Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo

Despite our advanced understanding of primary cancer development and progression, metastasis and the systemic spread of the disease to secondary sites remains the leading cause of cancer-associated death. The metastatic process is therefore a major potential therapeutic target area for cancer researchers and elucidating the key steps that are susceptible to therapeutic intervention will be critical to improve our treatment strategies. Recent advances in intravital imaging are rapidly improving our insight into this process and are helping in the design of stage-specific drug regimes for the treatment of metastatic cancer. Here we discuss current developments in intravital imaging and our recent use of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals to assess the efficacy of therapeutic intervention on early stages of tumor cell metastasis.Key words: in vivo imaging, photobleaching, photoactivation, biomarkersMetastasis is a complex process consisting of interactions between cancer cells and their surrounding extra-cellular matrix and stroma. To give rise to a secondary tumor, a primary tumor cell undergoes alterations to its cell-cell and cell-ECM contacts, allowing it to breach the basement membrane and intravasate into the vasculature or the lymphatic system. A tumor cell must survive in the circulation before extravasating at a secondary site and initiating new tumor growth and the development of its own blood supply. Imaging this process in live animals under native physiological conditions is inherently difficult due to poor sample stability, tissue penetration and autofluorescence of the tissue. However, new advances in fluorescent imaging, including the continued development of green fluorescent protein (GFP) and its variants, have facilitated the observation of this process and shed light on some key mechanisms that determine how and why cells metastasise. The use of fluorescent probes for in vivo imaging can be divided into two types (1) ‘passive’ markers or reporters used for direct visualization and tracking of cell movement in relation to extracellular structures and (2) more complex, ‘active’ reporters or biosensors for monitoring detailed processes such as biochemical activity or protein-protein interactions during metastasis.^1,2 In some cases there can be overlap between both types of imaging which will be addressed here.The majority of early intravital imaging studies focused on the stages of metastasis that occur after dissemination from the primary tumor and predominantly used a ‘passive’ reporter approach to assess tumor cell behavior. Models of circulating tumor cells have allowed for analysis at the single cell level of tumor cell velocity, persistence, shape change and interactions with the ECM and stroma in secondary tissue.^3–5 The use of fluorescently-labelled cells has also revealed some limiting factors that cause the arrest of cancer cells in target tissue such as trapping in small capillary networks due to tumor cell size or adhesion to surrounding vessel walls.^6,7 Furthermore, experimental models of metastasis such as intra-splenic, intra-cardial and tail vein injections in combination with fluorescently-tagged tumor cells has provided information on the colonisation, extravasation and dormancy of tumor cells in secondary sites (Fig. 1 and refs. ^{5, 8} and ⁹). Collectively, along with the rapid increase in tissue specific expression of GFP in mouse cancer models,¹⁰ a wealth of information on different steps of the metastatic process has begun to emerge.Open in a separate windowFigure 1(A) Whole body optical imaging of mCherry-expressing SW 620 colon cancer cell metastases after approximately six weeks post intra-splenic injection. Images were obtained using the Olympus OV100 whole body imaging system with an Olympus MT10, 150 w, Xenon light source, using a low magnification objective (macro lens) with a magnification of 0.14× and numerical aperture of 0.04. (B) mCherry expressing SW 620 colon cancer cells colonizing the liver 30 mins after intra-splenic injection. 1 × 10⁶ cells were injected into the spleen of an anesthetised CD-1 nude mouse and the incision sealed using ‘Clay Adams’ vetinary clips (VetTec). The mouse was placed on a heat pad for 30 mins then sacrificed. An incision was made in the abdomen to expose the liver and images of fluorescent cells within the liver were obtained using a 0.8× (0.22 NA) objective lens with variable zoom on the Olympus OV100.The departure of individual cells away from solid primary tumors into the blood stream has been a more difficult process to study using intravital imaging. It is a rare, sporadic event, requiring long acquisition and the inherent density and complex nature of the tumor tissue poses problems for imaging. Overcoming autofluorescence and light scattering has recently been improved due to advances in fluorophores^1,11 and the combined use of long-term multiphoton microscopy¹² has allowed greater resolution and tissue penetration than before. Multiphoton imaging can also provide additional detail regarding the interaction between cells and the surrounding extracellular environment using second harmonic signal generation (SHG) from collagen, elastin and other matrix proteins found in connective tissue.^13,14 In this regard, imaging the interaction of cancer cells with extracellular matrix has revealed distinct modes of cell locomotion adopted by cancer cells in vivo, such as ameboid or mesenchymal invasion, that depend upon the topography or density of the surrounding matrix.^3,13,15 A greater understanding of the initial cell movement and interaction with the extracellular environment will enhance our ability to pin-point cell-ECM targets that may be of clinical relevance in the future.Concurrent with the use of GFP as a ‘passive’ marker, a number of techniques have been developed that facilitate the visualization and localisation of GFP-tagged fusion proteins to quantify changes in protein expression, mobility and sub-cellular interactions during various processes in vitro. These include photobleaching (PB), photoactivation (PA), fluorescence resonance energy transfer (FRET) and fluorescent life time imaging microscopy (FLIM).^2,16,17 The adaptation of these techniques for in vivo imaging to examine the activity of key molecules will provide new ‘active’ markers or reporters that can be correlated with biological processes important in disease progression such as migration, proliferation and cell death. Other fluorescent probes such as MMPsense or Apotrace that measure ‘active’ processes such as metalloproteinase activity or apoptosis have also recently been used in animals.^18,19 In this way we can get closer to understanding how subcellular components or signal transduction pathways interact in real-time. The improved spatial and temporal detail will facilitate the ‘when and where’ we should target metastatic cancer cells for therapy.¹²In our recent paper we have adapted two techniques, photobleaching and photoactivation, for in vivo imaging and used them to assess the potential of E-cadherin as a molecular biosensor for cell migration in live tumors.²⁰ E-cadherin-based cell-cell contacts are prominent sites of remodelling during early stages of epithelial to mesenchymal transition (EMT). The disruption or deregulation of E-cadherin-based adhesions leads to the collapse of normal epithelial architecture that precedes the initial intravasation of cells from tumors.^21–23 In vitro photobleaching analysis of E-cadherin can be used as an ‘active’ molecular read-out of cell migration, as cells within a stationary colony show significantly reduced E-cadherin mobility compared to collectively migrating cells.²⁰ Moreover, as demonstrated in Figure 2 (reviewed in ref. ²⁰), E-cadherin mobility can also be spatially regulated within a population of tumor cells, as cells at the rear of a wound show impaired E-cadherin mobilisation compared to cells at the leading edge of the wound. This suggests a gradient of E-cadherin mobilisation within the local environment of a tumor may exist and could potentially be used in the future to map areas of weakened cell-cell adhesion from which cells are more likely to migrate. In vivo analysis of E-cadherin dynamics showed that changes in the mobility of E-cadherin can also be used as an ‘active’ marker of cell behavior in live animals, and may be useful in predicting cell mobilisation from primary tumors.²⁰Open in a separate windowFigure 2FRAP of GFP-E-cadherin at the rear or front of a wound heal assay. (A and B) Schematic and representative images of a wound heal assay depicting the area of cells selected for E-cadherin-based cell-cell junction FRAP analysis (red broken line). (C and D) Representative images of FRAP experiments performed at the rear or front of a wound heal assay respectively. White solid arrows represent area of photobleaching at the rear and white broken arrows represent area of photobleaching at the front of the wound. Red arrows indicate dynamics of cells at the front of the wound. Cells were classed to be at the front of the wound within the first three cells from the wound border (reviewed in ref. ²⁰).We also demonstrated the subcellular tracking of plasma membrane dynamics in vivo using the membrane-targeting sequence of H-Ras fused to photoactivatable-GFP.^24,25 Importantly, both the dynamics of cell-cell junctions, as visualised using E-cadherin:GFP, and the dynamics of the plasma membrane, which also plays a fundamental role in cell invasion and metastasis, are significantly different in vivo than in vitro.²⁰ Critically, this raises the possibility that many signalling axes and networks may function differently in vivo and therefore care must be taken when correlating in vitro information to the live setting. Lastly, we demonstrated the benefits of in vivo imaging in the assessment of molecular-based targeted therapeutics by using the Src inhibitor dasatinib, which impaired E-cadherin cell mobility in vivo but not in vitro.^20,26In the context of previous intravital imaging studies, our work suggests that we are at the beginning of a new stage of intravital imaging in which ‘active’ probes can help predict the efficacy of novel therapeutic treatments and also provide a context dependent read-out of oncogene-induced biological behavior in live animals. Importantly, not all molecules are adaptable for this type of in vivo imaging. Careful selection of candidate molecular markers that demonstrate clear changes attributable to a biological function, for example, subcellular relocalization or compartmentalisation, will be ideally suited for this type of intravital examination in the future.Here we have adopted two key fluorescent imaging techniques typically used in vitro and combined them with a fundamental biological question in vivo. The adaptation of other techniques for in vivo imaging such as FRET or FLIM-FRET probes will provide a detailed pixel by pixel map of the activity and behavior of key signalling proteins in live animals.^2,27 The use of these ‘active’ probes in vivo may hold further surprises concerning differences in molecular behavior in live animals compared to the traditional ‘snap-shot’ approach in vitro. Finally, one of the major challenges of in vivo imaging during drug discovery is the need for repeated imaging of the same animal in the presence or absence of drugs. The continued development of optical windows and observation chambers for non-invasive real-time imaging will facilitate this and allow for the assessment of drug response at the single cell level.²⁸ This, when combined with the subcellular optical techniques described here, will prove very useful in the future for in vivo imaging when evaluating the aetiology of the disease or during the drug discovery process.     

Worldwide population differentiation at disease-associated SNPs

Background

Recent genome-wide association (GWA) studies have provided compelling evidence of association between genetic variants and common complex diseases. These studies have made use of cases and controls almost exclusively from populations of European ancestry and little is known about the frequency of risk alleles in other populations. The present study addresses the transferability of disease associations across human populations by examining levels of population differentiation at disease-associated single nucleotide polymorphisms (SNPs).

Methods

We genotyped ~1000 individuals from 53 populations worldwide at 25 SNPs which show robust association with 6 complex human diseases (Crohn's disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, coronary artery disease and obesity). Allele frequency differences between populations for these SNPs were measured using Fst. The Fst values for the disease-associated SNPs were compared to Fst values from 2750 random SNPs typed in the same set of individuals.

Results

On average, disease SNPs are not significantly more differentiated between populations than random SNPs in the genome. Risk allele frequencies, however, do show substantial variation across human populations and may contribute to differences in disease prevalence between populations. We demonstrate that, in some cases, risk allele frequency differences are unusually high compared to random SNPs and may be due to the action of local (i.e. geographically-restricted) positive natural selection. Moreover, some risk alleles were absent or fixed in a population, which implies that risk alleles identified in one population do not necessarily account for disease prevalence in all human populations.

Conclusion

Although differences in risk allele frequencies between human populations are not unusually large and are thus likely not due to positive local selection, there is substantial variation in risk allele frequencies between populations which may account for differences in disease prevalence between human populations.     

Exploring the developmental overnutrition hypothesis using parental-offspring associations and FTO as an instrumental variable

Background

The developmental overnutrition hypothesis suggests that greater maternal obesity during pregnancy results in increased offspring adiposity in later life. If true, this would result in the obesity epidemic progressing across generations irrespective of environmental or genetic changes. It is therefore important to robustly test this hypothesis.

Methods and Findings

We explored this hypothesis by comparing the associations of maternal and paternal pre-pregnancy body mass index (BMI) with offspring dual energy X-ray absorptiometry (DXA)–determined fat mass measured at 9 to 11 y (4,091 parent–offspring trios) and by using maternal FTO genotype, controlling for offspring FTO genotype, as an instrument for maternal adiposity. Both maternal and paternal BMI were positively associated with offspring fat mass, but the maternal association effect size was larger than that in the paternal association in all models: mean difference in offspring sex- and age-standardised fat mass z-score per 1 standard deviation BMI 0.24 (95% confidence interval [CI]: 0.22 to 0.26) for maternal BMI versus 0.13 (95% CI: 0.11, 0.15) for paternal BMI; p-value for difference in effect < 0.001. The stronger maternal association was robust to sensitivity analyses assuming levels of non-paternity up to 20%. When maternal FTO, controlling for offspring FTO, was used as an instrument for the effect of maternal adiposity, the mean difference in offspring fat mass z-score per 1 standard deviation maternal BMI was −0.08 (95% CI: −0.56 to 0.41), with no strong statistical evidence that this differed from the observational ordinary least squares analyses (p = 0.17).

Conclusions

Neither our parental comparisons nor the use of FTO genotype as an instrumental variable, suggest that greater maternal BMI during offspring development has a marked effect on offspring fat mass at age 9–11 y. Developmental overnutrition related to greater maternal BMI is unlikely to have driven the recent obesity epidemic.     

Diel movement of smallmouth yellowfish Labeobarbus aeneus in the Vaal River,South Africa

Diel movements of Orange–Vaal smallmouth yellowfish Labeobarbus aeneus (Burchell, 1822) in the Vaal River, South Africa, were determined by externally attaching radio transmitters to 11 adult fish and manually tracking them between March and May 2012. Twenty-four radio telemetry monitoring surveys produced 2 304 diel tracks. At night, yellowfish displayed a preference for slow shallow (<0.3?m s?1, <0.5?m) and fast shallow habitats (>0.3?m s?1, <0.3?m), whereas by day they avoided these habitats, preferring fast deep areas (>0.3?m s?1, >0.3?m). The average total distance of 272?m moved per 24-hour period was three times greater than the diel range, and the average maximum displacement per minute was significantly higher in daytime (4?m) than at night (1.5?m). These findings suggest that L. aeneus is active primarily during the day in fast-flowing, deeper waters, and relatively inactive at night, when it occupies shallower habitats. This behaviour should be further explored to identify causal mechanisms underlying the diel habitat shifts in this species such as water temperature, foraging tactics and/or predator avoidance.     

Vitamin D Suppression of Endoplasmic Reticulum Stress Promotes an Antiatherogenic Monocyte/Macrophage Phenotype in Type 2 Diabetic Patients

Cardiovascular disease is the leading cause of morbidity/mortality in patients with type 2 diabetes mellitus (T2DM), but there is a lack of knowledge about the mechanism(s) of increased atherosclerosis in these patients. In patients with T2DM, the prevalence of 25-hydroxy vitamin D (25(OH)D) deficiency is almost twice that for nondiabetics and doubles the relative risk of developing cardiovascular disease compared with diabetic patients with normal 25(OH)D. We tested the hypothesis that monocytes from vitamin D-deficient subjects will have a proatherogenic phenotype compared with vitamin D-sufficient subjects in 43 patients with T2DM. Serum 25(OH)D level inversely correlated with monocyte adhesion to endothelial cells even after adjustment for demographic and comorbidity characteristics. Vitamin D-sufficient patients (≥30 ng/ml 25(OH)D) had lower monocyte endoplasmic reticulum (ER) stress, a predominance of M1 over M2 macrophage membrane receptors, and decreased mRNA expression of monocyte adhesion molecules PSGL-1, β₁-integrin, and β₂-integrin compared with patients with 25(OH)D levels of <30 ng/ml. In vitamin D-deficient macrophages, activation of ER stress increased adhesion and adhesion molecule expression and induced an M2-predominant phenotype. Moreover, adding 1,25(OH)₂D₃ to vitamin D-deficient macrophages shifted their phenotype toward an M1-predominant phenotype with suppressed adhesion. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients activated ER stress, accelerated adhesion, and increased adhesion molecule expression. The absence of ER stress protein CCAAT enhancer-binding protein homologous protein suppressed monocyte adhesion, adhesion molecule expression, and the M2-predominant phenotype induced by vitamin D deficiency. Thus, vitamin D is a natural ER stress reliever that induced an antiatherogenic monocyte/macrophage phenotype.     

Genome-wide association meta-analysis of cortical bone mineral density unravels allelic heterogeneity at the RANKL locus and potential pleiotropic effects on bone

Previous genome-wide association (GWA) studies have identified SNPs associated with areal bone mineral density (aBMD). However, this measure is influenced by several different skeletal parameters, such as periosteal expansion, cortical bone mineral density (BMD_C) cortical thickness, trabecular number, and trabecular thickness, which may be under distinct biological and genetic control. We have carried out a GWA and replication study of BMD_C, as measured by peripheral quantitative computed tomography (pQCT), a more homogenous and valid measure of actual volumetric bone density. After initial GWA meta-analysis of two cohorts (ALSPAC n = 999, aged ∼15 years and GOOD n = 935, aged ∼19 years), we attempted to replicate the BMD_C associations that had p<1×10⁻⁵ in an independent sample of ALSPAC children (n = 2803) and in a cohort of elderly men (MrOS Sweden, n = 1052). The rs1021188 SNP (near RANKL) was associated with BMD_C in all cohorts (overall p = 2×10⁻¹⁴, n = 5739). Each minor allele was associated with a decrease in BMD_C of ∼0.14SD. There was also evidence for an interaction between this variant and sex (p = 0.01), with a stronger effect in males than females (at age 15, males −6.77mg/cm³ per C allele, p = 2×10⁻⁶; females −2.79 mg/cm³ per C allele, p = 0.004). Furthermore, in a preliminary analysis, the rs1021188 minor C allele was associated with higher circulating levels of sRANKL (p<0.005). We show this variant to be independent from the previously aBMD associated SNP (rs9594738) and possibly from a third variant in the same RANKL region, which demonstrates important allelic heterogeneity at this locus. Associations with skeletal parameters reflecting bone dimensions were either not found or were much less pronounced. This finding implicates RANKL as a locus containing variation associated with volumetric bone density and provides further insight into the mechanism by which the RANK/RANKL/OPG pathway may be involved in skeletal development.     

Clustered environments and randomized genes: a fundamental distinction between conventional and genetic epidemiology

Background

In conventional epidemiology confounding of the exposure of interest with lifestyle or socioeconomic factors, and reverse causation whereby disease status influences exposure rather than vice versa, may invalidate causal interpretations of observed associations. Conversely, genetic variants should not be related to the confounding factors that distort associations in conventional observational epidemiological studies. Furthermore, disease onset will not influence genotype. Therefore, it has been suggested that genetic variants that are known to be associated with a modifiable (nongenetic) risk factor can be used to help determine the causal effect of this modifiable risk factor on disease outcomes. This approach, mendelian randomization, is increasingly being applied within epidemiological studies. However, there is debate about the underlying premise that associations between genotypes and disease outcomes are not confounded by other risk factors. We examined the extent to which genetic variants, on the one hand, and nongenetic environmental exposures or phenotypic characteristics on the other, tend to be associated with each other, to assess the degree of confounding that would exist in conventional epidemiological studies compared with mendelian randomization studies.

Methods and Findings

We estimated pairwise correlations between nongenetic baseline variables and genetic variables in a cross-sectional study comparing the number of correlations that were statistically significant at the 5%, 1%, and 0.01% level (α = 0.05, 0.01, and 0.0001, respectively) with the number expected by chance if all variables were in fact uncorrelated, using a two-sided binomial exact test. We demonstrate that behavioural, socioeconomic, and physiological factors are strongly interrelated, with 45% of all possible pairwise associations between 96 nongenetic characteristics (n = 4,560 correlations) being significant at the p < 0.01 level (the ratio of observed to expected significant associations was 45; p-value for difference between observed and expected < 0.000001). Similar findings were observed for other levels of significance. In contrast, genetic variants showed no greater association with each other, or with the 96 behavioural, socioeconomic, and physiological factors, than would be expected by chance.

Conclusions

These data illustrate why observational studies have produced misleading claims regarding potentially causal factors for disease. The findings demonstrate the potential power of a methodology that utilizes genetic variants as indicators of exposure level when studying environmentally modifiable risk factors.     

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

The use of small groups in a large lecture microbiology course

In the fall of 1997, we started using small groups in our large (100–200 students) junior level introductory microbiology course. Students form five-person groups early in the semester, and work on projects within these groups throughout the semester. These projects involve exploration of concepts such as metabolism, protein synthesis, and viral reproduction strategies and the submission of a poster describing a disease of their choice at the end of the semester. We have refined the use of the small groups during the last three semesters, and student acceptance and performance have improved steadily. In the fall semester of 1998, a comprehensive assessment of the effectiveness of these group projects was performed. Students were chosen at random to participate in student consultation groups to discuss group projects. Furthermore, we utilized a master teacher-in-residence from the Rocky Mountain Teachers Education Collaborative (RMTEC). This teacher-in-residence attended our classes, spoke with students, helped with student consultation groups, and provided observations of student responses to group work activities. RMTEC also provided funds to hire a research assistant to conduct student consultation groups, analyze student evaluations of our course, and compare evaluations from before and after the implementation of group examinations. Additionally, the Center for Teaching and Learning at Colorado State University assisted with mid-semester evaluations in each subsequent semester. The results of our analysis show that small groups in large lectures can be an effective learning tool provided students are given well-designed activities with clearly defined, obtainable goals and clearly articulated guidelines. Our experience also shows that the manner in which the instructor presents the process to students affects students' willingness to participate in the process. It must be clearly articulated to students why he has incorporated active learning strategies into the course, what he hopes students will gain from the experience, and how he expects students to participate in these activities. We recognize the increase in workload on ourselves as instructors, but the benefits seem worth the additional time and effort. This paper describes the group process that we use and provides an evaluation of the effort. Journal of Industrial Microbiology & Biotechnology (2000) 25, 121–126. Received 24 March 1999/ Accepted in revised form 23 November 1999     

Loss of Scar/WAVE Complex Promotes N-WASP- and FAK-Dependent Invasion

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Highlights? Arp2/3 complex drives epithelial cell invasion in the absence of WRC ? Loss of WRC promotes N-WASP-dependent invasion and degradative focal adhesions ? Loss of WRC promotes FAK activation and enhances anchorage-independent growth ? WRC suppresses cell transformation and tumor formation