Transcriptional profiling of tumor infiltrating neutrophils in a mouse model (MOC1) of oral cavity cancer

Neutrophils are abundant in the tumor microenvironment and frequently acquire immunosuppressive phenotypes, yet the mechanisms driving this transition remain incompletely defined. To investigate the role of the transcription factor Egr1 and MEK signaling in neutrophil-mediated immune suppression, we performed single-cell RNA sequencing (scRNA-seq) of tumor-infiltrating neutrophils isolated from a syngeneic oral cavity cancer model (MOC1) in mice. This dataset comprises four experimental groups: Egr1 wild-type (WT) vs. Egr1 knockout (KO) and vehicle control vs. trametinib (MEK inhibitor) treatment. Comparative transcriptional profiling revealed that Egr1-deficient neutrophils fail to upregulate key immunosuppressive and pro-inflammatory effectors, including Osm, Ptgs2, and Il1b, in response to the tumor microenvironment. Conversely, trametinib-treated neutrophils displayed reduced expression of hypoxia- and MEK-driven transcriptional programs, including suppression of Osm, Ptgs2, Il1b, and the AP-1 transcription factor Junb. Overall design: Tumors were harvested and digested into single cell suspensions using the Miltenyi Tumor Dissociation Kit per manufacturer recommendations. Total leukocytes were isolated using 80/40% Percoll centrifugation separation. Neutrophils were then isolated via positive magnetic isolation using Miltenyi anti-Ly6G+ microbeads per manufacturer recommendations. Neutrophils were isolated from three tumor-bearing mice and combined together for one single cell capture for each experimental condition: Egr1 wild-type vs. Egr1 knockout, and vehicle control vs. trametinib treatment. For each group, single-cell suspensions were processed using the 10x Chromium Next GEM Single Cell 3' v3.1 chemistry to generate scRNA-seq libraries. In total, four libraries were prepared and subsequently pooled for sequencing. Libraries were sequenced on an Illumina NextSeq 2000 P1 flow cell with a non-symmetric run configuration (28 cycles + 90 cycles). Each sample yielded more than 9 million reads, with sequencing quality exceeding Q30 in over 93.9% of barcode bases, 92.2% of read bases, and 95.6% of UMI bases. Demultiplexing was performed allowing one mismatch in barcodes. Alignment and gene quantification were carried out with Cell Ranger v8.0.1 using default parameters. Downstream analysis focused on transcriptional changes in neutrophils associated with Egr1 deficiency and pharmacologic MEK inhibition, enabling interrogation of hypoxia- and Egr1-dependent neutrophil states within the tumor microenvironment.