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.
Background & AimsOur 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.MethodsOrganoids 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.ResultsWhereas 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.ConclusionsThe 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.
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.
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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