Present and Future of Immunotherapy in Patients With Glioblastoma: Limitations and Opportunities.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor. Standard therapies, including surgical resection, chemoradiation, and tumor treating fields, have not resulted in major improvements in the survival outcomes of patients with GBM. The lack of effective strategies has led to an increasing interest in immunotherapic approaches, considering the success in other solid tumors. However, GBM is a highly immunosuppressive tumor, as documented by the presence of several mechanisms of immune escape, which may represent a reason why immunotherapy clinical trials failed in this kind of tumor. In this review, we examine the current landscape of immunotherapy strategies in GBM, focusing on the challenge of immunoresistance and potential mechanisms to overcome it. We discussed completed and ongoing clinical trials involving immune checkpoint inhibitors, oncolytic viruses, vaccines, and CAR T-cell therapies, to provide insights into the efficacy and outcomes of different immunotherapeutic interventions. We also explore the impact of radiotherapy on the immune system within the GBM microenvironment highlighting the complex interactions between radiation treatment and the immune response.

The immunosuppression pathway of tumor-associated macrophages is controlled by heme oxygenase-1 in glioblastoma patients.

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.

Targeting of immunosuppressive myeloid cells from glioblastoma patients by modulation of size and surface charge of lipid nanocapsules.

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.

Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immunosuppressive cells developing from myeloid progenitors, which are enriched in pathological conditions such as cancer, and are known to inhibit the functions of effector T cells. During aging, several changes occur both at the adaptive and innate immune system level, in a process defined as immunoscenescence. In particular, the low-grade inflammation state observed in the elderly appears to affect hematopoiesis. We previously demonstrated that the combination of GM-CSF and G-CSF drives the in vitro generation of bone marrow-derived MDSCs (BM-MDSCs) from precursors present in human bone marrow aspirates of healthy donors, and that these cells are endowed with a strong immune suppressive ability, resembling that of cancer-associated MDSCs. In the present work we investigated BM-MDSCs induction and functional ability in a cohort of pediatric versus elderly donors. To this aim, we analyzed the differences in maturation stages and ability to suppress T cell proliferation. We found that the ex vivo distribution of myeloid progenitors is similar between pediatric and elderly individuals, whereas after cytokine treatment a significant reduction in the more immature compartment is observed in the elderly. Despite the decreased frequency, BM-MDSCs maintain their suppressive capacity in aged donors. Taken together, these results indicate that in vitro induction of MDSCs from the BM is reduced with aging and opens new hypotheses on the role of age-related processes in myelopoiesis.

Correction to: Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors.

An amendment to this paper has been published and can be accessed via the original article.

Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor.

Tumor growth is associated with a profound alteration in myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). We showed that among factors produced by various experimental tumors, the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of CD8(+) T cells and allow long term acceptance of pancreatic islet allografts. Cytokines inducing MDSCs acted on a common molecular pathway and the immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on the C/EBPbeta transcription factor. Adoptive transfer of tumor antigen-specific CD8(+) T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in the myeloid compartment, suggesting that C/EBPbeta is a critical regulator of the immunosuppressive environment created by growing cancers.

Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study.

There is an increasing interest for monitoring circulating myeloid-derived suppressor cells (MDSCs) in cancer patients, but there are also divergences in their phenotypic definition. To overcome this obstacle, the Cancer Immunoguiding Program under the umbrella of the Association of Cancer Immunotherapy is coordinating a proficiency panel program that aims at harmonizing MDSC phenotyping. After a consultation period, a two-stage approach was designed to harmonize MDSC phenotype. In the first step, an international consortium of 23 laboratories immunophenotyped 10 putative MDSC subsets on pretested, peripheral blood mononuclear cells of healthy donors to assess the level of concordance and define robust marker combinations for the identification of circulating MDSCs. At this stage, no mandatory requirements to standardize reagents or protocols were introduced. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets, especially for the granulocytic subsets. In particular, the use of a dead-cell marker altered significantly the reported percentage of granulocytic MDSCs, confirming that these cells are especially sensitive to cryopreservation and/or thawing. Importantly, the gating strategy was heterogeneous and associated with high inter-center variance. Overall, our results document the high variability in MDSC phenotyping in the multicenter setting if no harmonization/standardization measures are applied. Although the observed variability depended on a number of identified parameters, the main parameter associated with variation was the gating strategy. Based on these findings, we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets.

Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion.

By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic DCs can control self-reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4-day culture with FDA-approved cytokines (recombinant human-GM-CSF and recombinant human-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fibrocyte-associated markers, promotes Treg-cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.

Metastatic lesions with and without interleukin-18-dependent genes in advanced-stage melanoma patients.

IL-18 is an immune-stimulating cytokine that promotes experimental melanoma metastasis via vascular endothelial growth factor (VEGF)-induced very late antigen (VLA)-4. We studied genes associated with the ability of melanoma cells to allow metastasis under IL-18 effects, and we verified their expression in metastatic lesions from patients with melanoma. Human melanoma cell lines with and without the IL-18 receptor (IL-18R)/VEGF/VLA-4-expressing phenotype were identified, and their metastatic potential was studied in nude mice. RNA from untreated and IL-18-treated melanoma phenotypes was hybridized to a cDNA microarray, and their signature genes were studied. RNA from primary and metastatic lesions from patients with melanoma was hybridized to a cDNA microarray to identify lesions with the transcript patterns of melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype. IL-18R/VEGF/VLA-4-expressing A375 and 1182 melanoma cells produced a higher metastasis number than 526 and 624.28 melanoma cells, not using this prometastatic pathway. Melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype had distinct transcript patterns. However, the type I transcriptional cluster, including cutaneous and lymph node metastases, but not the type II cluster, not including cutaneous metastases, had signature genes from IL-18-treated melanoma cells with, but not without, the IL-18R/VEGF/VLA-4 phenotype. Metastatic melanoma lesions with and without IL-18-dependent genes were identified, suggesting that melanoma metastasis developed via inflammation-dependent and inflammation-independent mechanisms. Signature genes from melanomas with and without the IL-18R/VEGF/VLA-4 phenotype may serve as diagnostic biomarkers of melanoma predisposition to prometastatic effects of IL-18.

Sodium fluorescein uptake by the tumor microenvironment in human gliomas and brain metastases.

OBJECTIVE: Intravenous sodium fluorescein (SF) is increasingly used during surgery of gliomas and brain metastases to improve tumor resection. Currently, SF is believed to permeate the brain regions where the blood-brain barrier (BBB) is damaged and to accumulate in the extracellular space but not in tumor or healthy cells, making it possible to demarcate tumor margins to guide resection. By evaluating the immune contexture of a number of freshly resected gliomas and brain metastases from patients undergoing SF-guided surgery, the authors recurrently observed fluorescence-positive cells. Therefore, the aim of this study was to determine if SF accumulates inside the cells of the tumor microenvironment (TME), and if so, in which type of cells, and whether incorporation can also be observed in the leukocytes of peripheral blood. METHODS: Freshly resected tumor specimens were dissociated to single cells and analyzed by multiparametric flow cytometry. Peripheral blood leukocytes, macrophages, and a glioma cell line were treated with SF in vitro, and their cell uptake was assessed by multiparametric and imaging flow cytometry and by confocal microscopy. RESULTS: The ex vivo and in vitro analyses revealed that SF accumulates intracellularly in leukocytes as well as in tumor cells, but with a high variability of incorporation in the different cell subsets analyzed. Myeloid cells showed the highest level of fluorescence. In vitro uptake experiments showed that SF accumulation increases over time. The imaging analyses confirmed the internalization of the compound inside the cells. CONCLUSIONS: SF is not just a marker of BBB damage, but its intracellular detection suggests that it selectively accumulates intracellularly. Future efforts should target the mechanisms of its differential uptake by the different TME cell types in depth.

Impact of microRNAs on regulatory networks and pathways in human colorectal carcinogenesis and development of metastasis.

BACKGROUND: Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed. RESULTS: Most changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples. CONCLUSIONS: Using a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development.

A human promyelocytic-like population is responsible for the immune suppression mediated by myeloid-derived suppressor cells.

We recently demonstrated that human BM cells can be treated in vitro with defined growth factors to induce the rapid generation of myeloid-derived suppressor cells (MDSCs), hereafter defined as BM-MDSCs. Indeed, combination of G-CSF + GM-CSF led to the development of a heterogeneous mixture of immature myeloid cells ranging from myeloblasts to band cells that were able to suppress alloantigen- and mitogen-stimulated T lymphocytes. Here, we further investigate the mechanism of suppression and define the cell subset that is fully responsible for BM-MDSC-mediated immune suppression. This population, which displays the structure and markers of promyelocytes, is however distinct from physiologic promyelocytes that, instead, are devoid of immuosuppressive function. In addition, we demonstrate that promyelocyte-like cells proliferate in the presence of activated lymphocytes and that, when these cells exert suppressive activity, they do not differentiate but rather maintain their immature phenotype. Finally, we show that promyelocyte-like BM-MDSCs are equivalent to MDSCs present in the blood of patients with breast cancer and patients with colorectal cancer and that increased circulating levels of these immunosuppressive myeloid cells correlate with worse prognosis and radiographic progression.

The immune cell landscape of glioblastoma patients highlights a myeloid-enriched and immune suppressed microenvironment compared to metastatic brain tumors.

Introduction: Brain metastases (BrM), which commonly arise in patients with melanoma, breast cancer and lung cancer, are associated with a poor clinical prognosis. In this context, the tumor microenvironment (TME) plays an important role since it either promotes or inhibits tumor progression. Our previous studies have characterized the immunosuppressive microenvironment of glioblastoma (GBM). The aim of this study is to compare the immune profiles of BrM and GBM in order to identify potential differences that may be exploited in their differential treatment. Methods: Tumor and/or blood samples were taken from 20 BrM patients and 19 GBM patients. Multi-parametric flow cytometry was used to evaluate myeloid and lymphoid cells, as well as the expression of immune checkpoints in the TME and blood. In selected cases, the immunosuppressive ability of sorted myeloid cells was tested, and the ex vivo proliferation of myeloid, lymphoid and tumor cell populations was analyzed. Results: High frequencies of myeloid cells dominated both the BrM and GBM landscapes, but a higher presence of tumor-associated macrophages was observed in GBM, while BrM were characterized by a significant presence of tumor-infiltrating lymphocytes. Exhaustion markers were highly expressed in all T cells from both primary and metastatic brain tumors. Ex vivo analysis of the cell cycle of a single sample of a BrM and of a GBM revealed subsets of proliferating tumor cells and blood-derived macrophages, but quiescent resident microglial cells and few proliferating lymphocytes. Macrophages sorted from a single lung BrM exhibited a strong immunosuppressive activity, as previously shown for primary GBM. Finally, a significant expansion of some myeloid cell subsets was observed in the blood of both GBM and BrM patients. Discussion: Our results define the main characteristics of the immune profile of BrM and GBM, which are distinguished by different levels of immunosuppressive myeloid cells and lymphocytes devoid of effector function. Understanding the role of the different cells in establishing the metastatic setting is critical for improving the therapeutic efficacy of new targeted immunotherapy strategies.

Neutralization of NET-associated human ARG1 enhances cancer immunotherapy.

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

Myeloid cell diversification and complexity: an old concept with new turns in oncology.

Tumour development is accompanied by an enhanced haematopoiesis. This is not a widespread activation since only cells belonging to the myelo-monocytic compartment are expanded and mobilized from primary sites of haematopoiesis to other organs, reaching also the tumour stroma. This process occurs early during tumour formation but becomes more evident in advanced disease. Far from being a simple, unwanted consequence of cancer development, accumulation of myelo-monocytitc cells plays a role in tumour vascularization, local spreading, establishment of metastasis at distant sites, and contribute to create an environment unfavourable for the adoptive immunity against tumour-associated antigens. Myeloid populations involved in these process are likely different but many cells, expanded in primary and secondary lymphoid organs of tumour-bearing mice, share various levels of the CD11b and Gr-1 (Ly6C/G) markers. CD11b(+)Gr-1(+) cells are currently named myeloid-derived suppressor cells for their ability to inhibit T lymphocyte responses in tumour-bearing hosts. In this manuscript, we review the recent literature on tumour-conditioned myeloid subsets that assist tumour growth, both in mice and humans.

Highlights on molecular mechanisms of MDSC-mediated immune suppression: paving the way for new working hypotheses.

MDSCs have been recognized in the last years as tolerogenic cells, potentially dangerous in the context of neoplasia, since they are able to induce tolerance to a variety of anti-tumor effectors, including CD4(+) and CD8(+) T cells. It is currently believed that the origin of MDSCs is due to an arrest of the myeloid differentiation process caused by tumor-secreted factors released in the tumor microenvironment that are able to exert an effect on myeloid progenitors, rendering them unable to terminally differentiate into dendritic cells, granulocytes and macrophages. As a consequence, these immature myeloid cells acquire suppressive activity through the activation of several mechanisms, controlled by different transcription factors. The lack of consensus about the phenotypical characterization of human MDSCs is the result of the existence of different MDSC subsets, most likely depending on the tumor in which they expand and on the tumor specific cytokine cocktail driving their activation. This, in turn, might also influence the mechanisms of MDSC-mediated immune suppression. In this review article we address the role of tumor-derived factors (TDFs) in MDSC-recruitment and activation, discuss the complex heterogeneity of MDSC phenotype and analyze the crosstalk between activated T cells and MDSCs.

Magnetic Resonance Imaging Correlates of Immune Microenvironment in Glioblastoma.

Background: Glioblastoma (GBM) is the most commonly occurring primary malignant brain tumor, and it carries a dismal prognosis. Focusing on the tumor microenvironment may provide new insights into pathogenesis, but no clinical tools are available to do this. We hypothesized that the infiltration of different leukocyte populations in the tumoral and peritumoral brain tissues may be measured by magnetic resonance imaging (MRI). Methods: Pre-operative MRI was combined with immune phenotyping of intraoperative tumor tissue based on flow cytometry of myeloid cell populations that are associated with immune suppression, namely, microglia and bone marrow-derived macrophages (BMDM). These cell populations were measured from the central and marginal areas of the lesion identified intraoperatively with 5-aminolevulinic acid-guided surgery. MRI features (volume, mean and standard deviation of signal intensity, and fractality) were derived from all MR sequences (T1w, Gd+ T1w, T2w, FLAIR) and ADC MR maps and from different tumor areas (contrast- and non-contrast-enhancing tumor, necrosis, and edema). The principal components of MRI features were correlated with different myeloid cell populations by Pearson's correlation. Results: We analyzed 126 samples from 62 GBM patients. The ratio between BMDM and microglia decreases significantly from the central core to the periphery. Several MRI-derived principal components were significantly correlated (p <0.05, r range: [-0.29, -0.41]) with the BMDM/microglia ratio collected in the central part of the tumor. Conclusions: We report a significant correlation between structural MRI clinical imaging and the ratio of recruited vs. resident macrophages with different immunomodulatory activities. MRI features may represent a novel tool for investigating the microenvironment of GBM.

IL4Ralpha+ myeloid-derived suppressor cell expansion in cancer patients.

Myeloid-derived suppressor cells (MDSC) contribute to immune dysfunctions induced by tumors both in experimental models and patients. In mice, MDSC are phenotypically heterogeneous cells that vary in their surface markers, likely depending on soluble factors produced by different tumors. We recently described a subset of inflammatory monocytes with immunosuppressive properties that can be found within the tumor mass, blood, and lymphoid organs of tumor-bearing mice. These cells expressed the alpha-chain of the receptor for IL-4 (IL4Ralpha) that was critical for their negative activity on CD8(+) T cells. In cancer patients, the nature of MDSC is still poorly defined because evidence exists for both monocytic and granulocytic features. We show in this study that myeloid cells with immunosuppressive properties accumulate both in mononuclear and polymorphonuclear fractions of circulating blood leukocytes of patients with colon cancer and melanoma, thus unveiling a generalized alteration in the homeostasis of the myeloid compartment. Similarly to mouse MDSC, IL4Ralpha is up-regulated in both myeloid populations but its presence correlates with an immunosuppressive phenotype only when mononuclear cells, but not granulocytes, of tumor-bearing patients are considered.

Sustained Accumulation of Blood-Derived Macrophages in the Immune Microenvironment of Patients with Recurrent Glioblastoma after Therapy.

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.

Myeloid Diagnostic and Prognostic Markers of Immune Suppression in the Blood of Glioma Patients.

Background: Although gliomas are confined to the central nervous system, their negative influence over the immune system extends to peripheral circulation. The immune suppression exerted by myeloid cells can affect both response to therapy and disease outcome. We analyzed the expansion of several myeloid parameters in the blood of low- and high-grade gliomas and assessed their relevance as biomarkers of disease and clinical outcome. Methods: Peripheral blood was obtained from 134 low- and high-grade glioma patients. CD14+, CD14+/p-STAT3+, CD14+/PD-L1+, CD15+ cells and four myeloid-derived suppressor cell (MDSC) subsets, were evaluated by flow cytometry. Arginase-1 (ARG1) quantity and activity was determined in the plasma. Multivariable logistic regression model was used to obtain a diagnostic score to discriminate glioma patients from healthy controls and between each glioma grade. A glioblastoma prognostic model was determined by multiple Cox regression using clinical and myeloid parameters. Results: Changes in myeloid parameters associated with immune suppression allowed to define a diagnostic score calculating the risk of being a glioma patient. The same parameters, together with age, permit to calculate the risk score in differentiating each glioma grade. A prognostic model for glioblastoma patients stemmed out from a Cox multiple analysis, highlighting the role of MDSC, p-STAT3, and ARG1 activity together with clinical parameters in predicting patient's outcome. Conclusions: This work emphasizes the role of systemic immune suppression carried out by myeloid cells in gliomas. The identification of biomarkers associated with immune landscape, diagnosis, and outcome of glioblastoma patients lays the ground for their clinical use.