Cátia Monteiro

Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities

In order to improve the efficacy of conventional radiotherapy, attention has been paid to immune cells, which not only modulate cancer cell response to therapy but are also highly recruited to tumours after irradiation. Particularly, the effect of ionizing radiation on macrophages, using therapeutically relevant doses, is not well understood. To evaluate how radiotherapy affects macrophage behaviour and macrophage-mediated cancer cell activity, human monocyte derived-macrophages were subjected, for a week, to cumulative ionizing radiation doses, as used during cancer treatment (2 Gy/fraction/day). Irradiated macrophages remained viable and metabolically active, despite DNA damage. NF-kappaB transcription activation and increased Bcl-xL expression evidenced the promotion of pro-survival activity. A significant increase of pro-inflammatory macrophage markers CD80, CD86 and HLA-DR, but not CCR7, TNF and IL1B was observed after 10 Gy cumulative doses, while anti-inflammatory markers CD163, MRC1, VCAN and IL-10 expression decreased, suggesting the modulation towards a more pro-inflammatory phenotype. Moreover, ionizing radiation induced macrophage morphological alterations and increased their phagocytic rate, without affecting matrix metalloproteases (MMP)2 and MMP9 activity. Importantly, irradiated macrophages promoted cancer cell-invasion and cancer cell-induced angiogenesis. Our work highlights macrophage ability to sustain cancer cell activities as a major concern that needs to be addressed to improve radiotherapy efficacy.

Human periprostatic white adipose tissue is rich in stromal progenitor cells and a potential source of prostate tumor stroma

A body of growing evidence now implicates white adipose tissue as a relevant source of stromal progenitor cells recruited to the tumor microenvironment to form supportive tumor stroma. While the role of periprostatic (PP) adipose tissue in prostate cancer progression has been barely appreciated, we sought to determine the progenitor cell population in PP adipose tissue and the association with prostate cancer. We isolated and characterized CD31−CD34+CD45−CD146− progenitor cells (adipose-derived stem cells [ASC]) in paired samples of PP and preperitoneal visceral adipose tissue from prostate tissue and peripheral blood mononuclear cells of prostate cancer and nodular prostatic hyperplasia patients. ASC were quantified by flow cytometry and confirmed through target gene expression. Here we show a significantly higher amount of ASC in PP than in visceral adipose tissue, independent of body mass index and prostatic disease. In the prostate, ASC are increased in cancer compared with prostatic nodular hyperplasia patients. Concordantly, adipsin gene (CFD) expression, which is known to be up-regulated in adipose stem cells, was overexpressed in PP adipose tissue, in the prostate of cancer patients and in prostate CD31−CD34+CD45−CD146− sorted cells. ASC were found at higher levels in the blood of prostate cancer patients simultaneously overweight/obese. Present findings indicate that PP adipose tissue is a reservoir of progenitor cells with the potential to migrate towards prostate tumors, although its clinical significance merits further evaluation.

The HIF1A functional genetic polymorphism at locus +1772 associates with progression to metastatic prostate cancer and refractoriness to hormonal castration.

The hypoxia inducible factor 1 alpha (HIF1a) is a key regulator of tumour cell response to hypoxia, orchestrating mechanisms known to be involved in cancer aggressiveness and metastatic behaviour. In this study we sought to evaluate the association of a functional genetic polymorphism in HIF1A with overall and metastatic prostate cancer (PCa) risk and with response to androgen deprivation therapy (ADT). The HIF1A +1772 C>T (rs11549465) polymorphism was genotyped, using DNA isolated from peripheral blood, in 1490 male subjects (754 with prostate cancer and 736 controls cancer-free) through Real-Time PCR. A nested group of cancer patients who were eligible for androgen deprivation therapy was followed up. Univariate and multivariate models were used to analyse the response to hormonal treatment and the risk for developing distant metastasis. Age-adjusted odds ratios were calculated to evaluate prostate cancer risk. Our results showed that patients under ADT carrying the HIF1A +1772 T-allele have increased risk for developing distant metastasis (OR, 2.0; 95%CI, 1.1-3.9) and an independent 6-fold increased risk for resistance to ADT after multivariate analysis (OR, 6.0; 95%CI, 2.2-16.8). This polymorphism was not associated with increased risk for being diagnosed with prostate cancer (OR, 0.9; 95%CI, 0.7-1.2). The HIF1A +1772 genetic polymorphism predicts a more aggressive prostate cancer behaviour, supporting the involvement of HIF1a in prostate cancer biological progression and ADT resistance. Molecular profiles using hypoxia markers may help predict clinically relevant prostate cancer and response to ADT.

An interferon-γ-delivery system based on chitosan/poly(γ-glutamic acid) polyelectrolyte complexes modulates macrophage-derived stimulation of cancer cell invasion in vitro

Macrophages represent a large component of the tumour microenvironment and are described to establish interactions with cancer cells, playing crucial roles in several stages of cancer progression. The functional plasticity of macrophages upon stimulation from the environment makes them susceptible to the influence of cancer cells and also renders them as promising therapeutic targets. In this work, we describe a drug delivery system to modulate the phenotype of macrophages, converting them from the pro-tumour M2 phenotype to the anti-tumour M1 phenotype, based on the incorporation of a pro-inflammatory cytokine (interferon-γ) in chitosan (Ch)/poly(γ-glutamic acid) (γ-PGA) complexes. Ch is a biocompatible cationic polysaccharide extensively studied and γ-PGA is a biodegradable, hydrophilic and negatively charged poly-amino acid. These components interact electrostatically, due to opposite charges, resulting in self-assembled structures that can be designed to deliver active molecules such as drugs and proteins. Ch and γ-PGA were self-assembled into polyelectrolyte multilayer films (PEMs) of 371nm thickness, using the layer-by-layer method. Interferon-γ (IFN-γ) was incorporated within the Ch layers at 100 and 500ng/mL. Ch/γ-PGA PEMs with IFN-γ were able to modulate the phenotype of IL-10-treated macrophages at the cell cytoskeleton and cytokine profile levels, inducing an increase of IL-6 and a decrease of IL-10 production. More interestingly, the pro-invasive role of IL-10-treated macrophages was hindered, as their stimulation of gastric cancer cell invasion in vitro decreased from 4 to 2-fold, upon modulation by Ch/γ-PGA PEMs with IFN-γ. This is the first report proposing Ch/γ-PGA PEMs as a suitable strategy to incorporate and release bioactive IFN-γ with the aim of modulating macrophage phenotype, counteracting their stimulating role on gastric cancer cell invasion.

Differentially expressed angiogenic genes in diabetic erectile tissue — Results from a microarray screening

Diabetes-induced metabolic derangements promote endothelial malfunction, contributing to erectile dysfunction (ED). However, it remains unclear which angiogenic molecular mechanisms are deregulated in diabetic corpus cavernosum (CC). We investigated early and late alterations in cavernosal angiogenic gene expression associated to diabetes. Angiogenic changes were assessed in penile tissue of streptozotocin-induced Wistar rats, in an early (2-week) and established stage (8-week) of diabetes. Differentially expressed genes were identified by microarrays and expression data validated by quantitative real-time PCR (qrt-PCR). At protein level, quantitative immunohistochemistry confirmed the arrays data and dual immunofluorescence for selected alterations and α-smooth muscle actin (α-SMA) identified the cellular location of target proteins. The selected differentially expressed genes were also evaluated in human non-diabetic and diabetic CC by quantitative immunolabeling. At 2-week diabetes there was no differential gene expression between non-diabetic and diabetic CC. At 8-week, 10 genes were found down-regulated in diabetics. The results were validated by qrt-PCR for the insulin-like growth factor-1 (Igf1) and the natriuretic peptide receptor-1 (Npr1) genes. Dual immunofluorescence for IGF-1/ α-SMA showed predominant localization of IGF-1 in SM. NPR-1 expression was diffuse and mostly present in trabecular fibroblasts and SM. Quantitative immunostaining confirmed the decreased expression of both proteins in diabetic tissues. Concordantly, we detected a significant reduction in IGF-1 and NPR-1 protein expressions in human diabetic samples. Microarray analysis identified 10 angiogenic-related molecules deregulated in CC of established diabetes. Among them, IGF-1 and NPR-1 were significantly down-regulated and might result in preventive/therapeutic targets for ED management.

Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour

BackgroundThe interactions established between macrophages and cancer cells are largely dependent on instructions from the tumour microenvironment. Macrophages may differentiate into populations with distinct inflammatory profiles, but knowledge on their role on cancer cell activities is still very scarce. In this work, we investigated the influence of pro-inflammatory (LPS-stimulated) and anti-inflammatory (IL-10-stimulated) macrophages on gastric and colorectal cancer cell invasion, motility/migration, angiogenesis and proteolysis, and the associated molecular mechanisms.MethodsFollowing exposure of gastric and colon cancer cell lines to LPS- and IL-10-stimulated human macrophages, either by indirect contact or conditioned media, we analyzed the effect of the different macrophage populations on cancer cell invasion, migration, motility and phosphorylation status of EGFR and several interacting partners. Cancer-cell induced angiogenesis upon the influence of conditioned media from both macrophage populations was assessed using the chick embryo chorioallantoic membrane assay. MMP activities were evaluated by gelatin zymograhy.ResultsOur results show that IL-10-stimulated macrophages are more efficient in promoting in vitro cancer cell invasion and migration. In addition, soluble factors produced by these macrophages enhanced in vivo cancer cell-induced angiogenesis, as opposed to their LPS-stimulated counterparts. We further demonstrate that differences in the ability of these macrophage populations to stimulate invasion or angiogenesis cannot be explained by the EGFR-mediated signalling, since both LPS- and IL-10-stimulated macrophages similarly induce the phosphorylation of cancer cell EGFR, c-Src, Akt, ERK1/2, and p38. Interestingly, both populations exert distinct proteolytic activities, being the IL-10-stimulated macrophages the most efficient in inducing matrix metalloprotease (MMP)-2 and MMP-9 activities. Using a broad-spectrum MMP inhibitor, we demonstrated that proteolysis was essential for macrophage-mediated cancer cell invasion and angiogenesis.ConclusionsWe propose that IL-10- and LPS-stimulated macrophages distinctly modulate gastric and colorectal cancer cell behaviour, as result of distinct proteolytic profiles that impact cell invasion and angiogenesis.

Performance of an Adipokine Pathway-Based Multilocus Genetic Risk Score for Prostate Cancer Risk Prediction

Few biomarkers are available to predict prostate cancer risk. Single nucleotide polymorphisms (SNPs) tend to have weak individual effects but, in combination, they have stronger predictive value. Adipokine pathways have been implicated in the pathogenesis. We used a candidate pathway approach to investigate 29 functional SNPs in key genes from relevant adipokine pathways in a sample of 1006 men eligible for prostate biopsy. We used stepwise multivariate logistic regression and bootstrapping to develop a multilocus genetic risk score by weighting each risk SNP empirically based on its association with disease. Seven common functional polymorphisms were associated with overall and high-grade prostate cancer (Gleason≥7), whereas three variants were associated with high metastatic-risk prostate cancer (PSA≥20 ng/mL and/or Gleason≥8). The addition of genetic variants to age and PSA improved the predictive accuracy for overall and high-grade prostate cancer, using either the area under the receiver-operating characteristics curves (P<0.02), the net reclassification improvement (P<0.001) and integrated discrimination improvement (P<0.001) measures. These results suggest that functional polymorphisms in adipokine pathways may act individually and cumulatively to affect risk and severity of prostate cancer, supporting the influence of adipokine pathways in the pathogenesis of prostate cancer. Use of such adipokine multilocus genetic risk score can enhance the predictive value of PSA and age in estimating absolute risk, which supports further evaluation of its clinical significance.

Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

Embryonic stem (ES)‐derived neural stem/progenitor cells (ES‐NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single‐cell suspensions of ES‐NSPCs, to obtain homogeneously‐sized ES‐NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES‐NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller‐sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES‐NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N‐cadherin and β‐catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase‐2 (MMP‐2) activity and percentage of neuronal cells. In line with the increased MMP‐2 activity levels found, hydrodynamically‐cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single‐cell suspensions of ES‐NSPCs, besides allowing the formation of homogeneously‐sized neurospheres, promoted ES‐NSPC differentiation and outward migration, possibly by influencing the expression of cell–cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly‐sized ES‐NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright