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.

Preliminary evidence on machine learning approaches for clusterizing students' cognitive profile.

Assessing the cognitive abilities of students in academic contexts can provide valuable insights for teachers to identify their cognitive profile and create personalized teaching strategies. While numerous studies have demonstrated promising outcomes in clustering students based on their cognitive profiles, effective comparisons between various clustering methods are lacking in the current literature. In this study, we aim to compare the effectiveness of two clustering techniques to group students based on their cognitive abilities including general intelligence, attention, visual perception, working memory, and phonological awareness. 292 students, aged 11-15 years, participated in the study. A two-level approach based on the joint use of Kohonen's Self-Organizing Map (SOMs) and k-means clustering algorithm was compared with an approach based on the k-means clustering algorithm only. The resulting profiles were then predicted via AdaBoost and ANN supervised algorithms. The results showed that the two-level approach provides the best solution for this problem while the ANN algorithm was the winner in the classification problem. These results laying the foundations for developing a useful instrument for predicting the students' cognitive profile.

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.

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.

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.

Developmental trajectories of global motion and global form perception from 4 years to adulthood.

Literature on the development of global motion and global form perception demonstrated their asynchronous developmental trajectories. However, former studies have failed to clearly establish the critical period of maturation for these specific abilities. This study aimed to analyze the developmental trajectories of global motion and global form discrimination abilities by controlling for basic visual functions and general cognitive ability and to present the global motion and global form normative scores. A sample of 456 children and adolescents (4-17 years of age) and 76 adults recruited from the Italian and Swedish general population participated in the study. Motion and form perception were evaluated by the motion coherence test and form coherence test, respectively. Raven's matrices were used to assess general cognitive ability, the Lea Hyvärinen chart test was used for full- and low-contrast visual acuity, and the TNO test was used for stereopsis. General cognitive ability and basic visual functions were strongly related to motion and form perception development. Global motion perception had an accelerated maturation compared with global form perception. For motion perception, an analysis of the oblique effect's development showed that it is present at 4 years of age. The standardized scores of global motion and form coherence tests can be used for clinical purposes.

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.

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.

Preliminary Validation of the CI-FRA Checklist: A Simple Screening Tool for Measuring the Early Signs of Reading and Spelling Disorders in Italian Primary Students.

Although several screening tests for recognizing early signs of reading and spelling difficulties have been developed, brief and methodologically grounded tools for teachers are very limited. The present study aimed to lay the foundation for a new screening tool for teachers: the Checklist for early Indicators of risk Factors in Reading Ability (CI-FRA). The proposed checklist consists of 20 items, based on a 7-point Likert scale, and it investigates five domains: reading, writing, attention, and motor skills. Six hundred sixty-seven children were evaluated by 40 teachers during the first year of primary school and, longitudinally, in the second year. Exploratory factor analysis and confirmatory factor analysis (CFA) were applied to verify structural validity. Concurrent validity was assessed by Spearman correlation to analyze the link between CI-FRA and reading and spelling standardized tests and cognitive tests. Reliability was assessed by Cronbach α and interclass correlation coefficient. The CFA reported a three-factor structure as the optimal solution, including language (reading and writing), visuospatial attention, and fine motor skills subscales. Good reliability, good internal consistency, and acceptable test-retest indices were found. Concurrent validity was confirmed by significant correlations between CI-FRA total score and standardized reading and spelling test, as well as by correlations between CI-FRA subscales and neuropsychological standardized test scores. Preliminary evaluation of sensitivity by receiver operating characteristic curves showed that the CI-FRA score has particularly high sensitivity and specificity for word reading speed deficit. In conclusion, the results confirm that CI-FRA is a theoretically grounded and statistically valid tool that could help the teachers to screen for early signs of reading and spelling difficulties.

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.

Internalizing Symptoms in Developmental Dyslexia: A Comparison Between Primary and Secondary School.

Although the relationship between developmental dyslexia (DD) and the risk of occurrence of internalizing symptomatology has been widely investigated in the extant literature, different findings have been reported. In this study, two experiments with two general purposes are presented. The first study investigates whether the differences in the severity of internalizing symptoms between DD and controls are greater in students attending secondary school than in those attending primary school. Sixty-five DD and 169 controls attending primary and secondary school took part in the first study. The diagnosis of dyslexia was obtained from standardized reading tests; internalizing symptom severity was assessed with the Self Administrated Psychiatric Scales for Children and Adolescents questionnaire. The results showed that adolescents with dyslexia had an increased level of self-perceived anxiety, depression and somatic symptoms, whereas no significant differences between DD and controls emerged in childhood. In the second study, a cohort of adolescents attending secondary school (DD = 44; controls = 51) was closely analyzed to clarify whether contextual and subjective factors could contribute toward exacerbating the risk of internalizing symptomatology at that age. Internalizing symptom severity was assessed with the Child Behavior Checklist, Youth Self Report questionnaire, decision-making factors were measured with the Melbourne Decision Making Questionnaire, and student's quality of life was gaged using the Clipper test. The results showed that high levels of internalizing symptoms in DD were associated with a low level of self-esteem and the tendency to react to problematic situations with hyperactivation. By contrast, positive relationships with peers were associated with low symptom severity. In conclusion, the intensified internalizing symptoms that could emerge in adolescents in association with the presence of dyslexia are predicted by social protective and risk factors that are associated with symptom severity. Accordingly, the results suggest that remediation programs for dyslexia should include implementing motivation strategies, self-esteem enhancement activities and building peers networks that, starting in childhood, can prevent the appearance of internalizing symptoms.

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.

The immune suppressive microenvironment of human gliomas depends on the accumulation of bone marrow-derived macrophages in the center of the lesion.

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.