Longitudinal evolution of the immune suppressive glioma microenvironment in different synchronous lesions during treatment

The role of immune suppression in glioma progression has been clearly established.1 We and others have recently demonstrated that myeloid cells play a major role in the tumor microenvironment of glioblastoma (GBM) patients,2,3 and that not only bone marrow-derived macrophages (BMDMs) have a higher intrinsic immune suppressive ability compared to resident microglial cells (MG), but also that this ability greatly increases going from the periphery to the tumor core.3 In lower grade gliomas (grades II and III), a much lower amount of BMDM is present, devoid of immune suppressive ability.3 We present here a longitudinal analysis of the immune infiltrate in a patient with a synchronous occurrence of GBM in the left temporal lobe, and a low-grade glioma (LGG) in the right frontal lobe, with discordant isocitrate dehydrogenase (IDH)-mutational status,4 followed by two GBM relapse

K+ accumulation and clearance in the calyx synaptic cleft of type I mouse vestibular hair cells

Vestibular organs of Amniotes contain two types of sensory cells, named Type I and Type II hair cells. While Type II hair cells are contacted by several small bouton nerve terminals, Type I hair cells receive a giant terminal, called a calyx, which encloses their basolateral membrane almost completely. Both hair cell types release glutamate, which depolarizes the afferent terminal by binding to AMPA post-synaptic receptors. However, there is evidence that non-vesicular signal transmission also occurs at the Type I hair cell-calyx synapse, possibly involving direct depolarization of the calyx by K+ exiting the hair cell. To better investigate this aspect, we performed whole-cell patch-clamp recordings from mouse Type I hair cells or their associated calyx. We found that [K+] in the calyceal synaptic cleft is elevated at rest relative to the interstitial (extracellular) solution and can increase or decrease during hair cell depolarization or repolarization, respectively. The change in [K+] was primarily driven by GK,L, the low-voltage-activated, non-inactivating K+ conductance specifically expressed by Type I hair cells. Simple diffusion of K+ between the cleft and the extracellular compartment appeared substantially restricted by the calyx inner membrane, with the ion channels and active transporters playing a crucial role in regulating intercellular [K+]. Calyx recordings were consistent with K+ leaving the synaptic cleft through postsynaptic voltage-gated K+ channels involving KV1 and KV7 subunits. The above scenario is consistent with direct depolarization and hyperpolarization of the calyx membrane potential by intercellular K+

Optimizing the vertebrate vestibular semicircular canal: could we balance any better?

The fluid-filled semicircular canals (SCCs) of the vestibular system are used by all vertebrates to sense angular rotation. Despite masses spanning seven decades, all mammalian SCCs are nearly the same size. We propose that the SCC represents a sensory organ that evolution has `optimally designed'. Four geometric parameters are used to characterize the SCC, and `building materials' of given physical properties are assumed. Identifying physical and physiological constraints on SCC operation, we find that the most sensitive SCC has dimensions consistent with available data.Comment: 4 pages, 3 figure

Row spacing and seed physiological quality of Crotalaria species.

Crotalaria crops have important uses, such as in green manuring, nematode control, biological nitrogen fixation and sugarcane reform in Savanna areas. Due to its strategic importance, knowledge about crotalaria seed production technology is a relevant factor to ensure the availability of high physiological quality seeds

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.[Figure not available: see fulltext.

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

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

The association between the tumor immune microenvironments and clinical outcome in low-grade, early-stage endometrial cancer patients

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out tomeasure CD8, CD68, FOXP3, PD-1,and PD-L1markers, aswell as cytokeratin (CK), on tissuemicroarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering.Most samples (69%) belonged to theimmune-desert subtype, characterized by lowimmune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8+ T-cell infiltration, as well as a high percentage of CD8/PD-1+ cells. Immune-exclusion samples (19%) displayed the lowest CD8+ infiltration combined with high PD-L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8+/PD-1+ andCK/PD-L1+ cells.FOXP3andmacrophage-rich phenotypes (3%and 4% of total samples) displayed relatively high levels of FOXP3+ regulatory T-cells and CD68+ macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8+ T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8+ T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomasCEA and IgM were funded by Fundación La Marató de TV3. This project was supported by grants from Partners of Choice Network from AstraZeneca and by the Instituto de Salud Carlos III (ISCIII) (PI17/01723 and PI21/00920), co-financed by the European Regional Development Fund ‘A way to achieve Europe’ (FEDER). We thank Marco Cassano (Lunaphore Technologies) for his help in writing the manuscrip

Proteomic analysis of low-grade, early-stage endometrial carcinoma reveals new dysregulated pathways associated with cell death and cell signaling

Low-grade, early-stage endometrial carcinoma (EC) is the most frequent malignant tumor of the uterine corpus. However, the molecular alterations that underlie these tumors are far from being fully understood. The purpose of this study is to describe dysregulated molecular pathways from EC patients. Sixteen samples of tumor tissue and paired healthy controls were collected and both were subjected to mass spectrometry (MS)/MS proteomic analysis. Gene ontology and pathway analysis was performed to discover dysregulated pathways and/or proteins using different databases and bioinformatic tools. Dysregulated pathways were cross-validated in an independent external cohort. Cell signaling, immune response, and cell death-associated pathways were robustly identified. The SLIT/ROBO signaling pathway demonstrated dysregulation at the proteomic and transcriptomic level. Necroptosis and ferroptosis were cell death-associated processes aberrantly regulated, in addition to apoptosis. Immune response-associated pathways showed a dominance of innate immune responses. Tumor immune infiltrates measured by immunofluorescence demonstrated diverse lymphoid and myeloid populations. Our results suggest a role of SLIT/ROBO, necroptosis, and ferroptosis, as well as a prominent role of innate immune response in low-grade, early-stage EC. These results could guide future research in this group of tumorsThis research was funded by the Instituto de Salud Carlos III (ISCIII) (PI17/01723), cofinanced by the European Development Regional Fund “A way to achieve Europe” (FEDER