Neutrophil-specific targeting of STAT3 impairs tumor progression via the expansion of cytotoxic CD8+ T cells

Neutrophils have emerged as key players in tumor progression, often associated with poor prognosis. Despite the ongoing efforts to target neutrophil functions in cancer, therapeutic success has been limited. In this study, we addressed the possibility of blocking STAT3 signaling in neutrophils as targeted therapeutic intervention in cancer. Conditional deletion of Stat3 in neutrophil-specific manner (Ly6GcreStat3fl/fl mice) significantly impaired tumor growth and metastasis in mice. Neutrophils isolated from these mice exhibited strong antitumoral phenotype, with increased MHCII, CD80/86 and ICAM-1 expression. Immune profiling of tumors and tumor-draining lymph nodes revealed a significant enrichment of CD8+ T cells (granzymeBhi, perforinhi and IFN-γhi) with strong cytotoxic activity. To further translate these findings to human settings, we blocked STAT3 signaling in patient neutrophils using a small molecule inhibitor LLL12 and assessed their effects on patient-derived cancer explants. In agreement with in vivo mouse data, we observed the expansion and activation of cytotoxic CD8+ T cells in such explants. To test the therapeutic applicability of STAT3 targeting, we utilized myeloid cell-selective STAT3 siRNA (CpG-STAT3ASO) to target neutrophils in vivo in tumor-bearing mice. Consistent with previous results, neutrophil-specific STAT3 knockdown impaired tumor growth and enhanced cytotoxic T cell activity in tumors and tumor-draining lymph nodes of treated mice. These findings highlight STAT3 signaling as a deleterious pathway supporting protumoral activity of neutrophils and suggest neutrophil-targeted STAT3 inhibition as a promising opportunity for cancer immunotherapy, providing novel insights in targeted therapeutic avenues. C57BL/6JCrl mice were used as wild type (WT) strain, and neutrophil-specific STAT3 knockout mouse strain (Ly6GcreSTAT3fl/fl, referred as NStat3-/-) were used for comparison. Both NStat3-/- and WT mice were subcutaneously engrafted with mouse oropharyngeal cancer cells (MOPC) by s.c. injection of 1x106 MOPC cells in 100 μL PBS into the right flank, and tumor growth had been observed for 21 days. At the day 21 mice were sacrificed and tissues were harvested under aseptic conditions. Tumors were harvested and stored in DMEMc medium on ice maximum 30 min before processing. Tumors were digested in an enzyme solution (DMEMc medium containing dispase 0.2 μg/mL, collagenase A 0.2 μg/mL, DNase I 100 μg/mL (Merck)), 1 mL per sample for 45 min at 500 rpm and 37°C. Cells were meshed through 100 μm sterile strainers (Cell Trics, Partec, Sysmex), centrifuged at 300g and 4°C for 5 min, supernatant was discarded. The pellet was resuspended in PBS (Dulbecco’s Phosphate Buffered Saline 1x, Gibco) containing Mouse BD Fc Block (BD Biosciences, Clone: 2.4G2, RRID:AB_394656) and incubated for 10 min at 20°C. The cells then stained for 30 min at 4°C with PE-anti-mouse Ly6G, Pacific Blue-anti-CD11b antibodies and APC/Cy7-viability dye for FACS of tumor-associated neutrophils (TANs). Cells were sorted using BD FACS Aria III Cell Sorter with the purity >95%.