Cancer Letters

Cancer Letters

Volume 518, 10 October 2021, Pages 59-71
Cancer Letters

Hedgehog transcriptional effector GLI mediates mTOR-Induced PD-L1 expression in gastric cancer organoids

https://doi.org/10.1016/j.canlet.2021.06.007Get rights and content

Highlights

  • Myeloid derived suppressor cells (MDSCs) accumulate within the tumor microenvironment (TME) and may contribute to resistance to immunotherapy.

  • The PI3K/AKT/mTOR pathway is activated in gastric cancer and may have immunomodulatory potential.

  • An autologous cancer PDO/immune cell co-culture system may be used to investigate PD-L1/PD-1 blockade together with the inhibition of immunosuppressive cells MDSCs.

  • The mTOR pathway mediates the non-canonical Hedgehog signaling cascade to induce PD-L1 expression.

  • The mTOR signaling pathway may be targeted to decrease PD-L1 expression and increase response to immunotherapy in gastric cancer.

Abstract

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.

Introduction

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide [1]. The 5-year survival rate varies in the range of 20–40% with a case fatality rate at approximately 70% because most gastric cancer patients are in the advanced stage at the time of diagnosis [2]. Treatment for advanced-stage gastric cancer primarily relies on combination chemotherapy [3]. However, outcomes are often hampered by severe side effects, hence better treatment is still required.

Early observations from our laboratory demonstrated that CagA stimulates Sonic Hedgehog (Shh) signaling within parietal cells, a response that is mediated by NFkB signaling [4,5]. We then showed that the gastric morphogen, Shh, is fundamental to the initiation of gastritis in response to H. pylori infection [4]. Following these studies, we used iPSC- and tissue-derived human gastric organoid cultures to report that H. pylori-induced immune checkpoint molecule programmed cell death 1 ligand (PD-L1), is mediated by Hedgehog (Hh) signaling [6]. In these studies, PD-L1 was localized to Spasmolytic Polypeptide (TFF2)-Expressing Metaplastic (SPEM) cells as a mechanism by which these cells may survive chronic inflammation, for the persistence of infection and progression of disease to cancer [6]. In gastric cancer, Hh signaling mediates PD-L1 expression within tumor cells [7], but, the exact mechanism is unclear. In gastric cancer, the mechanistic target of rapamycin (mTOR) is often deregulated and has been validated as one of the therapeutic targets [8]. Many mTOR inhibitors have been developed and more mTOR inhibitors are being evaluated in clinical trials. Clinical data have demonstrated that mTOR inhibitors, in combination with standard chemotherapy or immunotherapy, serve as effective targeted cancer therapeutics for gastric cancer [9]. Of relevance to the studies here, activation of the AKT/mTOR pathway plays a role in the regulation of PD-L1 [10], the pathogenesis of H. pylori [11] and the regulation of metaplasia [12]. In esophageal adenocarcinoma, there is an established a crosstalk between the mTOR/S6K1 and the Hh pathway, via smoothened-independent Gli1 activation, and provides a rationale for combination therapy [13]. Thus, here we hypothesize that mTOR may regulate PD-L1 via a non-canonical, smoothened-independent Hh pathway.

The application of checkpoint blockade immunotherapy has shown promise in several solid tumors, including gastric adenocarcinomas [[14], [15], [16]]. Although antibodies that inhibit programmed cell death 1 (PD-1) and programmed death-ligand 1 (PD-L1) have emerged as promising immunotherapy for patients with gastric cancer, only a subset of patients have benefitted from them and most of them who initially responded will eventually develop acquired resistance [[17], [18], [19], [20]]. There are currently no established selection criteria to predict whether a patient will benefit from immunotherapy alone or with combination therapy. Multiple suppressive immune cell types including macrophages, myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) accumulate within the tumor microenvironment (TME) of different malignancies. Of relevance to the studies presented here, MDSCs are known to block CD8+ cytotoxic T cell anti-tumor activity through l-arginine and l-cysteine sequestration as well as production of reactive oxygen species (ROS) [[21], [22], [23]]. Increased MDSC infiltration in the gastric cancer correlates with more advanced gastric cancer and reduced survival [24,25]. Thus, the depletion of MDSCs to impair tumor growth may be proposed as an anti-cancer therapeutic strategy [26]. However, methods to deplete MDSCs in gastric cancer are currently largely unknown.

One of the greatest advancements in cell culture technologies over the past decade is the development of three-dimensional organoids as in vitro models for drug screening and cancer therapeutics [[27], [28], [29], [30]]. Patient-derived organoids (PDOs) have been described for various tissues, but there are at present no gastric cancer PDOs that can be exploited as a high-fidelity preclinical model to predict the efficacy of immune checkpoint inhibition or elucidate the related molecular mechanisms of cancer immunology in vitro. In this study, we report the generation of an autologous cancer PDO/immune cell co-culture system that may be used to investigate PD-L1/PD-1 blockade together with the inhibition of immunosuppressive cells MDSCs for the development of therapeutic approaches for the treatment of gastric cancer.

Section snippets

Patient selection and study population

All patients underwent total or subtotal gastrectomy at the National University Hospital of Singapore. Patients were eligible for study enrollment if they had a histologically or cytologically confirmed diagnosis of primary gastric cancer, were 21 years or older, and had never undergone prior chemotherapy or radiotherapy. Patients were ineligible if they had symptomatic or progressive central nervous system metastases, or other uncontrolled medical disorders. Study protocols were reviewed and

PD-L1 and PMN-MDSCs are expressed within the gastric cancer TME and inhibit CTL proliferation and effector function

NanoString Digital Spatial Profiling (DSP) of immune-related protein markers using FFPE slide-mounted tissues from gastric cancer patients and normal stomachs was performed. We selected 6–12 regions of interest (ROIs) in the tumor microenvironment (Fig. 1B) and in normal stomach (Fig. 1A). Expressions of immunosuppressive cell markers were significantly higher in FFPE gastric cancer patient tissues (Fig. 1C; *p < 0.001), when compared to normal stomach controls. We also observed a significant

Discussion

Our analysis of the patient-derived gastric cancer organoid/immune cell co-cultures revealed the differentiation and polarization of the patient's MDSCs to an immune cell population expressing CD33+CD11b+CD15+CD14. In humans two distinct subset of MDSCs exist, monocytic-MDSCs (M-MDSCs) and polymorphonuclear (or granulocytic)-MDSCs (PMN-MDSCs) [22,23]. Infiltration of MDSCs within the gastric epithelium occurs early in response to a major risk factor of gastric cancer Helicobacter pylori (H.

Acknowledgements

This research is supported by: Singapore Ministry of Health National Medical Research Council under its Open Fund-Large Collaborative Grant (OF-LCG; MOH-OFLCG18May-0003) awarded to the Singapore Gastric Cancer Consortium; NIH (NIDDK) 2 R01 DK083402-06A1 grant, NIH 1U19AI116491-01 grant and Yeoh Ghim Seng Visiting Professorship in Surgery research fund from the National University of Singapore awarded to YZ; National Cancer Institute Singapore Center Grant Programme awarded to WPY. This project

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    These authors contributed equally.

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