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Single-cell RNA sequencing on peripheral blood PBMCs from 4T1 mouse model treated with STING agonist deliveredy by PEG biopsy marker does not reveal peripheral T cell clonal expansion.

ABSTRACT: To explore whether immune escape could be secondary to a failure of STING agonist to induce T-cell clonality and expansion, we performed single-cell RNA sequencing (scRNAseq) and T-cell receptor sequencing (TCR-seq) on peripheral PBMCs from animals treated with PEG markers with PBS or 100 µg ADU-S100, using the low tumor burden model of 4T1. Cell types were clustered based on gene signature analysis with 9 resultant clusters. We then analyzed the frequency of unique clonotypes that were expanded following PBS versus ADU-S100 treatment. In both groups, most of the cells that had a unique clonotype did not expand, showing a frequency of 1. There were no specific clonotypes that had a frequency over 5, indicating that ADU-S100 treatment of 4T1 mice does not result in peripheral T cell clonal expansion.

ORGANISM(S): Mus musculus

PROVIDER: GSE294846 | GEO | 2025/04/21

REPOSITORIES: GEO

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Project description:Purpose: Next-generation sequencing (NGS)-derived transcriptome profiling (RNA-seq) is a powerful tool for analysing cellular pathways involved in treatment responses. In this study, RNA-seq was used to perform differential gene expression (DEG) and gene ontology (GO) analyses in prostate tumors treated with different immunotherapies and their combination. This was in order to investigate which pathways are activated and mediate the therapeutic effect. Methods: Mice with TRAMP-C2 subcutaneous prostate tumors were treated with either HBSS (control) or cyto-IL-15 or the STING agonist ADU-S100 or combination of ADU-S100 and cyto-IL-15. At day 6, tumors were removed and RNA was harvested to be used for RNA sequencing with Illumina NovaSeq. Using DESeq2, a comparison of gene expression between the treatment cohorts was performed and the Wald test was used to generate p-values and Log2 fold changes. GO analysis was performed on the statistically significant genes by implementing GeneSCF software using Fisher exact test. The mgi GO list was used to cluster the set of genes based on their biological process. Results: DEG analysis shown that more than 1,000 genes were differentially expressed in tumors 6 days after treatment with ADU-S100 or its combination with cyto-IL-15 (1,040 genes for ADU-S100 compared to control; 1,141 genes for combination compared to control). Most of the differentially expressed genes were upregulated (>70% in both comparisons). cyto-IL-15 led to differential expression of 96 genes compared to control. Differential expressed genes included Cxcl11 involved in immune cell recruitment and activation, and genes encoding granzymes a and b, which are proteases released by cytotoxic T cells and natural killer (NK) cells to kill cancer cells. In the control versus ADU-S100 or combination comparisons, GO analysis revealed that the majority of genes were involved in processes such as cellular responses to interferon-γ and β, complement activation, innate immune response, phagocytosis, and B cell signalling and activation. Conclusions: This study provides valuable insights in differential expression and activation of genes and cellular pathways involved in eliciting responses to immunotherapies in mouse prostate tumors. Using NSG and more specifically RNAseq-based transcriptome characterization, a detailed quantitative and qualitative analysis on how novel therapies exert their efficacy in tumors can be achieved and uncover the specific biological processes and functions contributing to the anti-tumor effects.

Project description:Background: PD-1 pathway blockade has revolutionized cancer care, but many patients do not durably benefit from these therapies. Novel treatments to engage anti-tumor immunity in this setting are needed. The stimulator of interferon genes (STING) protein, an innate sensor of cytoplasmic DNA, is a promising target with several agonists in development; however, recent clinical trials of these agents have not demonstrated efficacy for most patients. Here, we present detailed biomarker analyses of a patient with anti-PD-L1 refractory Merkel cell carcinoma that had a durable (~1 year), abscopal response to ADU-S100 (intralesional STING agonist) combined with anti-PD-1 blockade. Methods: Tumor biopsies and peripheral blood acquired pre- and post-treatment were analyzed with single cell RNA sequencing, 27-color flow cytometry, T cell receptor sequencing and multiplexed immunohistochemistry (mIHC; post treatment tumor specimen only). Critically, cancer-specific CD8 T cells were identified using MHC-I tetramers containing peptides from the Merkel cell polyomavirus (MCPyV), the etiologic agent of this cancer. Cell lines and tumor specimens from 76 patients were used to evaluate STING expression and signaling in MCC. Results: High levels of antigen cancer-specific T cells were observed in the tumor prior to ADU-S100 treatment (12% of T cells recognized an MCPyV epitope). These cancer-specific CD8 T cells exhibited characteristics of exhaustion including high TOX and low TCF1. Following STING agonist treatment, MCPyV-specific CD8 T cells expanded 3-fold with minimal phenotypic changes. An increase in antigen presentation on MCC tumor cells was also observed (1.8% of tumor cells MHC-I positive before treatment, 8.2% after treatment). This was not a result of ADU-S100 acting directly on tumor cells as mIHC analysis of 88 tumor samples confirmed little or no STING expression in tumor cells, but high STING expression on infiltrating immune and stromal cells. Further in vitro studies showed MCPyV-positive MCC cell lines are STING deficient, and ADU-S100 did not induce interferon. Conclusions: Our study suggests that STING agonists act on intratumoral immune cells to release interferons, which upregulate tumor cell antigen presentation and reinvigorate the anti-tumor response from pre-existing cancer-specific T cells. Thus, patients with anti-PD-(L)1-refractory disease showing downregulation of antigen-presentation may benefit from intralesional STING agonists.

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Single-cell RNA sequencing on peripheral blood PBMCs from 4T1 mouse model treated with STING agonist deliveredy by PEG biopsy marker does not reveal peripheral T cell clonal expansion.

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