150 research outputs found
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
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
Low dose gemcitabine-loaded lipid nanocapsules target monocytic myeloid-derived suppressor cells and potentiate cancer immunotherapy
Tumor-induced expansion of myeloid-derived suppressor cells (MDSCs) is known to impair the efficacy of cancer immunotherapy. Among pharmacological approaches for MDSC modulation, chemotherapy with selected drugs has a considerable interest due to the possibility of a rapid translation to the clinic. However, such approach is poorly selective and may be associated with dose-dependent toxicities. In the present study, we showed that lipid nanocapsules (LNCs) loaded with a lauroyl-modified form of gemcitabine (GemC12) efficiently target the monocytic MDSC subset. Subcutaneous administration of GemC12-loaded LNCs reduced the percentage of spleen and tumor-infiltrating M-MDSCs in lymphoma and melanoma-bearing mice, with enhanced efficacy when compared to free gemcitabine. Consistently, fluorochrome-labeled LNCs were preferentially uptaken by monocytic cells rather than by other immune cells, in both tumor-bearing mice and human blood samples from healthy donors and melanoma patients. Very low dose administration of GemC12-loaded LNCs attenuated tumor-associated immunosuppression and increased the efficacy of adoptive T cell therapy. Overall, our results show that GemC12-LNCs have monocyte-targeting properties that can be useful for immunomodulatory purposes, and unveil new possibilities for the exploitation of nanoparticulate drug formulations in cancer immunotherapy
Modified antimetabolites-loaded lipid nanocapsules to enhance antitumor immunity
Introduction : Myeloidderived suppressor cells (MDSCs) are critical players of tumorinduced
immunosuppression in mouse models and cancer patients. They accumulate in the spleen and cancers of tumorbearing hosts where they suppress Tcell activation, proliferation and cytotoxic function [1]. Previous studies demonstrated that some anticancer agents, in addition to their cytotoxic effects on tumor cells, were able to affect MDSCs. This occurs for antimetabolites like 5fluorouracile (5FU) and Gemcitabine (Gem) [2]. In this work, the potential activity of novel lipophilic 5FU and Gem derivatives encapsulated into lipid nanocapsules (LNCs) to target monocytic (M)MDSC subset and tumor cells (pancreatic B6KPC3) was assessed. The aim was to study the immunogenic and anticancer properties of innovative nanosystems.
Methods: Gem and 5FU were modified to obtain monolauroylderivatives (GemC12 and 5FUC12). The
derivatives were purified by chromatography on silica column and characterized by nuclear magnetic
resonance. Blank and loadedLNCs were prepared using the phase inversion process [3]. Physicochemical characterization (size, dispersity, zeta potential and encapsulation efficiency) was performed. To study the in vitro induction of MMDSCs, the immunosuppressive activity and internalization assays of GemC12loaded LNCs, mouse bone marrow cells cultured in presence of GMCSF and IL6 were used. To investigate the efficacy of 5FUC12loaded LNCs, B6KPC3 cells were employed. Finally, as a preliminary
in vivo study, the biodistribution of fluorescentloaded LNCs (i.v. or s.c.) using tumorbearing mice (EG7OVA subcutaneous model) was evaluated.
Results: Lipophilic derivatives, 5FUC12 and GemC12, were synthetized. The yield of the products recovered was 60% and 40% for 5FUC12 and GemC12, respectively. Blank, 5FUC12 and GemC12loaded LNCs showed an average size of 60 nm, dispersity index below 0.1 and neutral surface charge. The encapsulation efficiency of drugs was close to 100%. In vitro and in vivo studies highlighted that GemC12loaded LNCs were internalized and depleted selectively MMDSCs. Using K6PC3, we demonstrated that 5FUC12loaded LNCs exerted a toxic effect comparable to the commercial 5FUsolution. In vivo studies following i.v. or s.c. administration of fluorescentloaded LNCs showed that LNCs reached peripheral tissues. As compared with i.v., following s.c. injection, fluorescent signal increased with time in the spleen, suggesting a slow LNCs absorption.
Conclusions : In the present study, lipophilic 5FUC12 and GemC12loaded LNC were obtained. GemC12
loaded LNCs were able to target MMDSCs in vivo and in vitro. Besides, 5FUC12loaded LNCs showed
efficacy as anticancer drug in a pancreatic cell line. Further in vitro and in vivo therapeutic evaluations would disclose the full potential of these novel LNCs.
Betaine-homocysteine S-methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated with plasma hyperhomocysteinemia
Betaine-homocysteine S-methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine; hence, their malfunction leads to hyperhomocysteinemia. Epidemiologic and experimental studies have revealed a correlation between hyperhomocysteinemia and hearing loss. Here, we have studied the expression of methionine cycle genes in the mouse cochlea and the impact of knocking out the Bhmt gene in the auditory receptor. We evaluated age-related changes in mouse hearing by recording auditory brainstem responses before and following exposure to noise. Also, we measured cochlear cytoarchitecture, gene expression by RNA-arrays and quantitative RT-PCR, and metabolite levels in liver and plasma by HPLC. Our results indicate that there is an age-dependent strain-specific expression of methionine cycle genes in the mouse cochlea and a further regulation during the response to noise damage. Loss of Bhmt did not cause an evident impact in the hearing acuity of young mice, but it produced higher threshold shifts and poorer recovery following noise challenge. Hearing loss was associated with increased cochlear injury, outer hair cell loss, altered expression of cochlear methionine cycle genes, and hyperhomocysteinemia. Our results suggest that BHMT plays a central role in the homeostasis of cochlear methionine metabolism and that Bhmt2 up-regulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT
Elevated myeloid-derived suppressor cells in pancreatic, esophageal and gastric cancer are an independent prognostic factor and are associated with significant elevation of the Th2 cytokine interleukin-13
We undertook a comprehensive analysis of circulating myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) in pancreatic, esophageal and gastric cancer patients and investigated whether MDSCs are an independent prognostic factor for survival. We evaluated a series of plasma cytokines and in particular re-evaluated the Th2 cytokine interleukin-13 (IL-13). Peripheral blood was collected from 131 cancer patients (46 pancreatic, 60 esophageal and 25 gastric) and 54 healthy controls. PBMC were harvested with subsequent flow cytometric analysis of MDSC (HLADR− Lin1low/− CD33+ CD11b+) and Treg (CD4+ CD25+ CD127low/− FoxP3+) percentages. Plasma IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17, G-CSF, IFN-γ, TNF-α and VEGF levels were analyzed by the Bio-Plex cytokine assay. Plasma arginase I levels were analyzed by ELISA. MDSCs and Tregs were statistically significantly elevated in pancreatic, esophageal and gastric cancer compared with controls, and MDSC numbers correlated with Treg levels. Increasing MDSC percentage was associated with increased risk of death, and in a multivariate analysis, MDSC level was an independent prognostic factor for survival. A unit increase in MDSC percentage was associated with a 22% increased risk of death (hazard ratio 1.22, 95% confidence interval 1.06–1.41). Arginase I levels were also statistically significantly elevated in upper gastrointestinal cancer patients compared with controls. There was Th2 skewing for cytokine production in all three diseases, and importantly there were significant elevations of the pivotal Th2 cytokine interleukin-13, an increase that correlated with MDSC levels
Phenotypic and transcriptomic characterization of canine myeloid-derived suppressor cells
Myeloid-derived suppressor cells (MDSCs) are key players in immune evasion, tumor progression and metastasis. MDSCs accumulate under various pathological states and fall into two functionally and phenotypically distinct subsets that have been identified in humans and mice: polymorphonuclear (PMN)-MDSCs and monocytic (M)-MDSCs. As dogs are an excellent model for human tumor development and progression, we set out to identify PMN-MDSCs and M-MDSCs in clinical canine oncology patients. Canine hypodense MHC class II-CD5-CD21-CD11b+ cells can be subdivided into polymorphonuclear (CADO48A+CD14-) and monocytic (CADO48A-CD14+) MDSC subsets. The transcriptomic signatures of PMN-MDSCs and M-MDSCs are distinct, and moreover reveal a statistically significant similarity between canine and previously published human PMN-MDSC gene expression patterns. As in humans, peripheral blood frequencies of canine PMN-MDSCs and M-MDSCs are significantly higher in dogs with cancer compared to healthy control dogs (PMN-MDSCs: p < 0.001; M-MDSCs: p < 0.01). By leveraging the power of evolution, we also identified additional conserved genes in PMN-MDSCs of multiple species that may play a role in MDSC function. Our findings therefore validate the dog as a model for studying MDSCs in the context of cancer
Oxidative stress in pregnancy and fertility pathologies
Oxidative stress designates the state of imbalance between reactive oxygen species (ROS) production and antioxidant levels. In a healthy placenta, there is an increase in ROS production, due to formation of new tissues and inherent metabolism, but this is balanced by higher levels of antioxidants. However, this balance is lost in some situations, with a consequent increase in oxidative stress levels. Oxidative stress has been implicated in several placental disorders and pregnancy pathologies. The present review intends to summarize what is known about the relationship between oxidative stress and well-known pregnancy disorders
Cell Cycle Gene Networks Are Associated with Melanoma Prognosis
BACKGROUND: Our understanding of the molecular pathways that underlie melanoma remains incomplete. Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies. Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules. Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients. The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis. An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study. Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active. Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro. CONCLUSIONS/SIGNIFICANCE: This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis. Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response). The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets
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