Immunophenotypic and transcriptomic profiling of dendritic cell subsets in high-grade glioma patients

High grade gliomas (HGGs) are aggressive brain tumors with poor prognosis and high propensity to foster an immunosuppressive microenvironment. Among several immunotherapeutic approaches aimed at boosting antitumor immune responses, strategies based on the administration of dendritic cells (DCs) represent a promise in the treatment of HGG, which is the human cancer characterized by the highest susceptibility to DC-based vaccines. However, the mechanisms underlying the efficacy of DC vaccines, as well as the effects of HGG microenvironment on DCs, and the subsets of DCs specifically recruited to the tumor site are poorly understood. Yet, a detailed characterization of tumor-infiltrating DCs is now possible based on the recent molecular definition of distinct DC subsets characterized by transcriptional and functional specialization, together with the increasing availability of high-dimensional technologies like multicolor flow cytometry and single-cell RNA-sequencing (scRNAseq) that enable complex multiparameter analysis. In this study, we enrolled 26 patients affected by HGGs (III and IV grade), followed at the unit of Oncologic Neurosurgery at Humanitas Cancer Center, and 12 sex- and age-matched healthy donors (HDs). Flow-cytometric characterization of circulating and infiltrating DC subsets was performed by applying an 18-color panel recently optimized in our lab that allows the identification of distinct DC subsets and their activatory/inhibitory phenotype. scRNAseq was performed on tumor and healthy brain tissue samples obtained from HGG patients, by using 10x Genomics technology. Ingenuity Pathway Analysis (IPA) software was used to investigate the pathways differentially activated or inhibited in DCs. Our results indicated that the frequency of circulating pDCs, cDC1s and cDC2s was significantly lower in HGG patients than healthy donors. The reduction of all circulating DC subsets was evident only in patients affected by IV IDH-WT gliomas that are the most severe HGGs. Patients undergoing steroid treatment (dexamethasone) to reduce cerebral oedema showed a significant reduction in circulating DCs, independent from glioma grade. The analysis of tissue DCs revealed that DCs did not infiltrate healthy brain parenchyma, but were abundant in HGG tumor tissue. Notably, tumor-infiltrating DCs were markedly reduced in steroid-receiving patients. By performing scRNAseq, we confirmed the presence of DCs in tumor samples, whereas DCs were mostly absent in healthy brain parenchyma. By IPA analysis, we observed that the largest population of HGG-infiltrating DCs was characterized by a transcriptomic profile suggestive of severe functional impairment that was more severe in patients undergoing perioperative steroid treatment. In conclusion, in this study we observed that all DC-lineage DC subsets infiltrate HGG but their function is subverted by the tumor microenvironment. We also observed a negative impact of steroids on tumor-infiltrating DCs that may contribute to the clinical HGG progression observed in steroid-treated patients.