Glucocorticoid receptor regulates PD-L1 and MHC-I in pancreatic cancer cells to promote immune evasion and immunotherapy resistance

Despite unprecedented responses of some cancers to immune checkpoint blockade (ICB) therapies, the application of checkpoint inhibitors in pancreatic cancer has been unsuccessful. Glucocorticoids and glucocorticoid receptor (GR) signaling are long thought to suppress immunity by acting on immune cells. Here we demonstrate a previously undescribed tumor cell-intrinsic role for GR in activating PD-L1 expression and repressing the major histocompatibility complex class I (MHC-I) expression in pancreatic ductal adenocarcinoma (PDAC) cells through transcriptional regulation. In mouse models of PDAC, either tumor cell-specific depletion or pharmacologic inhibition of GR leads to PD-L1 downregulation and MHC-I upregulation in tumor cells, which in turn promotes the infiltration and activity of cytotoxic T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. In patients with PDAC, GR expression correlates with high PD-L1 expression, low MHC-I expression, and poor survival. Our results reveal GR signaling in cancer cells as a tumor-intrinsic mechanism of immunosuppression and suggest that therapeutic targeting of GR is a promising way to sensitize pancreatic cancer to immunotherapy.


Statistics
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Software and code
Policy information about availability of computer code Data collection Flow cytometry data were collected using the Invitrogen Attune NxT Acoustic Focusing Cytometer. Quantitative PCR data were obtained with the Bio-Rad CFX96 Real-Time System. Fluorescent images were obtained using the Vectra Polaris Automated Quantitative Pathology Imaging System. Immunoblotting images were obtained using the ChemiDoc Touch Imaging System (Bio-Rad) and Image Lab Touch software (Bio-Rad, version 2.3.0.07). IHC images were obtained using a fully automated digital pathology slide-system (KFBIO, KF-PRO-005).

Data analysis
Flow cytometry data were analysed using FlowJo (FlowJo, version 10.4). Immunofluorescent images were analyzed using ImageJ (version 1.52p) or Phenochart (version 1.0.12). Statistical analysis was performed using Graphpad Prism (GraphPad software, version 8). CyTOF data were analyzed using Cytobank (https://premium.cytobank.org/cytobank/login). The correlation of expression levels of two genes was analyzed using the R corrplot package and the cor function. Survival analysis was performed using the R survival package.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy GR (encoded by NR3C1) mRNA levels in paired normal pancreatic tissue and PDAC were obtained from the dataset GSE15471 in the Gene Expression Omnibus

March 2021
(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15471). TCGA gene expression data were obtained from The Cancer Genome Atlas data portal (https:// tcga-data.nci.nih.gov/tcga/dataAccessMatrix.htm). The source data that support the findings of this study are available with no restrictions. The uncropped blots are shown in Supplementary Fig. 9. Source data are provided with this paper.

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Sample size
No sample size calculation was done either for in vitro or in vivo studies. For in vivo and in vitro studies, sample sizes were determined based on our preliminary experiments. In our experience, n = 5-7 mice per group (in vivo) and n = 3-4 samples per group (in vitro) are sufficient to detect meaningful biological differences with good reproducibility.
Data exclusions No data or animals were excluded from analysis.

Replication
Except for the animal studies (one time), chemokine array analysis (one time), and tissue microarray analysis (one time), each experiment was repeated at least three times with similar results.
Randomization Mice were randomly assigned to different treatment groups.

Blinding
For cell-based experiments, Western blotting, flow cytometry, and in vitro assays, blinding was not performed, because the investigator had to know the groups to load the samples or perform the assay. Blinding was not performed in mouse experiments. The investigator needed to know the treatment groups in order to perform the study.

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Validation
All antibodies used are commercially available and validated by the manufacturers. Pre-validated antibodies were purchased from reputable sources. All proteins are well studied and all antibodies are widely used in the literature. The experiments included appropriate controls. We validated the GR-specific antibody by using two independent GR shRNAs.