Metabolic flux increases glycoprotein sialylation: implications for cell adhesion and cancer metastasis.

This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ≈ 31% showed no change in sialylation, ≈ 29% exhibited a modest increase, whereas ≈ 40% experienced an increase of greater than twofold. Increased sialylation of specific glycoproteins resulted in changes to the adhesive properties of SW1990 pancreatic cancer cells (e.g. increased CD44-mediated adhesion to selectins under physiological flow and enhanced integrin-mediated cell mobility on collagen and fibronectin). These results indicate that cancer cells can become more aggressively malignant by controlling the sialylation of proteins implicated in metastatic transformation via metabolic flux.

Mucin 16 is a functional selectin ligand on pancreatic cancer cells.

Selectins promote metastasis by mediating specific interactions between selectin ligands on tumor cells and selectin-expressing host cells in the microvasculature. Using affinity chromatography in conjunction with tandem mass spectrometry and bioinformatics tools, we identified mucin 16 (MUC16) as a novel selectin ligand expressed by metastatic pancreatic cancer cells. While up-regulated in many pancreatic cancers, the biological function of sialofucosylated MUC16 has yet to be fully elucidated. To address this, we employed blot rolling and cell-free flow-based adhesion assays using MUC16 immunopurified from pancreatic cancer cells and found that it efficiently binds E- and L- but not P-selectin. The selectin-binding determinants are sialofucosylated structures displayed on O- and N-linked glycans. Silencing MUC16 expression by RNAi markedly reduces pancreatic cancer cell binding to E- and L-selectin under flow. These findings provide a novel integrated perspective on the enhanced metastatic potential associated with MUC16 overexpression and the role of selectins in metastasis.

Divergent roles of CD44 and carcinoembryonic antigen in colon cancer metastasis.

After separating from a primary tumor, metastasizing cells enter the circulatory system and interact with host cells before lodging in secondary organs. Previous studies have implicated the surface glycoproteins CD44 and carcinoembryonic antigen (CEA) in adhesion, migration, and invasion, suggesting that they may influence metastatic progression. To elucidate the role of these multifunctional molecules while avoiding the potential drawbacks of ectopic expression or monoclonal antibody treatments, we silenced the expression of CD44 and/or CEA in LS174T colon carcinoma cells and analyzed their ability to metastasize in 2 independent mouse models. Quantitative PCR revealed that CD44 knockdown increased lung and liver metastasis >10-fold, while metastasis was decreased by >50% following CEA knockdown. These findings were corroborated by in vitro experiments assessing the metastatic potential of LS174T cells. Cell migration was decreased as a result of silencing CEA but was enhanced in CD44-knockdown cells. In addition, CD44 silencing promoted homotypic aggregation of LS147T cells, a phenotype that was reversed by additional CEA knockdown. Finally, CD44-knockdown cells exhibited greater mechanical compliance than control cells, a property that correlates with increased metastatic potential. Collectively, these data indicate that CEA, but not CD44, is a viable target for therapeutics aimed at curbing colon carcinoma metastasis.

Sialofucosylated podocalyxin is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells.

Selectin-mediated interactions in the vasculature promote metastatic spread by facilitating circulating tumor cell binding to selectin-expressing host cells. Therefore, identifying the selectin ligand(s) on tumor cells is critical to the prevention of blood-borne metastasis. A current challenge is to distinguish between structures expressed by circulating tumor cells that can bind selectins in vitro from the functional ligands whose depletion suppresses selectin-dependent binding under flow in vivo. Interestingly, podocalyxin (PODXL), which can bind E- and L-selectin, is upregulated in a number of cancers, including those of the breast, colon, and pancreas. In this work, we show that metastatic pancreatic cancer cells overexpress PODXL compared with nonmalignant pancreatic epithelial cells. We further demonstrate via tissue microarray that 69% of pancreatic ductal adenocarcinomas stain positive for PODXL. In cases of focal expression, positive staining is restricted to the invasive front of primary tumors. By combining immunoblot, immunodepletion, short-hairpin RNA-mediated gene silencing, and flow-based adhesion assays, we evaluated the functional role of sialofucosylated PODXL in selectin-mediated adhesion under flow. Our data indicate that sialofucosylated PODXL is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells, as specific depletion of this molecule from the cell surface significantly interferes with selectin-dependent interactions. Cumulatively, these data support a correlation between sialofucosylated PODXL expression and enhanced binding to selectins by metastatic pancreatic cancer cells and offer additional perspective on the upregulation of PODXL in aggressive cancers.

Quantitative molecular profiling of biomarkers for pancreatic cancer with functionalized quantum dots.

Applications in nanomedicine, such as diagnostics and targeted therapeutics, rely on the detection and targeting of membrane biomarkers. In this article we demonstrate absolute quantitative profiling, spatial mapping, and multiplexing of cancer biomarkers using functionalized quantum dots (QDs). We demonstrate highly selective targeting molecular markers for pancreatic cancer with extremely low levels of nonspecific binding. We confirm that we have saturated all biomarkers on the cell surface, and, in conjunction with control experiments, extract absolute quantitative values for the biomarker density in terms of the number of molecules per square micron on the cell surface. We show that we can obtain quantitative spatial information of biomarker distribution on a single cell, important because tumors' cell populations are inherently heterogeneous. We validate our quantitative measurements (number of molecules per square micron) using flow cytometry and demonstrate multiplexed quantitative profiling using color-coded QDs. FROM THE CLINICAL EDITOR: This paper demonstrates a nice example for quantum dot-based molecular targeting of pancreatic cancer cells for advanced high sensitivity diagnostics and potential future selective therapeutic purposes.

Establishing quasi-steady state operations of microphysiological systems (MPS) using tissue-specific metabolic dependencies.

Microphysiological systems (MPS), consisting of tissue constructs, biomaterials, and culture media, aim to recapitulate relevant organ functions in vitro. MPS components are housed in fluidic hardware with operational protocols, such as periodic complete media replacement. Such batch-like operations provide relevant nutrients and remove waste products but also reset cell-secreted mediators (e.g. cytokines, hormones) and potentially limit exposure to drugs (and metabolites). While each component plays an essential role for tissue functionality, MPS-specific nutrient needs are not yet well-characterized nor utilized to operate MPSs at more physiologically-relevant conditions. MPS-specific nutrient needs for gut (immortalized cancer cells), liver (human primary hepatocytes) and cardiac (iPSC-derived cardiomyocytes) MPSs were experimentally quantified. In a long-term study of the gut MPS (10 days), this knowledge was used to design operational protocols to maintain glucose and lactate at desired levels. This quasi-steady state operation was experimentally validated by monitoring glucose and lactate as well as MPS functionality. In a theoretical study, nutrient needs of an integrated multi-MPS platform (gut, liver, cardiac MPSs) were computationally simulated to identify long-term quasi-steady state operations. This integrative experimental and computational approach demonstrates the utilization of quantitative multi-scale characterization of MPSs and incorporating MPS-specific information to establish more physiologically-relevant experimental operations.

Dieting practices, weight perceptions, and body composition: a comparison of normal weight, overweight, and obese college females.

BACKGROUND: Of concern to health educators is the suggestion that college females practice diet and health behaviors that contradict the 2005 dietary guidelines for Americans. In this regard, there remain gaps in the research related to dieting among college females. Namely, do normal weight individuals diet differently from those who are overweight or obese, and are there dieting practices used by females that can be adapted to promote a healthy body weight? Since it is well recognized that females diet, this study seeks to determine the dieting practices used among normal, overweight, and obese college females (do they diet differently) and identify dieting practices that could be pursued to help these females more appropriately achieve and maintain a healthy body weight. METHODS: A total of 185 female college students aged 18 to 24 years participated in this study. Height, weight, waist and hip circumferences, and skinfold thickness were measured to assess body composition. Surveys included a dieting practices questionnaire and a 30-day physical activity recall. Participants were classified according to body mass index (BMI) as normal weight (n = 113), overweight (n = 35), or obese (n = 21). Data were analyzed using JMP IN(R) software. Descriptive statistics included means, standard deviations, and frequency. Subsequent data analysis involved Pearson X2 and one-way analysis of variance with comparison for all pairs that were significantly different using Tukey-Kramer honestly significant difference test. RESULTS: Outcomes of this study indicate the majority of participants (83%) used dieting for weight loss and believed they would be 2% to 6% greater than current weight if they did not diet; normal weight, overweight, and obese groups perceived attractive weight to be 94%, 85%, and 74%, respectively, of current weight; 80% of participants reported using physical activity to control weight, although only 19% exercised at a level that would promote weight loss; only two of 15 dieting behaviors assessed differed in terms of prevalence of use among groups, which were consciously eating less than you want (44% normal weight, 57% overweight, 81% obese) and using artificial sweeteners (31% normal weight and overweight, 5% obese); and the most prevalent explicit maladaptive weight loss behavior was smoking cigarettes (used by 9% of participants) and most unhealthy was skipping breakfast (32%). CONCLUSION: Collectively, results indicate female college students, regardless of weight status, would benefit from open discussions with health educators regarding healthy and effective dieting practices to achieve/maintain a healthy body weight. The results are subject to replication among high school, middle-aged, and older females.

Functional interplay between the cell cycle and cell phenotypes.

Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes. By perturbing the cell cycle through inhibition of cell-cycle regulators or changing nuclear morphology by depletion of structural proteins, our results reveal that cell cycle regulators and structural proteins can significantly interfere with each other's prima facie functions. This study introduces a high-throughput method to simultaneously measure the cell cycle and phenotypes at single-cell resolution, which reveals a complex functional interplay between the cell cycle and cell phenotypes.

A physical sciences network characterization of non-tumorigenic and metastatic cells.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

Chemotaxis of cell populations through confined spaces at single-cell resolution.

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications.