An objective comparison of cell-tracking algorithms.

We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.

Regional variation in arterial stiffening and dysfunction in Western diet-induced obesity.

Increased central vascular stiffening, assessed in vivo by determination of pulse wave velocity (PWV), is an independent predictor of cardiovascular event risk. Recent evidence demonstrates that accelerated aortic stiffening occurs in obesity; however, little is known regarding stiffening of other disease-relevant arteries or whether regional variation in arterial stiffening occurs in this setting. We addressed this gap in knowledge by assessing femoral PWV in vivo in conjunction with ex vivo analyses of femoral and coronary structure and function in a mouse model of Western diet (WD; high-fat/high-sugar)-induced obesity and insulin resistance. WD feeding resulted in increased femoral PWV in vivo. Ex vivo analysis of femoral arteries revealed a leftward shift in the strain-stress relationship, increased modulus of elasticity, and decreased compliance indicative of increased stiffness following WD feeding. Confocal and multiphoton fluorescence microscopy revealed increased femoral stiffness involving decreased elastin/collagen ratio in conjunction with increased femoral transforming growth factor-ß (TGF-ß) content in WD-fed mice. Further analysis of the femoral internal elastic lamina (IEL) revealed a significant reduction in the number and size of fenestrae with WD feeding. Coronary artery stiffness and structure was unchanged by WD feeding. Functionally, femoral, but not coronary, arteries exhibited endothelial dysfunction, whereas coronary arteries exhibited increased vasoconstrictor responsiveness not present in femoral arteries. Taken together, our data highlight important regional variations in the development of arterial stiffness and dysfunction associated with WD feeding. Furthermore, our results suggest TGF-ß signaling and IEL fenestrae remodeling as potential contributors to femoral artery stiffening in obesity.

Cxcl8 (IL-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response.

Neutrophils play a pivotal role in the innate immune response. The small cytokine CXCL8 (also known as IL-8) is known to be one of the most potent chemoattractant molecules that, among several other functions, is responsible for guiding neutrophils through the tissue matrix until they reach sites of injury. Unlike mice and rats that lack a CXCL8 homolog, zebrafish has two distinct CXCL8 homologs: Cxcl8-l1 and Cxcl8-l2. Cxcl8-l1 is known to be upregulated under inflammatory conditions caused by bacterial or chemical insult but until now the role of Cxcl8s in neutrophil recruitment has not been studied. In this study we show that both Cxcl8 genes are upregulated in response to an acute inflammatory stimulus, and that both are crucial for normal neutrophil recruitment to the wound and normal resolution of inflammation. Additionally, we have analyzed neutrophil migratory behavior through tissues to the site of injury in vivo, using open-access phagocyte tracking software PhagoSight. Surprisingly, we observed that in the absence of these chemokines, the speed of the neutrophils migrating to the wound was significantly increased in comparison with control neutrophils, although the directionality was not affected. Our analysis suggests that zebrafish may possess a subpopulation of neutrophils whose recruitment to inflamed areas occurs independently of Cxcl8 chemokines. Moreover, we report that Cxcl8-l2 signaled through Cxcr2 for inducing neutrophil recruitment. Our study, therefore, confirms the zebrafish as an excellent in vivo model to shed light on the roles of CXCL8 in neutrophil biology.

Influence of soluble or matrix-bound isoforms of vascular endothelial growth factor-A on tumor response to vascular-targeted strategies.

Antiangiogenic therapy based on blocking the actions of vascular endothelial growth factor-A (VEGF) can lead to "normalization" of blood vessels in both animal and human tumors. Differential expression of VEGF isoforms affects tumor vascular maturity, which could influence the normalization process and response to subsequent treatment. Fibrosarcoma cells expressing only VEGF120 or VEGF188 isoforms were implanted either subcutaneously (s.c.) or in dorsal skin-fold "window" chambers in SCID mice. VEGF120 was associated with vascular fragility and hemorrhage. Tumor-bearing mice were treated with repeat doses of SU5416, an indolinone receptor tyrosine kinase inhibitor with activity against VEGFR-2 and proven preclinical ability to induce tumor vascular normalization. SU5416 reduced vascularization in s.c. implants of both VEGF120 and VEGF188 tumors. However, in the window chamber, SU5416 treatment increased red cell velocity in VEGF120 (representing vascular normalization) but not VEGF188 tumors. SU5416 treatment had no effect on growth or necrosis levels in either tumor type but tended to counteract the increase in interstitial fluid pressure seen with growth of VEGF120 tumors. SU5416 pretreatment resulted in the normally fragile blood vessels in VEGF120-expressing tumors becoming resistant to the vascular damaging effects of the tubulin-binding vascular disrupting agent (VDA), combretastatin A4 3-O-phosphate (CA4P). Thus, vascular normalization induced by antiangiogenic treatment can reduce the efficacy of subsequent VDA treatment. Expression of VEGF120 made tumors particularly susceptible to vascular normalization by SU5416, which in turn made them resistant to CA4P. Therefore, VEGF isoform expression may be useful for predicting response to both antiangiogenic and vascular-disrupting therapy.

Vascular effects dominate solid tumor response to treatment with combretastatin A-4-phosphate.

Vascular-targeted therapeutics are increasingly used in the clinic. However, less is known about the direct response of tumor cells to these agents. We have developed a combretastatin-A-4-phosphate (CA4P) resistant variant of SW1222 human colorectal carcinoma cells to examine the relative importance of vascular versus tumor cell targeting in the ultimate treatment response. SW1222(Res) cells were generated through exposure of wild-type cells (SW1222(WT) ) to increasing CA4P concentrations in vitro. Increased resistance was confirmed through analyses of cell viability, apoptosis and multidrug-resistance (MDR) protein expression. In vivo, comparative studies examined tumor cell necrosis, apoptosis, vessel morphology and functional vascular end-points following treatment with CA4P (single 100 mg/kg dose). Tumor response to repeated CA4P dosing (50 mg/kg/day, 5 days/week for 2 weeks) was examined through growth measurement, and ultimate tumor cell survival was studied by ex vivo clonogenic assay. In vitro, SW1222(Res) cells showed reduced CA4P sensitivity, enhanced MDR protein expression and a reduced apoptotic index. In vivo, CA4P induced significantly lower apoptotic cell death in SW1222(Res) versus SW1222(WT) tumors indicating maintenance of resistance characteristics. However, CA4P-induced tumor necrosis was equivalent in both lines. Similarly, rapid CA4P-mediated vessel disruption and blood flow shut-down were observed in both lines. Cell surviving fraction was comparable in the two tumor types following single dose CA4P and SW1222(Res) tumors were at least as sensitive as SW1222(WT) tumors to repeated dosing. Despite tumor cell resistance to CA4P, SW1222(Res) response in vivo was not impaired, strongly supporting the view that vascular damage dominates the therapeutic response to this agent.

Arterial Stiffening in Western Diet-Fed Mice Is Associated with Increased Vascular Elastin, Transforming Growth Factor-ß, and Plasma Neuraminidase.

Consumption of excess fat and carbohydrate (Western diet, WD) is associated with alterations in the structural characteristics of blood vessels. This vascular remodeling contributes to the development of cardiovascular disease, particularly as it affects conduit and resistance arteries. Vascular remodeling is often associated with changes in the elastin-rich internal elastic lamina (IEL) and the activation of transforming growth factor (TGF)-ß. In addition, obesity and type II diabetes have been associated with increased serum neuraminidase, an enzyme known to increase TGF-ß cellular output. Therefore, we hypothesized that WD-feeding would induce structural modifications to the IEL of mesenteric resistance arteries in mice, and that these changes would be associated with increased levels of circulating neuraminidase and the up-regulation of elastin and TGF-ß in the arterial wall. To test this hypothesis, a WD, high in fat and sugar, was used to induce obesity in mice, and the effect of this diet on the structure of mesenteric resistance arteries was investigated. 4-week old, Post-weaning mice were fed either a normal diet (ND) or WD for 16 weeks. Mechanically, arteries from WD-fed mice were stiffer and less distensible, with marginally increased wall stress for a given strain, and a significantly increased Young's modulus of elasticity. Structurally, the wall cross-sectional area and the number of fenestrae found in the internal elastic lamina (IEL) of mesenteric arteries from mice fed a WD were significantly smaller than those of arteries from the ND-fed mice. There was also a significant increase in the volume of elastin, but not collagen in arteries from the WD cohort. Plasma levels of neuraminidase and the amount of TGF-ß in mesenteric arteries were elevated in mice fed a WD, while ex vivo, cultured vascular smooth muscle cells exposed to neuraminidase secreted greater amounts of tropoelastin and TGF-ß than those exposed to vehicle. These data suggest that consumption of a diet high in fat and sugar causes stiffening of the vascular wall in resistance arteries through a process that may involve increased neuraminidase and TGF-ß activity, elevated production of elastin, and a reduction in the size and number of fenestrae in the arterial IEL.

Maternal Hyperleptinemia Is Associated with Male Offspring's Altered Vascular Function and Structure in Mice.

Children of mothers with gestational diabetes have greater risk of developing hypertension but little is known about the mechanisms by which this occurs. The objective of this study was to test the hypothesis that high maternal concentrations of leptin during pregnancy, which are present in mothers with gestational diabetes and/or obesity, alter blood pressure, vascular structure and vascular function in offspring. Wildtype (WT) offspring of hyperleptinemic, normoglycemic, Leprdb/+ dams were compared to genotype matched offspring of WT-control dams. Vascular function was assessed in male offspring at 6, and at 31 weeks of age after half the offspring had been fed a high fat, high sucrose diet (HFD) for 6 weeks. Blood pressure was increased by HFD but not affected by maternal hyperleptinemia. On a standard diet, offspring of hyperleptinemic dams had outwardly remodeled mesenteric arteries and an enhanced vasodilatory response to insulin. In offspring of WT but not Leprdb/+ dams, HFD induced vessel hypertrophy and enhanced vasodilatory responses to acetylcholine, while HFD reduced insulin responsiveness in offspring of hyperleptinemic dams. Offspring of hyperleptinemic dams had stiffer arteries regardless of diet. Therefore, while maternal hyperleptinemia was largely beneficial to offspring vascular health under a standard diet, it had detrimental effects in offspring fed HFD. These results suggest that circulating maternal leptin concentrations may interact with other factors in the pre- and post -natal environments to contribute to altered vascular function in offspring of diabetic pregnancies.

Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions.

The neutrophil's eye-view: inference and visualisation of the chemoattractant field driving cell chemotaxis in vivo.

As we begin to understand the signals that drive chemotaxis in vivo, it is becoming clear that there is a complex interplay of chemotactic factors, which changes over time as the inflammatory response evolves. New animal models such as transgenic lines of zebrafish, which are near transparent and where the neutrophils express a green fluorescent protein, have the potential to greatly increase our understanding of the chemotactic process under conditions of wounding and infection from video microscopy data. Measurement of the chemoattractants over space (and their evolution over time) is a key objective for understanding the signals driving neutrophil chemotaxis. However, it is not possible to measure and visualise the most important contributors to in vivo chemotaxis, and in fact the understanding of the main contributors at any particular time is incomplete. The key insight that we make in this investigation is that the neutrophils themselves are sensing the underlying field that is driving their action and we can use the observations of neutrophil movement to infer the hidden net chemoattractant field by use of a novel computational framework. We apply the methodology to multiple in vivo neutrophil recruitment data sets to demonstrate this new technique and find that the method provides consistent estimates of the chemoattractant field across the majority of experiments. The framework that we derive represents an important new methodology for cell biologists investigating the signalling processes driving cell chemotaxis, which we label the neutrophils eye-view of the chemoattractant field.

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors.

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.