Testing anti-cancer drugs with holographic incoherent-light-source quantitative phase imaging.

Quantitative Phase Imaging is becoming an important tool in the objective evaluation of cellular responses to experimental treatment. The technique is based on interferometric measurements of the optical thickness of cells in tissue culture reporting on the distribution of dry mass inside the cells. As the measurement of the optical thickness is interferometric, it is not subjected to the Abbe resolution limit, and the use of an incoherent-light source further increases the accuracy practically achieving 0.93nm in optical path difference corresponding to 4.6 femtograms/µm2. Holographic mode reduces the exposure in comparison to phase-shifting or phase-stepping interference microscopy and allows observation of faster dynamics. An attractive application is in the development of novel anti-cancer drugs and there is an important potential for pretesting chemotherapeutic drugs with biopsy material for personalized cancer treatment. The procedure involves the preparation of live cells in tissue culture, seeding them into suitable observation chambers, and time-lapse recording with an adjusted microscope. Subsequent image processing and statistical analysis are essential last steps producing the results, which include rapid measurements of cell growth in terms of dry-mass increase in individual cells, speed of cell motility and other dynamic morphometric parameters.

Replica-mold nanopatterned PHEMA hydrogel surfaces for ophthalmic applications.

Biomimicking native tissues and organs require the development of advanced hydrogels. The patterning of hydrogel surfaces may enhance the cellular functionality and therapeutic efficacy of implants. For example, nanopatterning of the intraocular lens (IOL) surface can suppress the upregulation of cytoskeleton proteins (actin and actinin) within the cells in contact with the IOL surface and, hence, prevent secondary cataracts causing blurry or opaque vision. Here we introduce a fast and efficient method for fabricating arrays consisting of millions of individual nanostructures on the hydrogel surface. In particular, we have prepared the randomly distributed nanopillars on poly(2-hydroxyethyl methacrylate) hydrogel using replica molding and show that the number, shape, and arrangement of nanostructures are fully adjustable. Characterization by atomic force microscopy revealed that all nanopillars were of similar shape, narrow size distribution, and without significant defects. In imprint lithography, choosing the appropriate hydrogel composition is critical. As hydrogels with imprinted nanostructures mimic the natural cell environment, they can find applications in fundamental cell biology research, e.g., they can tune cell attachment and inhibit or promote cell clustering by a specific arrangement of protrusive nanostructures on the hydrogel surface.

Anisotropic solid-state PLA foaming templated by crystal phase pre-oriented with 3D printing: cell supporting structures with directional capillary transfer function.

Bones represent a superb biomaterial that combines high mechanical stiffness with nutrition delivery to its osteocyte cells through the microscopical Haversian canals and bone canaliculi. Such a structure is hard to reproduce artificially, though. 3D printing delivers an unprecedented shape control of the created objects. Yet the resolution limit of the most common 3D printers is insufficient, lying between tens to hundreds of microns, while more precise 3D printing techniques fail in fabricating larger objects. We present a unique, yet simple and versatile method for preparing hierarchically aligned microporous canals using a biocompatible polymer polylactic acid (PLA) with their structure controlled at the submicron to macro scale. The layout was inspired by the microscopical Haversian canals and bone canaliculi of cortical bone. This procedure takes advantage of the PLA complex crystalline behavior, which was pre-oriented by 3D printing, orientedly crystallized in CO2, foamed, and cryo-shrunk. The canal formation was assessed via WAXS, FTIR spectroscopy, and DSC and complemented by the evaluation of mechanical properties, biocompatibility, and directionally selective capillary transport in the final structure. The fine dimensions of the canals unmatched by the common 3D printing techniques capable of making macroscale objects combined with the abovementioned properties represent promising potential for various applications, such as advanced cell-supporting designs with a built-in nutrition function.

Addressing cancer invasion and cell motility with quantitative light microscopy.

The incidence of death caused by cancer has been increasing worldwide. The growth of cancer cells is not the main problem. The majority of deaths are due to invasion and metastasis, where cancer cells actively spread from primary tumors. Our inbred rat model of spontaneous metastasis revealed dynamic phenotype changes in vitro correlating with the metastatic potential in vivo and led to a discovery of a metastasis suppressor, protein 4.1B, which affects their 2D motility on flat substrates. Subsequently, others confirmed 4.1B as metastasis suppressor using knock-out mice and patient data suggesting mechanism involving apoptosis. There is evidence that 2D motility may be differentially controlled to the 3D situation. Here we show that 4.1B affects cell motility in an invasion assay similarly to the 2D system, further supporting our original hypothesis that the role of 4.1B as metastasis suppressor is primarily mediated by its effect on motility. This is encouraging for the validity of the 2D analysis, and we propose Quantitative Phase Imaging with incoherent light source for rapid and accurate testing of cancer cell motility and growth to be of interest for personalized cancer treatment as illustrated in experiments measuring responses of human adenocarcinoma cells to selected chemotherapeutic drugs.

FoxO1-GAB1 axis regulates homing capacity and tonic AKT activity in chronic lymphocytic leukemia.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

Automated classification of cell morphology by coherence-controlled holographic microscopy.

In the last few years, classification of cells by machine learning has become frequently used in biology. However, most of the approaches are based on morphometric (MO) features, which are not quantitative in terms of cell mass. This may result in poor classification accuracy. Here, we study the potential contribution of coherence-controlled holographic microscopy enabling quantitative phase imaging for the classification of cell morphologies. We compare our approach with the commonly used method based on MO features. We tested both classification approaches in an experiment with nutritionally deprived cancer tissue cells, while employing several supervised machine learning algorithms. Most of the classifiers provided higher performance when quantitative phase features were employed. Based on the results, it can be concluded that the quantitative phase features played an important role in improving the performance of the classification. The methodology could be valuable help in refining the monitoring of live cells in an automated fashion. We believe that coherence-controlled holographic microscopy, as a tool for quantitative phase imaging, offers all preconditions for the accurate automated analysis of live cell behavior while enabling noninvasive label-free imaging with sufficient contrast and high-spatiotemporal phase sensitivity.

Mitotic catenation is monitored and resolved by a PKCε-regulated pathway.

Exit from mitosis is controlled by silencing of the spindle assembly checkpoint (SAC). It is important that preceding exit, all sister chromatid pairs are correctly bioriented, and that residual catenation is resolved, permitting complete sister chromatid separation in the ensuing anaphase. Here we determine that the metaphase response to catenation in mammalian cells operates through PKCε. The PKCε-controlled pathway regulates exit from the SAC only when mitotic cells are challenged by retained catenation and this delayed exit is characterized by BubR1-high and Mad2-low kinetochores. In addition, we show that this pathway is necessary to facilitate resolution of retained catenanes in mitosis. When delayed by catenation in mitosis, inhibition of PKCε results in premature entry into anaphase with PICH-positive strands and chromosome bridging. These findings demonstrate the importance of PKCε-mediated regulation in protection from loss of chromosome integrity in cells failing to resolve catenation in G2.

Novel automated tracking analysis of particles subjected to shear flow: kindlin-3 role in B cells.

Shear flow assays are used to mimic the influence of physiological shear force in diverse situations such as leukocyte rolling and arrest on the vasculature, capture of nanoparticles, and bacterial adhesion. Analysis of such assays usually involves manual counting, is labor-intensive, and is subject to bias. We have developed the Leukotrack program that incorporates a novel (to our knowledge) segmentation routine capable of reliable detection of cells in phase contrast images. The program also automatically tracks rolling cells in addition to those that are more firmly attached and migrating in random directions. We demonstrate its use in the analysis of lymphocyte arrest mediated by one or more active conformations of the integrin LFA-1. Activation of LFA-1 is a multistep process that depends on several proteins including kindlin-3, the protein that is mutated in leukocyte adhesion deficiency-III patients. We find that the very first stage of LFA-1-mediated attaching is unable to proceed in the absence of kindlin-3. Our evidence indicates that kindlin-3-mediated high-affinity LFA-1 controls both the early transient integrin-dependent adhesions in addition to the final stable adhesions made under flow conditions.

Killing of Kras-mutant colon cancer cells via Rac-independent actin remodeling by the ßGBP cytokine, a physiological PI3K inhibitor therapeutically effective in vivo.

Activating mutations in Kras are the most frequent mutations in human cancer. They define a subset of patients who do not respond to current therapies and for whom prognosis is poor. Oncogenic Kras has been shown to deregulate numerous signaling pathways of which the most intensively studied are the Ras/extracellular signal-regulated kinase cascade and the phosphoinositide 3-kinase (PI3K)/Akt cascade. However, to date, there are no effective targeted therapies in the clinic against Kras-mutant cancers. Here, we report that the ß-galactoside-binding protein (ßGBP) cytokine, a physiologic inhibitor of class I PI3Ks, is a potent activator of apoptosis in Kras-mutant colorectal cancer cells, even when coharboring mutant-activated PIK3CA. Our study unveils an elective route to intrinsic and extrinsic apoptosis, which involves the cytoskeleton. Early events are inhibition of PI3K activity and Rac-independent actin rearrangement assignable to phosphoinositide changes at the plasma membrane. Cyclin E deregulation, arrest of DNA synthesis, and checkpoint kinase 2 activation underscore events critical to the activation of an intrinsic apoptotic program. Clustering of CD95/Fas death receptors underscore events critical to the activation of extrinsic apoptosis. In nude mice, we present the first evidence that xenograft tumor development is strongly inhibited by Hu-r-ßGBP. Taken together, our results open a new therapeutic opportunity to a subset of patients refractive to current treatments. This first demonstration of therapeutic efficacy against Kras-mutant colon cancer suggests that Hu-r-ßGBP may also be therapeutically effective against other cancers harboring activating Ras mutations as well as PIK3CA mutations.

Regulation of lymphatic-blood vessel separation by endothelial Rac1.

Sprouting angiogenesis and lymphatic-blood vessel segregation both involve the migration of endothelial cells, but the precise migratory molecules that govern the decision of blood vascular endothelial cells to segregate into lymphatic vasculature are unknown. Here, we deleted endothelial Rac1 in mice (Tie1-Cre(+);Rac1(fl/fl)) and revealed, unexpectedly, that whereas blood vessel morphology appeared normal, lymphatic-blood vessel separation was impaired, with corresponding edema, haemorrhage and embryonic lethality. Importantly, normal levels of Rac1 were essential for directed endothelial cell migratory responses to lymphatic-inductive signals. Our studies identify Rac1 as a crucial part of the migratory machinery required for endothelial cells to separate and form lymphatic vasculature.

EKV mutant connexin 31 associated cell death is mediated by ER stress.

The epidermis expresses a number of connexin (Cx) proteins that are implicated in gap junction-mediated cell communication. Distinct dominantly inherited mutations in Cx31 cause the skin disease erythrokeratoderma variabilis (EKV) and hearing loss with or without neuropathy. Functional studies reveal tissue-specific effects of these Cx31 disease-associated mutations. The Cx31 mutants (R42P)Cx31, (C86S)Cx31 and (G12D)Cx31 are associated with EKV and the mutant (66delD)Cx31 with peripheral neuropathy and hearing loss, however the mechanisms of pathogenesis remain to be elucidated. Expression of (R42P)Cx31, (C86S)Cx31 and (G12D)Cx31 in vitro, but not (WT)Cx31 or (66delD)Cx31, cause elevated levels of cell-type specific cell death. Previous studies suggest that Cx-associated cell death may be related to abnormal 'leaky' hemichannels but we produced direct evidence against that being the major mechanism. Additionally, our immunocytochemistry showed upregulation of components of the unfolded protein response (UPR) in cells expressing the EKV-associated Cx31 mutants but not (WT)Cx31 or (66delD)Cx31. We conclude that the endoplasmic reticulum (ER) stress leading to the UPR is the main mechanism of mutant Cx31-associated cell death. These results indicate that, in vivo, ER stress may lead to abnormal keratinocyte differentiation and hyperproliferation in EKV patient skin.

Cdc42 and Rac family GTPases regulate mode and speed but not direction of primary fibroblast migration during platelet-derived growth factor-dependent chemotaxis.

Cdc42 and Rac family GTPases are important regulators of morphology, motility, and polarity in a variety of mammalian cell types. However, comprehensive analysis of their roles in the morphological and behavioral aspects of chemotaxis within a single experimental system is still lacking. Here we demonstrate using a direct viewing chemotaxis assay that of all of the Cdc42/Rac1-related GTPases expressed in primary fibroblasts, Cdc42, Rac1, and RhoG are required for efficient migration towards platelet-derived growth factor (PDGF). During migration, Cdc42-, Rac1-, and RhoG-deficient cells show aberrant morphology characterized as cell elongation and cell body rounding, loss of lamellipodia, and formation of thick membrane extensions, respectively. Analysis of individual cell trajectories reveals that cell speed is significantly reduced, as well as persistence, but to a smaller degree, while the directional response to the gradient of PDGF is not affected. Combined knockdown of Cdc42, Rac1, and RhoG results in greater inhibition of cell speed than when each protein is knocked down alone, but the cells are still capable of migrating toward PDGF. We conclude that, Cdc42, Rac1, and RhoG function cooperatively during cell migration and that, while each GTPase is implicated in the control of morphology and cell speed, these and other Cdc42/Rac-related GTPases are not essential for the directional response toward PDGF.

Novel shear flow assay provides evidence for non-linear modulation of cancer invasion.

Cancer is a frequent disease in western countries and there is no effective treatment for metastasis, the main cause of death in cancer patients. The situation can be improved by a better understanding of the cancer invasion process. In order to reveal new aspects of this dynamic process, we developed a novel direct viewing cancer cell invasion assay with shear flow in vitro. This assay comprised of a custom-made flow chamber, specially developed cell labelling, high-resolution wide-field microscopy and image-processing-based quantitation. We applied this assay to metastatic rat sarcoma cells which invaded monolayers of rat endothelial cells. Our findings showed that after adhesion, the sarcoma cells initially invaded significantly faster under flow conditions compared to situations without shear stress. Later, however, the rate of invasion under flow decreased and the sarcoma cells without shear stress achieved significantly higher levels of invasion. Our observations thus revealed the non-linear modulation of a cancer cell invasion process by shear flow, demonstrating that cancer cells can respond to flow by enhancement of invasiveness similarly to white blood cells.

Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell.

T lymphocytes use LFA-1 to migrate into lymph nodes and inflammatory sites. To investigate the mechanisms regulating this migration, we utilize mAbs selective for conformational epitopes as probes for active LFA-1. Expression of the KIM127 epitope, but not the 24 epitope, defines the extended conformation of LFA-1, which has intermediate affinity for ligand ICAM-1. A key finding is that KIM127-positive LFA-1 forms new adhesions at the T lymphocyte leading edge. This LFA-1 links to the cytoskeleton through alpha-actinin-1 and disruption at the level of integrin or actin results in loss of cell spreading and migratory speed due to a failure of attachment at the leading edge. The KIM127 pattern contrasts with high-affinity LFA-1 that expresses both 24 and KIM127 epitopes, is restricted to the mid-cell focal zone and controls ICAM-1 attachment. Identification of distinctive roles for intermediate- and high-affinity LFA-1 in T lymphocyte migration provides a biological function for two active conformations of this integrin for the first time.

Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector.

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.

Inhibition of invasion and induction of apoptosis by selenium in human malignant brain tumour cells in vitro.

Selenium is considered to be one of the most promising micronutrients for cancer prevention and therapy, based on evidence from epidemiological studies, laboratory-based research and clinical trial intervention. There are ample reports of selenium methionine and sodium selenite's ability to induce apoptosis in various cancers in vitro. There are a few reports in the literature on the effects of selenium on established glioma cell lines but none on biopsy-derived short-term brain tumour cultures. In this in vitro study the effects of a range of concentrations (2-10 microg/ml) of sodium selenite were investigated in one low-passage culture of biopsy-derived glioma cells (IPSB-18, an anaplastic astrocytoma, P 18-22) and a normal human brain cell culture (CC2565, P11). Results from 2 viability assays, 3[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulphorodamine B (SRB) consistently showed that the IC50 for selenium in the astrocytoma was approximately 5 microg/ml whilst the normal brain cells were unaffected by selenium in the range of concentrations studied. Time-lapse video microscopy revealed that, while at 4 microg/ml selenium, the time taken to achieve 100% cell death was 17 h, with increasing concentrations of selenium from 6 to 8 microg/ml and finally at 10 microg/ml the IPSB-18 cells rounded up and died much more quickly. The time taken to achieve 100% cell death was 7 h, 7 h and 6 h, respectively, suggesting that the effect was similar at higher concentrations. Flow cytometry indicated that cell death was by apoptosis. RT-PCR results showed downregulation of the gene expression of 6 matrix metalloproteases (MMP2, 9, 14, 15, 16, 24), their inhibitors, TIMPs and epidermal growth factor receptor, in IPSB-18 cells treated with 2, 4 and 8 microg/ml of selenium. Collectively, the data in this study suggests that selenium, not only induces tumour cell-specific apoptosis but also has anti-invasive potential.

Evidence for protein 4.1B acting as a metastasis suppressor.

We compared a non-metastasising sarcoma cell population with three related populations of increasing metastatic potential. The metastatic cells in vitro exhibited a significantly reduced incidence of actin stress fibres but enhanced motility and chemotaxis. We also investigated gene expression underlying progression to a metastatic phenotype by performing a microarray analysis of the four sarcoma populations. We identified a subset of genes with significantly altered expression levels between non-metastasising and metastasising cells in tissue culture and in primary tumours. One such gene, encoding protein 4.1B, is downregulated in the metastatic sarcoma populations. To investigate possible roles of 4.1B in the mechanisms of metastasis, we used RNA interference (RNAi) to reduce its expression in the non-metastatic cells. Cells with reduced 4.1B expression displayed an altered F-actin morphology, with significantly fewer stress fibres. We also found that the 4.1B RNAi cells migrated at twice the speed of the untreated cells. Metastatic cells exogenously expressing 4.1B migrated at half the speed of control metastatic cells and displayed suppressed chemotaxis. Therefore, we propose that the reduction of 4.1B in the metastatic cells promotes the metastatic phenotype as a result of inducing a loss of actin stress fibres and a concomitant increase in cell motility.

Guidance of bidirectional motor complexes by mRNA cargoes through control of dynein number and activity.

During asymmetric cytoplasmic mRNA transport, cis-acting localization signals are widely assumed to tether a specific subset of transcripts to motor complexes that have intrinsic directionality. Here we provide evidence that mRNA transcripts control their sorting by regulating the relative activities of opposing motors on microtubules. We show in Drosophila embryos that all mRNAs undergo bidirectional transport on microtubules and that cis-acting elements produce a range of polarized transcript distributions by regulating the frequency, velocity, and duration of minus-end-directed runs. Increased minus-end motility is dependent on the dosage of RNA elements and the proteins Egalitarian (Egl) and Bicaudal-D (BicD). We show that these proteins, together with the dynein motor, are recruited differentially to different RNA signals. Cytoplasmic transfer experiments reveal that, once assembled, cargo/motor complexes are insensitive to reduced cytoplasmic levels of transport proteins. Thus, the concentration of these proteins is only critical at the onset of transport. This work suggests that the architecture of RNA elements, through Egl and BicD, regulates directional transport by controlling the relative numbers of opposite polarity motors assembled. Our data raise the possibility that recruitment of different numbers of motors and regulatory proteins is a general strategy by which microtubule-based cargoes control their sorting.

Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells.

The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P2 in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis. Using a novel quantitative immunofluorescence technique, we report that plasma membrane PtdIns(4,5)P2 becomes transiently depleted upon activation of exocytosis, and is not detected on the membranes of fusing granules. Depletion is caused by phospholipase C activity, and is mandatory for exocytosis. Although phospholipase C is required for Ca2+ release from internal stores, the majority of the requirement for PtdIns(4,5)P2 hydrolysis occurs downstream of Ca2+ signalling - as shown in permeabilised cells, where the inositol (1,4,5)-trisphosphate-Ca2+ pathway is bypassed. Neither generation of the PtdIns(4,5)P2 metabolite, diacylglycerol (DAG) or simple removal and/or sequestration of PtdIns(4,5)P2 are sufficient for exocytosis to occur. However, treatment of permeabilised cells with DAG induces a small potentiation of exocytosis, indicating that it may be required. We propose that a cycle of PtdIns(4,5)P2 synthesis and breakdown is crucial for exocytosis to occur in mast cells, and may have a more general role in all professional secretory cells.

Cluster analysis of networks generated through homology: automatic identification of important protein communities involved in cancer metastasis.

BACKGROUND: Protein-protein interactions have traditionally been studied on a small scale, using classical biochemical methods to investigate the proteins of interest. More recently large-scale methods, such as two-hybrid screens, have been utilised to survey extensive portions of genomes. Current high-throughput approaches have a relatively high rate of errors, whereas in-depth biochemical studies are too expensive and time-consuming to be practical for extensive studies. As a result, there are gaps in our knowledge of many key biological networks, for which computational approaches are particularly suitable. RESULTS: We constructed networks, or 'interactomes', of putative protein-protein interactions in the rat proteome--the rat being an organism extensively used for cancer studies. This was achieved by integrating experimental protein-protein interaction data from many species and translating this data into the reference frame of the rat. The putative rat protein interactions were given confidence scores based on their homology to proteins that have been experimentally observed to interact. The confidence score was furthermore weighted according to the extent of the experimental evidence, giving a higher weight to more frequently observed interactions. The scoring function was subsequently validated and networks constructed around key proteins, identified as being highly up- or down-regulated in rat cell lines of high metastatic potential. Using clustering methods on the networks, we have identified key protein communities involved in cancer metastasis. CONCLUSION: The protein network generation and subsequent network analysis used here, were shown to be useful for highlighting key proteins involved in metastasis. This approach, in conjunction with microarray expression data, can be extended to other species, thereby suggesting possible pathways around proteins of interest.