Probing neutrophils heterogeneity in Glioblastoma: how the metabolism shapes the function of immunosuppressive CD71+Neutrophils

Glioblastoma (GBM) is the most aggressive and lethal form of brain cancer and do not benefit from immunotherapies. These disappointing results may be explained by the strong immunosuppressive tumor microenvironment (TME) that characterize this form of cancer. Neutrophils (Neu) have emerged as key contributors to the TME in different types of cancers, including brain cancers, where their presence is associated with a poor prognosis and a worse response to therapies. Neu represent a heterogeneous population and different subsets of Neu can co-exist in the same cancer patient. However, their characterization remains mainly descriptive in the context of GBM. Moreover, targeting immunosuppressive Neu has proven to be extremely difficult, mostly due to the incomplete understanding of the mechanisms by which Neu can shape the immunosuppressive TME. Histone lactylation (Kla) is a recently described post-translational modification driven by lactate production and able to control the expression of homeostatic and regulative genes during macrophages differentiation. However, the role of Kla in Neu has never been investigated With this project, we proved that Neu acquire immunosuppressive features within the GBM TME in both human patients and mice models. Single cell RNA sequencing analysis on blood versus tumor derived Neu isolated from GBM-bearing mice, revealed the expansion of a glycolytic subpopulation of Neu within the tumor, characterized by the expression of the transferrin receptor CD71 and by a strong hypoxic signature. Based on the expression of CD71, we identified two different populations of Neu: CD71-Neu and CD71+Neu. CD71+Neu, but not CD71-Neu, suppressed T cells responses in mouse and human GBM tumors. Hypoxia induced suppressive activity in CD71+Neu via glucose- and lactate-driven histone Kla, which promoted the expression of the potent immunosuppressive enzyme Arginase-1 (ARG-1). CD71+Neu were massively mobilized from the bone marrow during tumor progression and quickly infiltrated GBM tumors, where they acquired a proliferative potential and resistance to ferroptosis, thus being able to persist for long periods within the GBM TME. Finally, targeting Kla in vivo through IFNα- or Isosafrole-mediated inhibition of lactate dehydrogenase A (LDHA), resulted in a dramatic improvement of the survival of GBM-bearing mice. For the first time, these findings bring evidence on Neu heterogeneity in the context of GBM, showing insights on the mechanism that governs the immunosuppressive functions of pro-tumoral Neu. The detrimental role of lactate and Kla in controlling these functions opens a new era for the treatment of GBM patients, offering a novel target to improve the survival and the response to immunotherapies in this cohort of patients.