In a separate series of experiments, the role of Hh transcription factor GLI1 was identified using hPITOiPSC treated with mifepristone with or without GANT61 (GLI inhibitor) or ketoconazole (Smoothened, SMO inhibitor). CRISPR-Cas9 gene editing of hPITOiPSC pituitary organoids were used to model the development of pituitary corticotroph adenomas in the presence of BRAF, USP48 and USP8 mutations. Human pituitary adenoma tissue harvested fresh during pituitary surgery was used to generate pituitary corticotroph subtype adenoma organoids (hPITOs). Dose responses using standard of care drugs were performed using the hPITOs. Results 1) At baseline SSTR2 was abundantly expressed within hPITOiPSC. Mifepristone induced significant increases in ACTH secretion and POMC expression that correlated with induced SSTR2 and 5. Mifepristone-induced SSTR2 and 5 expression was significantly inhibited in the presence of GANT61, whereas SMO inhibitor ketoconazole had a minimal effect on mifepristone-induced SSTR2/5, POMC expression and ACTH secretion. Dexamethasone alone significantly inhibited both GLI1 and SSTR2 and 5. 2) While pituitary organoids that were differentiated from control iPSCs (iPSCCtrl) expressed all major hormone-producing cell lineages, there was a significant increase in ACTH expression with loss of PIT1, GH, FSH, LH and PRL in iPCSs expressing mutated BRAF, USP48 and USP8. Organoids expressing a mutation in BRAF had significantly higher SSTR2 expression levels compared to controls. 3) HPITOs generated from pituitary adenomas of CD patients showed differential organoid responses to pasireotide, mifepristone, and ketoconazole. Conclusion Within pituitary adenomas, the SSTR is a transcriptional target of Gli1, and this response is blocked by the activation of the GR. These data form the basis of combination therapy for CD with mifepristone and pasireotide. Presentation: Saturday, June 11, 2022 1:37 p.m. - 1:42 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Cushing's disease (CD) is a serious endocrine disorder caused by dysregulated adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, obesity, cognitive impairment, anxiety, depression, and death. The first-line treatment for CD is pituitary surgery, which is followed by disease remission. Current surgical remission rates reported range from 47–85% depending on several remission criteria. The lack of specificity, poor tolerability, and low efficacy of the subsequent second-line medical therapies makes CD a medical therapeutic challenge. One major limitation that hinders the development of specific medical therapies is the lack of human relevant model systems that recapitulate the cellular composition of pituitary adenomas. Human pituitary adenoma tissue was harvested during transsphenoidal surgery from CD patients to generate organoids (hPITOs). hPITOs generated from corticotroph, lactotroph, gonadotroph and somatotroph adenomas exhibited morphological diversity among the organoid lines between individual patients and amongst subtypes. The similarity in cell lineages between the organoid line and the patient’s tumor was validated by comparing the neuropathology report to the expression pattern of pituitary adenoma specific markers using spectral flow cytometry and exome sequencing. A high-throughput drug screen demonstrated patient-specific drug responses of hPITOs amongst each adenoma subtype. Generation of induced pluripotent stem cells (iPSCs) from patients carrying germline mutations relevant to CD exhibited dysregulated cell lineage commitment. The human pituitary adenoma organoids represent a novel approach in how we model complex pathologies in CD patients that may enable more effective personalized medicine for these patients.

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM).ObjectiveTo identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM.DesignWe performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4– cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b.ResultsMicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth.ConclusionTaken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.

Background and AimsA subset of MDSCs that express murine Schlafen4 (SLFN4) or its human ortholog SLFN12L polarize in the Helicobacter-inflamed stomach coincident with intestinal or spasmolytic polypeptide-expressing metaplasia (SPEM). We propose that individuals with a more robust response to damage-activated molecular patterns (DAMPs) and increased Toll-like receptor (TLR9) expression are predisposed to the neoplastic complications of Helicobacter infection.MethodsA mouse or human Transwell™ co-culture system comprised of dendritic cells (DCs), 2-dimensional gastric epithelial monolayers and Helicobacter were used to dissect the cellular source of interferon (IFNα) in the stomach by flow cytometry. Conditioned media from the cocultures polarized primary myeloid cells. Myeloid-derived suppressor cell (MDSC) activity was determined by T cell suppression assays. In human subjects with intestinal metaplasia or gastric cancer, the rs5743836 TLR9T>C variant was genotyped and linked to TLR9, IFNα and SLFN12L expression by immunohistochemistry. NFκB binding to the TLR9 C allele was determined by electrophoretic mobility shift assays.ResultsHelicobacter infection induced gastric epithelial and plasmacytoid DC expression of TLR9 and IFNα. Co-culturing primary mouse or human cells with DCs and Helicobacter induced TLR9, IFNα secretion and SLFN+-MDSC polarization. Neutralizing IFNα in vivo mitigated Helicobacter-induced SPEM. The TLR9 minor C allele creates an NFκb binding site associated with higher levels of TLR9, IFNα and SLFN12L in Helicobacter-infected stomachs that correlated with a greater incidence of metaplasias and cancer.ConclusionTLR9 plays an essential role in the production of IFNα and polarization of SLFN+-MDSCs upon Helicobacter infection. Subjects carrying the rs5743836 TLR9 minor C allele are predisposed to neoplastic complications if chronically infected.

Simple Summary HER2 may contribute to immune evasion in gastric cancer that is associated with PD-L1 expression. Autologous organoid/immune cell co-cultures serve as an appropriate in vitro model to study the effects of anti-HER2 targeted therapy in combination with anti-PD1 immune checkpoint inhibition and may be used as an ex vivo tool for precision medicine. Abstract (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.

A subset of myeloid-derived suppressor cells (MDSCs) that express murine Schlafen4 (SLFN4) or its human ortholog SLFN12L polarize in the Helicobacter-inflamed stomach coincident with intestinal or spasmolytic polypeptide-expressing metaplasia. We propose that individuals with a more robust response to damage-activated molecular patterns and increased Toll-like receptor 9 (TLR9) expression are predisposed to the neoplastic complications of Helicobacter infection.A mouse or human Transwell co-culture system composed of dendritic cells (DCs), 2-dimensional gastric epithelial monolayers, and Helicobacter were used to dissect the cellular source of interferon-α (IFNα) in the stomach by flow cytometry. Conditioned media from the co-cultures polarized primary myeloid cells. MDSC activity was determined by T-cell suppression assays. In human subjects with intestinal metaplasia or gastric cancer, the rs5743836 TLR9TC variant was genotyped and linked to TLR9, IFNα, and SLFN12L expression by immunohistochemistry. Nuclear factor-κB binding to the TLR9 C allele was determined by electrophoretic mobility shift assays.Helicobacter infection induced gastric epithelial and plasmacytoid DC expression of TLR9 and IFNα. Co-culturing primary mouse or human cells with DCs and Helicobacter induced TLR9, IFNα secretion, and SLFNTLR9 plays an essential role in the production of IFNα and polarization of SLFN

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.

Gastric cancer is the third most common cause of cancer-related death worldwide with a 5-year survival rate of only 29%. The incidence of gastric cancer in the United States is relatively low due to the diagnosis and treatment of the major risk factor Helicobacter pylori (H. pylori). Even after H. pylori infection has been eradicated, there is still a risk of developing gastric cancer. Gastric cancer is the final clinical outcome that is often initiated by a sustained inflammatory response to H. pylori infection and immune cell-epithelial crosstalk. The review focuses on reporting the mechanisms by which the bacterium modulates the host’s innate and adaptive immune response and the gastric epithelium as part of a strategy to create an immunosuppressive microenvironment that ultimately leads to gastric cancer.

Maintaining the correct ratio of cellular niches in space and time allows tissues to perform their complex functions and create patterns. This maintenance of tissue patterning is often dysregulated during cancer development. In order to study tissue patterning, temporal and spatial resolution of communication between single cells is required. To explore how this complex process is controlled, we developed a patient-derived organoid screening platform to study both tissue patterning and kinase pathway dynamics in single cells across hundreds of conditions. Unlike normal and polyp-derived organoids, tumor organoids displayed a lack of patterning reminiscent of in vivo dysplasia. Using this system, we discovered that waves of Erk signaling induced by apoptotic cells are critical for maintaining tissue patterning in human colonic organoids. Using a small molecule-based screen, we discovered novel kinases which limit Erk activation across tissues and help maintain patterning. Perturbation of Erk restricting kinases results in dysfunctional colonic wound repair and re-establishment of tissue patterns in vivo. Citation Format: Kelvin W. Pond, Jayati Chakrabarti, Curtis A. Thorne, Andrew L. Paek. ERK dynamics are essential for colon tissue patterning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 171.

ObjectiveThe absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of AIM2 in cancer is ill-defined.DesignThe expression of AIM2 and its clinical significance was assessed in human gastric cancer (GC) patient cohorts. Genetic or therapeutic manipulation of AIM2 expression and activity was performed in the genetically engineered gp130F/F spontaneous GC mouse model, as well as human GC cell line xenografts. The biological role and mechanism of action of AIM2 in gastric tumourigenesis, including its involvement in inflammasome activity and functional interaction with microtubule-associated end-binding protein 1 (EB1), was determined in vitro and in vivo.ResultsAIM2 expression is upregulated by interleukin-11 cytokine-mediated activation of the oncogenic latent transcription factor STAT3 in the tumour epithelium of GC mouse models and patients with GC. Genetic and therapeutic targeting of AIM2 in gp130F/F mice suppressed tumourigenesis. Conversely, AIM2 overexpression augmented the tumour load of human GC cell line xenografts. The protumourigenic function of AIM2 was independent of inflammasome activity and inflammation. Rather, in vivo and in vitro AIM2 physically interacted with EB1 to promote epithelial cell migration and tumourigenesis. Furthermore, upregulated expression of AIM2 and EB1 in the tumour epithelium of patients with GC was independently associated with poor patient survival.ConclusionAIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.

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.

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.

H. pylori infection is one of the leading causes of gastric cancer and the pathogenicity of H. pylori infection is associated with its ability to induce chronic inflammation and apoptosis resistance. While H. pylori infection-induced expression of pro-inflammatory cytokines for chronic inflammation is well studied, the molecular mechanism underlying the apoptosis resistance in infected cells is not well understood. In this study, we demonstrated that H. pylori infection-induced apoptosis resistance in gastric epithelial cells triggered by Raptinal, a drug that directly activates caspase-3. This resistance resulted from the induction of cIAP2 (encoded by BIRC3) since depletion of BIRC3 by siRNA or inhibition of cIAP2 via BV6 reversed H. pylori-suppressed caspase-3 activation. The induction of cIAP2 was regulated by H. pylori-induced BIRC3 eRNA synthesis. Depletion of BIRC3 eRNA decreased H. pylori-induced cIAP2 and reversed H. pylori-suppressed caspase-3 activation. Mechanistically, H. pylori stimulated the recruitment of bromodomain-containing factor Brd4 to the enhancer of BIRC3 and promoted BIRC3 eRNA and mRNA synthesis. Inhibition of Brd4 diminished the expression of BIRC3 eRNA and the anti-apoptotic response to H. pylori infection. Importantly, H. pylori isogenic cagA-deficient mutant failed to activate the synthesis of BIRC3 eRNA and the associated apoptosis resistance. Finally, in primary human gastric epithelial cells, H. pylori also induced resistance to Raptinal-triggered caspase-3 activation by activating the Brd4-dependent BIRC3 eRNA synthesis in a CagA-dependent manner. These results identify a novel function of Brd4 in H. pylori-mediated apoptosis resistance via activating BIRC3 eRNA synthesis, suggesting that Brd4 could be a potential therapeutic target for H. pylori-induced gastric cancer.

While the incidence of gastric cancer in the United States is relatively low due to the diagnosis and treatment of the major risk factor Helicobacter pylori (H. pylori), 5-year patient survival is only approximately 29%. Even after H. pylori infection has been eradicated there is still a risk of developing gastric cancer. Gastric cancer is the final clinical outcome that is often initiated by a sustained inflammatory response and altered epithelial cell differentiation and metaplasia in response to H. pylori infection. Identifying the early epithelial responses to H. pylori infection is important in advancing our understanding of the events that shape a conducive environment for the progression of gastric cancer. Thus, we developed a human gastric tissue-derived organoid-based approach to identify the initiating molecular events that trigger gastric cancer development in response to bacterial infection.

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.

While the incidence of gastric cancer in the United States is relatively low due to the diagnosis and treatment of the major risk factor Helicobacter pylori (H. pylori), 5-year patient survival is only approximately 29%. Even after H. pylori infection has been eradicated there is still a risk of developing gastric cancer. Gastric cancer is the final clinical outcome that is often initiated by a sustained inflammatory response and altered epithelial cell differentiation and metaplasia in response to H. pylori infection. Identifying the early epithelial responses to H. pylori infection is important in advancing our understanding of the events that shape a conducive environment for the progression of gastric cancer. Thus, we developed a human gastric tissue-derived organoid-based approach to identify the initiating molecular events that trigger gastric cancer development in response to bacterial infection.

ObjectiveGastroenteropancreatic neuroendocrine tumours (GEP-NETs) encompass a diverse group of neoplasms that vary in their secretory products and in their location within the gastrointestinal tract. Their prevalence in the USA is increasing among all adult age groups.AimTo identify the possible derivation of GEP-NETs using genome-wide analyses to distinguish small intestinal neuroendocrine tumours, specifically duodenal gastrinomas (DGASTs), from pancreatic neuroendocrine tumours.DesignWhole exome sequencing and RNA-sequencing were performed on surgically resected GEP-NETs (discovery cohort). RNA transcript profiles available in the Gene Expression Omnibus were analysed using R integrated software (validation cohort). Digital spatial profiling (DSP) was used to analyse paraffin-embedded GEP-NETs. Human duodenal organoids were treated with 5 or 10 ng/mL of tumor necrosis factor alpha (TNFα) prior to qPCR and western blot analysis of neuroendocrine cell specification genes.ResultsBoth the discovery and validation cohorts of small intestinal neuroendocrine tumours induced expression of mesenchymal and calcium signalling pathways coincident with a decrease in intestine-specific genes. In particular, calcium-related, smooth muscle and cytoskeletal genes increased in DGASTs, but did not correlate with MEN1 mutation status. Interleukin 17 (IL-17) and tumor necrosis factor alpha (TNFα) signalling pathways were elevated in the DGAST RNA-sequencing. However, DSP analysis confirmed a paucity of immune cells in DGASTs compared with the adjacent tumour-associated Brunner’s glands. Immunofluorescent analysis showed production of these proinflammatory cytokines and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) by the tumours and stroma. Human duodenal organoids treated with TNFα induced neuroendocrine tumour genes, SYP, CHGA and NKX6.3.ConclusionsStromal–epithelial interactions induce proinflammatory cytokines that promote Brunner’s gland reprogramming.

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl−channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET691, of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP]i), EP2, or EP4receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl−current activation (increasing EC50s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA655/RGEA691) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]iin hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl−currents without increasing [cAMP]i,while PGE2and forskolin-IBMX increased both. Fatty acids were not agonists of EP2or EP4receptors. L-161,982, a supposed EP4-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl−currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET691of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]i, and EP2or EP4receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.

Helicobacter pylori infection is a proven carcinogen for gastric cancer. Its virulence factor vacuolating cytotoxin A (VacA) promotes more severe disease and gastric colonization. VacA, by an unknown mechanism, usurps lysosomal and autophagy pathways to generate a protected reservoir for H. pylori that confers bacterial survival in vitro. Here, we show the existence of a VacA-generated intracellular niche in vivo that protects the bacteria from antibiotic treatment and leads to infection recrudescence after therapy. Furthermore, we report that VacA targets the lysosomal calcium channel TRPML1 to disrupt endolysosomal trafficking and mediate these effects. Remarkably, H. pylori that lack toxigenic VacA colonize enlarged dysfunctional lysosomes in the gastric epithelium of trpml1-null mice, where they are protected from eradication therapy. Furthermore, a small molecule agonist directed against TRPML1 reversed the toxic effects of VacA on endolysosomal trafficking, culminating in the clearance of intracellular bacteria. These results suggest that TRPML1 may represent a therapeutic target for chronic H. pylori infection.

Background/Hypothesis Hedgehog (Hh) signaling not only plays a crucial role in embryonic development, but also controls gastric physiological events including epithelial cell differentiation, regen...

SUMMARY While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential., Graphical Abstract, In brief Watson et al. demonstrate that mechanical conditioning by stiff microenvironments in breast tumors is maintained in cancer cells after dissemination to softer microenvironments, including bone marrow. They show that mechanical conditioning promotes invasion and osteolysis and establish a mechanical conditioning (MeCo) score, associated with bone metastasis in patients.

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.

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.

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.