Single-Cell RNA-Seq Analysis of Patient Myeloid-Derived Suppressor Cells and the Response to Inhibition of Bruton's Tyrosine Kinase

Abstract

Myeloid-derived suppressor cell (MDSC) levels are elevated in patients with cancer and contribute to reduced efficacy of immune checkpoint therapy. MDSC express Bruton's tyrosine kinase (BTK) and BTK inhibition with ibrutinib, an FDA-approved irreversible inhibitor of BTK, leads to reduced MDSC expansion/function in mice and significantly improves the antitumor activity of anti-PD-1 antibody treatments. Single-cell RNA sequencing (scRNA-seq) was used to characterize the effect of ibrutinib on gene expression of fluorescence-activated cell sorting-enriched MDSC from patients with different cancer types [breast, melanoma, head and neck squamous cell cancer (HNSCC)]. Melanoma patient MDSC were treated in vitro for 4 hours with 5 μmol/L ibrutinib or DMSO, processed for scRNA-seq using the Chromium 10× Genomics platform, and analyzed via the Seurat v4 standard integrative workflow. Baseline gene expression of MDSC from patients with breast, melanoma, and HNSCC cancer revealed similarities among the top expressed genes. In vitro ibrutinib treatment of MDSC from patients with melanoma resulted in significant changes in gene expression. GBP1, IL-1β, and CXCL8 were among the top downregulated genes whereas RGS2 and ABHD5 were among the top upregulated genes (P < 0.001). Double positive CD14+CD15+ MDSC and PMN-MDSC responded similarly to BTK inhibition and exhibited more pronounced gene changes compared with early MDSC and M-MDSC. Pathway analysis revealed significantly downregulated pathways including TREM1, nitric oxide signaling, and IL-6 signaling (P < 0.004).

Implications: scRNA-seq revealed characteristic gene expression patterns for MDSC from different patients with cancer and BTK inhibition led to the downregulation of multiple genes and pathways important to MDSC function and migration.

Figure 1.. Cell groupings identified by single cell RNA-seq analysis of cancer PBMC enriched for MDSC.

(A) Schematic representation of the study workflow. PBMC were isolated from the blood of melanoma (n=3), head & neck (n=1), and breast (n=1) cancer patients. MDSC (CD11b⁺, CD33⁺, HLA-DR^lo/-) were isolated using fluorescence-activated cell sorting (FACS) and transcriptome analysis was performed. (B) UMAP plot of all samples. (C) Heatmap of canonical gene markers used to verify identity of cell clusters (pangloDB).

Figure 2.. Baseline gene expression of MDSC from different cancer types.

(A) UMAP plots of genes expressed by MDSC relative to other cell populations in different cancers (breast, head and neck, melanoma) n=1 each cancer. (B) Volcano plot of differentially expressed (DE) genes in breast MDSC relative to head & neck MDSC. Top 10 upregulated genes in MDSC cluster in each cancer type are listed in the table.

Figure 3.. In vitro ibrutinib treatment of MDSC from melanoma patients results in significant changes in gene expression.

(A) UMAP plots of melanoma MDSC (n=2) treated with control (DMSO) or ibrutinib 5 μM for 4 hours. (B) Volcano plot of DE genes in melanoma MDSC after treatment with ibrutinib. (C) Table listing upregulated and downregulated genes in MDSC after ibrutinib treatment (0.5 absolute log₂-fold change cutoff).

Figure 4.. Pathway analysis of gene expression changes in MDSC following BTK inhibition.

Significantly DE genes (p<0.01) in the melanoma MDSC cluster after ibrutinib treatment (n=2) were analyzed via Ingenuity Pathway Analysis (IPA). (A) Bar chart displaying regulated pathways after ibrutinib treatment. Colors indicate directionality of affected pathway with positive z scores displayed as blue/upregulated and negative z scores displayed as red/downregulated. (B) Log expression ratio of individual genes in each pathway after ibrutinib treatment. (C) Log expression ratio of genes involved in cellular movement and activation after ibrutinib treatment.

Figure 5.. MDSC cell cluster interaction with CD8⁺ T cells, CD4⁺ T cells, and NK cells.

(A) Circle plot displaying MDSC cell cluster communication with others cell clusters (CD8⁺ T cells, CD4⁺ T cells, NK cells, dendritic cells) from DMSO and ibrutinib treated MDSC. The weight of the arrows indicates a greater number of ligands-receptor pairs. (B) Receptor-ligand information flow enrichment of MDSC interaction with each cluster in DMSO treatment and ibrutinib treatment p<0.01. The probability of communication is displayed as minimal communication (blue) and maximum communication (red).

Figure 6.. MDSC subset gene expression mirrors total MDSC gene changes after in vitro ibrutinib treatment.

(A) Distribution of MDSC subsets in melanoma MDSC. M-MDSC (CD14⁺CD15⁻, 59.9%), PMN-MDSC (CD14⁻CD15⁺, 2.8%), double positives (CD14⁺CD15⁺, 7.9%), and early-MDSC (CD14⁻CD15⁻, 29.4%). (B) Volcano plot of DE genes in melanoma MDSC subsets after treatment with ibrutinib. (C) Heatmap of average log₂-fold change of select genes after ibrutinib treatment in MDSC subsets.

Figure 7.. PCR and protein analysis of select genes mirrors single cell analysis gene changes in MDSC after BTK inhibition.

PBMC were isolated from melanoma patients (n=3–4) and purified for MDSC using FACS. MDSC were treated for 6h with 1 μM ibrutinib and RNA was isolated. Gene expression of selected genes was quantified using PCR with β-actin as housekeeping gene. (A) CXCL10 (*p<0.01, n=3), (B) IL-1β (p=0.08, n=4), (C) GBP1 (*p<0.05, n=3), and (D) CXCL8 (*p<0.05, n=3). (E) MDSC were treated for 24h with DMSO or ibrutinib (1–5 μM), and CXCL8 was measured in the supernatant via ELISA. (F-G) MDSC treated as in E and expression of (F) ICAM (*p<0.05 n=3), and (G) ALCAM (*p<0.05 n=3) was measured by flow cytometry. Median fluorescence intensity (MFI) for ICAM and ALCAM was calculated and graphed. Representative histograms of the expression of ICAM and ALCAM by MDSC after treatment. Bar graphs quantifying MFI. (Statistics: Paired students t tests for all comparisons)