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Cushing's disease (CD) is a serious endocrine disorder attributed to an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that that subsequently leads to 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 a 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 Cushing's disease patients.
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Hipersecreção Hipofisária de ACTH , Neoplasias Hipofisárias , Humanos , Corticotrofos/metabolismo , Corticotrofos/patologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/uso terapêutico , Hipersecreção Hipofisária de ACTH/tratamento farmacológico , Hipersecreção Hipofisária de ACTH/metabolismo , Hipersecreção Hipofisária de ACTH/patologia , Mifepristona/farmacologia , Mifepristona/metabolismo , Mifepristona/uso terapêutico , Proteínas Hedgehog , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Hormônio Adrenocorticotrópico/farmacologia , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/uso terapêuticoRESUMO
(1) Background: Cushing's disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) 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. Current surgical remission rates reported in only 56% of patients depending on several criteria. The lack of specificity, poor tolerability, and low efficacy of the subsequent second-line medical therapies make CD a medical therapeutic challenge. One major limitation that hinders the development of specific medical therapies is the lack of relevant human model systems that recapitulate the cellular composition of PitNET microenvironment. (2) Methods: human pituitary tumor tissue was harvested during transsphenoidal surgery from CD patients to generate organoids (hPITOs). (3) Results: hPITOs generated from corticotroph, lactotroph, gonadotroph, and somatotroph tumors 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 PitNET specific markers, using spectral flow cytometry and exome sequencing. A high-throughput drug screen demonstrated patient-specific drug responses of hPITOs amongst each tumor subtype. Generation of induced pluripotent stem cells (iPSCs) from a CD patient carrying germline mutation CDH23 exhibited dysregulated cell lineage commitment. (4) Conclusions: The human pituitary neuroendocrine tumor organoids represent a novel approach in how we model complex pathologies in CD patients, which will enable effective personalized medicine for these patients.
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Tumores Neuroendócrinos , Hipersecreção Hipofisária de ACTH , Neoplasias Hipofisárias , Humanos , Hipersecreção Hipofisária de ACTH/tratamento farmacológico , Hipersecreção Hipofisária de ACTH/cirurgia , Organoides , Tumores Neuroendócrinos/tratamento farmacológico , Hidrocortisona , Microambiente TumoralRESUMO
BACKGROUND & AIMS: 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. METHODS: 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 TLR9T>C 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. RESULTS: 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 SLFN+-MDSC polarization. Neutralizing IFNα in vivo mitigated Helicobacter-induced spasmolytic polypeptide-expressing metaplasia. The TLR9 minor C allele creates a nuclear factor-κB 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. CONCLUSIONS: TLR9 plays an essential role in the production of IFNα and polarization of SLFN+ MDSCs on Helicobacter infection. Subjects carrying the rs5743836 TLR9 minor C allele are predisposed to neoplastic complications if chronically infected.
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Infecções por Helicobacter , Células Supressoras Mieloides , Neoplasias Gástricas , Receptor Toll-Like 9 , Animais , Helicobacter , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Humanos , Interferon-alfa , Metaplasia , Camundongos , NF-kappa B/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Receptor 4 Toll-Like , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoRESUMO
OBJECTIVE: The 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. DESIGN: The 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 gp130 F/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. RESULTS: AIM2 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 gp130 F/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. CONCLUSION: AIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.
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Melanoma , Neoplasias Gástricas , Animais , Carcinogênese/genética , Movimento Celular/genética , Receptor gp130 de Citocina/metabolismo , DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Imunidade Inata/genética , Inflamassomos/genética , Inflamassomos/metabolismo , Camundongos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/patologia , Regulação para CimaRESUMO
(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.
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OBJECTIVE: Gastroenteropancreatic 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. AIM: To 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. DESIGN: Whole 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. RESULTS: Both 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. CONCLUSIONS: Stromal-epithelial interactions induce proinflammatory cytokines that promote Brunner's gland reprogramming.
Assuntos
Gastrinoma , Neoplasias Intestinais , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Adulto , Cálcio , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA , Fator de Necrose Tumoral alfa/genéticaRESUMO
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.
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Neoplasias Gástricas , Antígeno B7-H1 , Técnicas de Cocultura , Humanos , Organoides , Neoplasias Gástricas/terapia , Linfócitos T CitotóxicosRESUMO
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.
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Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Fenômenos Biomecânicos , Medula Óssea/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Mecanotransdução Celular , Invasividade Neoplásica , Microambiente TumoralRESUMO
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. 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. 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. In conclusion, the PDO/immune cell co-cultures may be used to study immunosuppressive MDSC function within the gastric tumor microenvironment. The mTOR signaling pathway mediates GLI-induced PD-L1 expression in gastric cancer.
Assuntos
Antígeno B7-H1/genética , Proteínas Hedgehog/genética , Organoides/metabolismo , Neoplasias Gástricas/genética , Serina-Treonina Quinases TOR/genética , Transcrição Gênica/genética , Proteína GLI1 em Dedos de Zinco/genética , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Helicobacter pylori/patogenicidade , Humanos , Imunoterapia/métodos , Transdução de Sinais/genética , Neoplasias Gástricas/microbiologia , Linfócitos T Citotóxicos/metabolismo , Microambiente Tumoral/genéticaRESUMO
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.
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Neoplasias , Organoides , Trato Gastrointestinal , HumanosRESUMO
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.
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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.
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Apoptose/fisiologia , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Elementos Facilitadores Genéticos/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteína 3 com Repetições IAP de Baculovírus/fisiologia , Caspase 3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/patogenicidade , Humanos , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Estômago/patologia , Neoplasias Gástricas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
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Helicobacter pylori/fisiologia , Organoides/fisiologia , Estômago/fisiologia , Técnicas de Cultura de Tecidos/métodos , Animais , Técnicas de Cocultura , Humanos , Microinjeções , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Ratos , Linfócitos T Citotóxicos/citologiaRESUMO
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). OBJECTIVE: To identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM. DESIGN: We 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. RESULTS: MicroRNA 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. CONCLUSION: Taken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.
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Proteínas de Transporte/metabolismo , Infecções por Helicobacter , MicroRNAs/metabolismo , Neoplasias Gástricas , Animais , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Helicobacter pylori/fisiologia , Interferon-alfa/metabolismo , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/metabolismo , Lesões Pré-Cancerosas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologiaRESUMO
Multiple myeloma (MM) drug resistance highlights a need for alternative therapeutic strategies. In this study, we show that CASIN, a selective inhibitor of cell division cycle 42 (Cdc42) GTPase, inhibited proliferation and survival of melphalan/bortezomib-resistant MM cells more profoundly than that of the sensitive cells. Furthermore, CASIN was more potent than melphalan/bortezomib in inhibiting melphalan/bortezomib-resistant cells. In addition, CASIN sensitized melphalan/bortezomib-resistant cells to this drug combination. Mechanistically, Cdc42 activity was higher in melphalan/bortezomib-resistant cells than that in the sensitive cells. CASIN inhibited mono-ubiquitination of Fanconi anemia (FA) complementation group D2 (FANCD2) of the FA DNA damage repair pathway in melphalan-resistant but not melphalan-sensitive cells, thereby sensitizing melphalan-resistant cells to DNA damage. CASIN suppressed epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and extracellular signal-regulated kinase (ERK) activities to a larger extent in bortezomib-resistant than in melphalan-sensitive cells. Reconstitution of ERK activity partially protected CASIN-treated bortezomib-resistant cells from death, suggesting that CASIN-induced killing is attributable to suppression of ERK. Importantly, CASIN extended the lifespan of mouse xenografts of bortezomib-resistant cells and caused apoptosis of myeloma cells from bortezomib-resistant MM patients. Finally, CASIN had negligible side effects on peripheral blood mononuclear cells (PBMC) from healthy human subjects and normal B cells. Our data provide a proof of concept demonstration that rational targeting of Cdc42 represents a promising approach to overcome MM drug resistance.
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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.
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Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Lisossomos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Antibacterianos/farmacologia , Autofagia , Canais de Cálcio/metabolismo , Modelos Animais de Doenças , Infecções por Helicobacter/patologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Viabilidade Microbiana , Transporte Proteico , Estômago/microbiologia , Estômago/patologia , Canais de Potencial de Receptor Transitório/genéticaRESUMO
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.
Assuntos
Antígeno B7-H1/metabolismo , Infecções por Helicobacter/imunologia , Adolescente , Antígenos de Bactérias/genética , Antígeno B7-H1/genética , Células Epiteliais/metabolismo , Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Regulação da Expressão Gênica/genética , Proteínas Hedgehog/metabolismo , Infecções por Helicobacter/genética , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Humanos , Organoides/microbiologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Estômago , Adulto JovemRESUMO
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.
Assuntos
Organoides/patologia , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Epirubicina/farmacologia , Epirubicina/uso terapêutico , Epitélio/efeitos dos fármacos , Epitélio/patologia , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Ontologia Genética , Humanos , Concentração Inibidora 50 , Camundongos , Organoides/efeitos dos fármacos , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Fenótipo , Receptor ErbB-2/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Neoplasias Gástricas/tratamento farmacológicoRESUMO
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.
Assuntos
Mucosa Gástrica/citologia , Organoides/citologia , Cultura Primária de Células/métodos , Animais , Células Epiteliais/citologia , Humanos , CamundongosRESUMO
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.