DENDRITIC CELL SUBSETS IN THE PATHOGENESIS OF HIGH GRADE GLIOMAS

Abstract Background and aims. High grade gliomas (HGGs) are aggressive brain tumours characterized by a poor prognosis and the ability to promote an immunosuppressive tumour microenvironment that impairs anti-tumor immune responses. Therefore, there is increasing interest in developing new immunotherapeutic approaches, aimed at boosting anti-tumor immune responses in HGG patients. Because HGG has shown the highest susceptibility to dendritic cell (DC) vaccines amongst other human cancers, DC-based immunotherapeutic strategies may be particularly promising in these patients. DCs are antigen presenting cells that have the unique ability to initiate antitumor immune responses, making these cells crucial in cancer immunosurveillance. They are a rare population composed of different subsets that differ each other in origin, immunophenotype and function. The differential role of different DC subsets in HGG, and in particular the subsets specifically recruited into the tumour site and the impact of HGG on the activatory/tolerogenic properties of DCs have been poorly investigated, so far. For these reasons, in this study we performed a deep characterization of circulating and tumour-infiltrating DC subsets, and investigated possible correlations between DC parameters and histopathological and molecular HGG features, patient outcome and response to treatment. To this aim, we used multiparameter flow-cytometry and single-cell RNA sequencing (scRNAseq), which allow complex analyses on high-dimensional data. Materials and methods. In this cross-sectional study we enrolled HGG patients undergoing surgery at their first diagnosis, and we applied an 18-colour flow-cytometry panel that allows the identification of DC-lineage DCs (pDCs, cDC1s, cDC2s) and inflammatory DCs (slanDCs, moDCs), and the characterisation of their activatory/inhibitory state. This panel was applied to DC characterization in the peripheral blood (n=23) and the tumour lesion (n=10) of HGG patients. Twelve whole blood samples obtained from healthy donors (HDs) and 3 healthy brain tissue samples were included as controls. scRNAseq experiments were performed on 7 tumoral samples and 2 healthy brain tissues obtained from HGG patients, by using 10x Genomics technology. Ingenuity Pathway Analysis (IPA) software was used to investigate the pathways and functions differentially activated or inhibited in infiltrating DCs. We also performed a longitudinal study on a second cohort of patients, diagnosed with recurrent HGG and enrolled in different immunotherapeutic early clinical trials (ieCTs), mainly containing immune checkpoint inhibitors (n=17). In these patients, we assessed the count and phenotype of circulating DC subsets before and at different time points after immunotherapy, by using the same 18-colour flow-cytometry panel described above. Multivariate analyses were used to correlate DC parameters with the patient outcome. Results. In the cross-sectional study, we observed by flow-cytometry that the frequency of circulating pDCs, cDC1s, cDC2s and slanDCs was significantly lower in HGG patients than HDs. DC reduction was evident only in patients affected by the most severe form of HGGs (IDHwt IV grade gliomas). The analysis of tissue DCs revealed that DC subsets were absent in healthy brain parenchyma, whereas they infiltrated HGG tumour tissues. In particular all subsets of myeloid DCs (including cDC1s, cDC2s, slanDCs, and moDCs) were observed in the tumours, whereas pDCs were observed only in a few patients. Tumour-infiltrating DCs were markedly reduced in corticosteroid-receiving patients. By performing scRNAseq, we confirmed that DCs were mostly absent in healthy brain parenchyma whereas they were present in tumour samples and could be sub-divided in 2 sub-clusters. By IPA analysis, we observed a functional dichotomy between these clusters, with the largest one being characterised by an impaired/dormancy state, as assessed by the down-regulation of pathways and functions related to pro-inflammatory responses, cell motility and cell interactions, compared with the smallest cluster characterised, on the contrary, by a more active profile. In the longitudinal study performed on relapsed HGG patients enrolled in ieCTs, we observed that patients with a positive clinical response to immunotherapeutic agents, as assessed by an increased overall survival, showed an increase in the number of circulating cDCs. Conclusions. This study demonstrated that different subsets of DCs infiltrate human HGGs, but are mainly characterized by a transcriptomic profile suggestive of a functional impairment. These results provide novel insights into the comprehension of the molecular mechanisms of DC impairment in HGG microenvironment, and pave the way for the development of novel strategies aimed at restoring the ability of DCs to activate cytotoxic anti-tumour immune cells. Our observation in the longitudinal study that an increase of cDCs correlated with a better clinical response to immunotherapy seems to support the relevant role played by DCs in the control of HGG growth. On the other hand, our study also demonstrated that corticosteroid treatment, commonly used in HGG patients for the management of cerebral oedema, reduces the number of tumour-infiltrating DCs. Based on the above considerations, this finding may suggest a negative impact of corticosteroid treatment on anti-tumour immune responses, thus supporting the use of alternative approaches to control this clinical complication. Altogether, our results support and encourage the study of DCs in HGG, in order to improve our knowledge on the role played by DCs within the immunosuppressive tumour microenvironment that characterizes this human cancer. To this aim, in the near future we plan to apply new bioinformatic tools to the analysis of single-cell data collected in HGG tumour environment that may be particularly useful for investigating the intricate interactions occurring between DCs and other HGG-infiltrating immune cells or malignant glioma cells.