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

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

Placental vascularity and markers of angiogenesis in relation to prenatal growth status in overnourished adolescent ewes.

INTRODUCTION: Placental vascularity may be important in the development of fetal growth restriction (FGR). The overnourished adolescent ewe is a robust model of the condition, with ∼50% of offspring demonstrating FGR (birthweight >2 standard deviations below optimally-fed control mean). We studied whether placental vascularity, angiogenesis and glucose transport reflect FGR severity. METHODS: Singleton pregnancies were established in adolescent ewes either overnourished to putatively restrict fetoplacental growth (n = 27) or control-fed (n = 12). At 131d (term = 145d) pregnancies were interrupted and fetuses classified as FGR (n = 17,  Non-FGR > FGR and fetal:placental weight ratios were higher in overnourished versus Control groups. COT vascular indices were Non-FGR > FGR > Control. COT-CAD, CSD and APC were significantly greater in Non-FGR overnourished versus Control and intermediate in FGR groups. CAR vascularity did not differ. CAR-VEGFA/FLT1/KDR/ANGPT1/ANGPT2/SLC2A1/SLC2A3 mRNA was lower and COT-ANGPT2 higher in overnourished versus Control groups. DISCUSSION: Relative to control-intake pregnancy, overnourished pregnancies are characterised by higher COT vascularity, potentially a compensatory response to reduced nutrient supply, reflected by higher fetal:placental weight ratios. Compared with overnourished pregnancies where fetal growth is relatively preserved, overnourished pregnancies culminating in marked FGR have less placental vascularity, suggesting incomplete adaptation to the prenatal insult

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

A gene expression signature associated with survival in metastatic melanoma

BACKGROUND: Current clinical and histopathological criteria used to define the prognosis of melanoma patients are inadequate for accurate prediction of clinical outcome. We investigated whether genome screening by means of high-throughput gene microarray might provide clinically useful information on patient survival. METHODS: Forty-three tumor tissues from 38 patients with stage III and stage IV melanoma were profiled with a 17,500 element cDNA microarray. Expression data were analyzed using significance analysis of microarrays (SAM) to identify genes associated with patient survival, and supervised principal components (SPC) to determine survival prediction. RESULTS: SAM analysis revealed a set of 80 probes, corresponding to 70 genes, associated with survival, i.e. 45 probes characterizing longer and 35 shorter survival times, respectively. These transcripts were included in a survival prediction model designed using SPC and cross-validation which allowed identifying 30 predicting probes out of the 80 associated with survival. CONCLUSION: The longer-survival group of genes included those expressed in immune cells, both innate and acquired, confirming the interplay between immunological mechanisms and the natural history of melanoma. Genes linked to immune cells were totally lacking in the poor-survival group, which was instead associated with a number of genes related to highly proliferative and invasive tumor cells

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

The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis

<p>Abstract</p> <p>Background</p> <p>The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.</p> <p>Methods</p> <p>We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.</p> <p>Results</p> <p>We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including <it>SPRR1A/B</it>, <it>KRT16/17</it>, <it>CD24</it>, <it>LOR</it>, <it>GATA3</it>, <it>MUC15</it>, and <it>TMPRSS4</it>, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as <it>MAGE</it>, <it>GPR19</it>, <it>BCL2A1</it>, <it>MMP14</it>, <it>SOX5</it>, <it>BUB1</it>, <it>RGS20</it>, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (<it>SPP-1</it>, <it>MITF</it>, <it>CITED-1</it>, <it>GDF-15</it>, <it>c-Met</it>, <it>HOX </it>loci) and suppressor genes (<it>PITX-1</it>, <it>CST-6</it>, <it>PDGFRL</it>, <it>DSC-3</it>, <it>POU2F3</it>, <it>CLCA2</it>, <it>ST7L</it>), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.</p> <p>Conclusion</p> <p>The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.</p

Transcriptome Profiling of Whole Blood Cells Identifies PLEK2 and C1QB in Human Melanoma

Developing analytical methodologies to identify biomarkers in easily accessible body fluids is highly valuable for the early diagnosis and management of cancer patients. Peripheral whole blood is a "nucleic acid-rich" and "inflammatory cell-rich" information reservoir and represents systemic processes altered by the presence of cancer cells.We conducted transcriptome profiling of whole blood cells from melanoma patients. To overcome challenges associated with blood-based transcriptome analysis, we used a PAXgene™ tube and NuGEN Ovation™ globin reduction system. The combined use of these systems in microarray resulted in the identification of 78 unique genes differentially expressed in the blood of melanoma patients. Of these, 68 genes were further analyzed by quantitative reverse transcriptase PCR using blood samples from 45 newly diagnosed melanoma patients (stage I to IV) and 50 healthy control individuals. Thirty-nine genes were verified to be differentially expressed in blood samples from melanoma patients. A stepwise logit analysis selected eighteen 2-gene signatures that distinguish melanoma from healthy controls. Of these, a 2-gene signature consisting of PLEK2 and C1QB led to the best result that correctly classified 93.3% melanoma patients and 90% healthy controls. Both genes were upregulated in blood samples of melanoma patients from all stages. Further analysis using blood fractionation showed that CD45(-) and CD45(+) populations were responsible for the altered expression levels of PLEK2 and C1QB, respectively.The current study provides the first analysis of whole blood-based transcriptome biomarkers for malignant melanoma. The expression of PLEK2, the strongest gene to classify melanoma patients, in CD45(-) subsets illustrates the importance of analyzing whole blood cells for biomarker studies. The study suggests that transcriptome profiling of blood cells could be used for both early detection of melanoma and monitoring of patients for residual disease