Deciphering neutrophil heterogeneity to enhance therapeutic efficacy of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) in triple negative breast cancers

TRAIL induces apoptosis in many preclinical cancer models including breast cancer models and has been extensively studied as a potential cancer therapeutic target. However, the clinical efficacy of TRAIL agonists is limited, suggesting that there are unknown modulatory mechanisms responsible for the lack of activity in patients. Our prior work demonstrated that TRAIL induces various cytokines (CXCL1, CXCL2, CXCL3, CXCL8, CXCL11, IL6) via NFKB2-dependent pathway in triple negative breast cancers (TNBC), promoting the recruitment of immunosuppressive neutrophils, suggesting that neutrophil-mediated regulation of the tumor immune microenvironment modulates TRAIL activity in TNBC. Thus, this study aims 1) to characterize the neutrophil heterogeneity induced by TRAIL as well as derived factors from TRAIL treated TNBC, and 2) to identify targetable pathways that could reprogram neutrophils towards an anti-tumor phenotype, thus enhancing TRAIL efficacy. To address this, neutrophils isolated from a healthy donor were incubated with 4 different conditions: serum-free media (SFM), SFM supplemented with TRAIL (SFM-T), conditioned media from TNBC (CM), or conditioned media from TRAIL-treated TNBC (T-CM). Single cell RNA sequencing (scRNA-seq) of neutrophils (43,420 cells) across the 4 conditions indicated that there are 10 transcriptionally distinct clusters. These findings were further validated with scRNA-seq of pooled neutrophils from 5 different healthy individuals (35,500 cells). t -SNE analysis identified similar transcriptionally distinct states encompassing inflammation, antigen presentation, and ribosome biogenesis programs, demonstrating remarkable neutrophil heterogeneity. Based on literature and functional relevance, we focused on 2 key clusters that were increased upon treatment of the neutrophils with T-CM identified in both experiments. One is enriched for antigen-presentation genes and immunosuppressive markers such as CCL4 , CD274 , IL1A , and IL1B , and a second enriched for ribosome biogenesis-related genes including DDX21 , UTP18 , LAGE3 , and NPM1 . These clusters also exhibited low expression of IFN-gamma/alpha-responsive genes, further suggesting the immunosuppressive role of T-CM. In contrast, SFM-T treatment resulted in a population with markers regulating neutrophil-specific function (e.g. NETosis, degranulation). The Sc-RNA-seq findings are currently being confirmed by gene expression analysis and surface staining methods. Collectively, our study delineates a potential mechanistic role of supernatants from TRAIL treated TNBC cells and neutrophil plasticity. These findings imply that targeting the innate immune system may modulate the effects of TRAIL on TNBC tumors enhancing the therapeutic outcomes.