Background Systemic and local immune suppression plays a significant role in glioma progression. Glioma microenvironment contains both brain-resident microglial cells (MG) and bone marrow-derived macrophages (BMDM), but the study of their functional and immune regulatory activity has been hampered until now by the lack of markers allowing a proper identification and isolation to collect pure populations. Methods Myeloid and lymphoid infiltrate were characterized in grade II, III and IV gliomas by multicolor flow cytometry, along with the composition of the cell subsets of circulating myeloid cells. Macrophages were sorted and tested for their immunosuppressive ability. Moreover, following preoperative administration of 5-aminolevulinic acid to patients, distinct areas of tumor lesion were surgically removed and analyzed, based on protoporphyrin IX fluorescence emission. Results The immune microenvironment of grade II to grade IV gliomas contains a large proportion of myeloid cells and a small proportion of lymphocytes expressing markers of dysfunctional activity. BMDM and resident MG cells were characterized through a combination of markers, thus permitting their geographical identification in the lesions, their sorting and subsequent analysis of the functional characteristics. The infiltration by BMDM reached the highest percentages in grade IV gliomas, and it increased from the periphery to the center of the lesion, where it exerted a strong immunosuppression that was, instead, absent in the marginal area. By contrast, MG showed little or no suppression. Functional differences, such as iron metabolism and phagocytosis, characterized resident versus blood-derived macrophages. Significant alterations in circulating monocytes were present in grade IV patients, correlating with accumulation of tumor macrophages. Conclusions Grade IV gliomas have an alteration in both circulating and tumor-associated myeloid cells and, differently from grade II and III gliomas, show a significant presence of blood-derived, immune suppressive macrophages. BMDM and MG have different functional properties.

Background: Myeloid derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are two of the major players involved in the inhibition of anti-tumor immune response in cancer patients, leading to poor prognosis. Selective targeting of myeloid cells has therefore become an attractive therapeutic strategy to relieve immunosuppression and, in this frame, we previously demonstrated that lipid nanocapsules (LNCs) loaded with lauroyl-modified gemcitabine efficiently target monocytic MDSCs in melanoma patients. In this study, we investigated the impact of the physico-chemical characteristics of LNCs, namely size and surface potential, towards immunosuppressive cell targeting. We exploited myeloid cells isolated from glioblastoma patients, which play a relevant role in the immunosuppression, to demonstrate that tailored nanosystems can target not only tumor cells but also tumor-promoting cells, thus constituting an efficient system that could be used to inhibit their function. Results: The incorporation of different LNC formulations with a size of 100 nm, carrying overall positive, neutral or negative charge, was evaluated on leukocytes and tumor-infiltrating cells freshly isolated from glioblastoma patients. We observed that the maximum LNC uptake was obtained in monocytes with neutral 100 nm LNCs, while positively charged 100 nm LNCs were more effective on macrophages and tumor cells, maintaining at low level the incorporation by T cells. The mechanism of uptake was elucidated, demonstrating that LNCs are incorporated mainly by caveolae-mediated endocytosis. Conclusions: We demonstrated that LNCs can be directed towards immunosuppressive cells by simply modulating their size and charge thus providing a novel approach to exploit nanosystems for anticancer treatment in the frame of immunotherapy.

The immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) is mainly driven by tumor‐associated macrophages (TAMs). We explored whether their sustained iron metabolism and immunosuppressive activity were correlated, and whether blocking the central enzyme of the heme catabolism pathway, heme oxygenase‐1 (HO‐1), could reverse their tolerogenic activity. To this end, we investigated iron metabolism in bone marrow‐derived macrophages (BMDMs) isolated from GBM specimens and in in vitro‐derived macrophages (Mφ) from healthy donor (HD) blood monocytes. We found that HO‐1 inhibition abrogated the immunosuppressive activity of both BMDMs and Mφ, and that immunosuppression requires both cell‐to‐cell contact and soluble factors, as HO‐1 inhibition abolished IL‐10 release, and significantly reduced STAT3 activation as well as PD‐L1 expression. Interestingly, not only did HO‐1 inhibition downregulate IDO1 and ARG‐2 gene expression, but also reduced IDO1 enzymatic activity. Moreover, T cell activation status affected PD‐L1 expression and IDO1 activity, which were upregulated in the presence of activated, but not resting, T cells. Our results highlight the crucial role of HO‐1 in the immunosuppressive activity of macrophages in the GBM TME and demonstrate the feasibility of reprogramming them as an alternative therapeutic strategy for restoring immune surveillance.

The cell composition of the glioblastoma (GBM) microenvironment depends on the recruitment of myeloid cells from the blood, promoting tumor progression by inducing immunosuppression. This phenomenon hampers immunotherapies and investigating its complexity may help to tailor new treatments. Peripheral blood and tissue specimens from the central and marginal tumor areas were collected from 44 primary and 19 recurrent GBM patients. Myeloid and lymphoid cell subsets and the levels of immunosuppressive markers were defined by multiparametric flow cytometry. Multiplexed immunohistochemistry was used to confirm the differences in the immune infiltrate and to analyze the cell spatial distribution. Relapsing GBM showed an increased presence of blood-derived macrophages in both tumor areas and a higher frequency of infiltrating lymphocytes, with a high level of exhaustion markers. The expansion of some myeloid-derived suppressor cell (MDSC) subsets in the blood was found in both primary and recurrent GBM patients. A significant inverse correlation between infiltrating T cells and an MDSC subset was also found. In patients with recurrent GBM after standard first-line therapy, the immune-hostile tumor microenvironment and the levels of some MDSC subsets in the blood persisted. Analysis of the immune landscape in GBM relapses aids in the definition of more appropriate stratification and treatment.

Glioblastoma multiforme (GBM) is one of the most malignant primary brain tumor and is known to create an immunosuppressive microenvironment that hampers the efficacy of therapies. We demonstrated that GBM microenvironment is infiltrated with a large proportion of bone marrow-derived macrophages (BMDMs) that possess a strong immune suppressive activity. Following 5-aminolevulinic acid (5-ALA) administration to patients before surgery, BMDMs show the highest protoporphyrin IX (PPIX) fluorescence emission, compared to immune and non-immune cells. Since PPIX is the precursor of heme we wondered if the presence of this metabolite could be the consequence of an altered iron metabolism in BMDMs, and if this pathway could be linked to their immunosuppressive activity. GBM tissues were dissociated and subjected to immunomagnetic or FACS sorting for the separation of BMDMs. The expression of HMOX1, the gene coding for heme oxygenase-1 (HO-1) which is a central enzyme in the iron pathway, was analyzed by RT-PCR. BMDM immune suppressive activity was evaluated as the ability to suppress the proliferation of T lymphocytes also in the presence of HO-1 inhibitors. In order to optimize the experimental conditions, immunosuppressive macrophages derived from M-CSF treated monocytes from healthy donors were set up. Another in vitro model of immunosuppressive cells, bone marrow derived-myeloid derived suppressor cells (BM-MDSCs), was tested. HMOX1 expression was found to be upregulated in BMDMs compared to other cells. The treatment of BMDMs with a HO-1 inhibitor was able to restore T cells proliferation in immune suppressive assays. The recovery of immunosuppression following treatment with the inhibitor has also been demonstrated in macrophages and in BM-MDSCs. Our results show that the connection between iron metabolism and immune response could be exploited from a therapeutic point of view. In particular, HO-1 plays a role in BMDM-induced immunosuppression and could represent a new target to relieve the immunosuppressive microenvironment present in GBM patients. University of Padua. AIRC. All authors have declared no conflicts of interest.