Deterministic reprogramming of neutrophils within tumors

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies. Editor’s summary Neutrophils are the most abundant white blood cell population in the body. Although different types of neutrophils frequently gather in the tumor microenvironment, it is currently unclear how they might coordinate to support tumor growth. Using an experimental model of pancreatic cancer, Ng et al. report populations of tumor-infiltrating neutrophils that converged to develop into a single long-lived “T3” cell subset. T3 neutrophils prompted the growth of new blood vessels, which enhanced tumor survival in areas with low oxygen and limited nutrients. Depleting T3 neutrophils or inhibiting their angiogenic function reduced tumor growth. The researchers suggest that neutrophils could be reprogrammed within the tumor microenvironment into a single functional state, and modulation of protumoral neutrophil responses may have therapeutic potential. —Priscilla N. Kelly Reprogramming of tumor-infiltrating neutrophils drives their convergent differentiation within the tumor microenvironment. INTRODUCTION Neutrophils are the first responders to infection and injury and are rapidly recruited to affected tissues in large numbers to enact their protective function. As such, neutrophils were historically perceived as a homogeneous and transient population. Recently, however, a diverse array of neutrophil states has been reported in cancer, varying in their maturation, surface marker expression, and transcript profiles. The relationship between these neutrophil states and their organization into a unified protumoral response have yet to be elucidated, limiting the therapeutic targeting of neutrophils in cancer. RATIONALE To identify the mechanisms by which disparate neutrophil states are coordinated into a concerted protumoral response, we used single-cell RNA sequencing and ATACseq (assay for transposable chromatin sequencing) on neutrophils from various organs and tumors in a murine orthotopic model of pancreatic cancer. Tumor neutrophil states identified from these analyses were validated by multiparametric flow cytometry, and spatial mapping at the RNA and protein levels were performed to reveal their localization within the pancreatic tumors. In vitro and in vivo approaches were then used to examine how the tumor environment shapes neutrophil phenotype, lifespan, and protumoral functions. RESULTS We identified three distinct neutrophil states within the tumor microenvironment, T1, T2, and T3, which were epigenetically and transcriptionally distinct from neutrophils in the bone marrow, spleen, and blood. By assessing nuclear morphology and maturation status, we determined that immature and mature neutrophils infiltrating the tumor differentiated into transitional T1 and T2 populations, respectively. T1 and T2 neutrophils underwent further reprogramming to converge into the T3 neutrophil state, which was terminally differentiated and expressed the surface marker dcTRAIL-R1. dcTRAIL-R1 up-regulation in tumor-naïve neutrophils could be induced by exposure to tumor-conditioned medium in vitro or entry into the tumor in vivo, and was accompanied by the expression of T3-specific genes. More importantly, this phenomenon was independent of their initial maturation phenotype. These findings thus underscore the capability of neutrophils to adopt a new functional phenotype, overlaying it onto their existing differentiation stage. The T3 phenotype was strongly correlated with a prolonged lifespan, with dcTRAIL-R1+ neutrophils persisting for more than 5 days within the tumor. Furthermore, T3 neutrophils were mainly localized to a unique hypoxic-glycolytic niche within the tumor, where they optimally exerted their pro-angiogenic function. This indicated that neutrophil reprogramming plays a critical role in enabling their survival under hypoxia, oxidative stress, and metabolic perturbations within the tumor microenvironment. Specifically, T3 neutrophils expressed high levels of vascular endothelial growth factor alpha (VEGFα) and substantially enhanced blood vessel formation within the tumor core, and only coinjection of T3 neutrophils with tumor cells accelerated tumor growth. Therefore, the ablation of either T3 neutrophils or VEGFα inhibits this growth enhancement. Finally, all three tumor neutrophil states were observed across mouse models and in multiple human cancers, with the T3 signature predicting poorer patient outcomes in two independent human pancreatic cancer cohorts and other solid tumor types. CONCLUSION By examining neutrophils in the context of their ontogeny, we uncovered their intrinsic flexibility in adapting to environmental signals regardless of their initial maturation stage. This implies that neutrophils infiltrating a tissue niche follow a common path, merging their different functional states into a single terminal phenotype as guided by the tissue. Within the tumor, this deterministic program likely ensures a continual supply of pro-angiogenic T3 neutrophils that fuel tumor growth. Our findings thus demonstrate how short-lived effector cells such as neutrophils effectively tailor their functions to accommodate tissue requirements, highlighting the untapped possibilities of targeting the local neutrophil response as immunotherapy. Tumor-infiltrating neutrophils undergo convergent reprogramming into pro-angiogenic neutrophils that support tumor growth. In cancer, both immature and mature neutrophils infiltrate the tumor. After entering the tumor microenvironment, these neutrophils undergo differentiation, leading to the formation of transitional populations. Through reprogramming, these populations ultimately converge into a terminal neutrophil state. Reprogrammed neutrophils strongly express VEGFα and localize to a unique hypoxic-glycolytic niche near the tumor core. This places them in an optimal position to exert their pro-angiogenic function within hypoxic and nutrient-poor tumor regions, thereby promoting tumor growth. The emergence of tumor reprogramming reflects the adaptability of neutrophils to environmental cues, allowing them to consolidate their protumoral responses.