Differentiation of Col I and Col III Isoforms in Stromal Models of Ovarian Cancer by Analysis of Second Harmonic Generation Polarization and Emission Directionality

AbstractA profound remodeling of the extracellular matrix occurs in many epithelial cancers. In ovarian cancer, the minor collagen isoform of Col III becomes upregulated in invasive disease. Here we use second harmonic generation (SHG) imaging microscopy to probe structural differences in fibrillar models of the ovarian stroma comprised of mixtures of Col I and III. The SHG intensity and forward-backward ratios decrease with increasing Col III content, consistent with decreased phasematching due to more randomized structures. We further probe the net collagen α-helix pitch angle within the gel mixtures using what is believed to be a new pixel-based polarization-resolved approach that combines and extends previous analyses. The extracted pitch angles are consistent with those of peptide models and the method has sufficient sensitivity to differentiate Col I from the Col I/Col III mixtures. We further developed the pixel-based approach to extract the SHG signal polarization anisotropy from the same polarization-resolved image matrix. Using this approach, we found that increased Col III results in decreased alignment of the dipole moments within the focal volume. Collectively, the SHG measurements and analysis all indicate that incorporation of Col III results in decreased organization across several levels of collagen organization. Furthermore, the findings suggest that the collagen isoforms comingle within the same fibrils, in good agreement with ultrastructural data. The pixel-based polarization analyses (both excitation and emission) afford determination of structural properties without the previous requirement of having well-aligned fibers, and the approaches should be generally applicable in tissue

Complement C1q Activates Tumor Suppressor WWOX to Induce Apoptosis in Prostate Cancer Cells

BACKGROUND:Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells. METHODOLOGY/PRINCIPAL FINDINGS:DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. CONCLUSIONS/SIGNIFICANCE:We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation

Visualization 2.mp4

The rendering process of Fig. 4(a)-4(d

Visualization 3.mp4

The rendering process of Fig. 5(a), 5(b), 5(d), and 5(f

Visualization 1.mp4

The resulting 4D image for a 10-µm fluorescent bea

Surface-Enhanced Raman Scattering Microchip Fabricated by Femtosecond Laser

We report a surface-enhanced Raman scattering (SERS) microchip that is capable of measuring SERS signals of liquid samples with high sensitivity. The microdevice is an integration of a silicon-based SERS substrate, a multimode optical fiber (MMF), and a microchannel embedded in the photosensitive glass fabricated by the femtosecond laser followed by thermal treatment, wet etching, and annealing. The performance of the SERS microchip is evaluated by measuring rhodamine 6G using a 632.8nm He-Ne laser at 4.3mW excitation power, which reveals that the detection limit is lower than 10 -10M at a 1s short accumulation time

Ovarian Cancer Cell Adhesion/Migration Dynamics on Micro-Structured Laminin Gradients Fabricated by Multiphoton Excited Photochemistry

Haptotaxis, i.e., cell migration in response to adhesive gradients, has been previously implicated in cancer metastasis. A better understanding of cell migration dynamics and their regulation could ultimately lead to new drug targets, especially for cancers with poor prognoses, such as ovarian cancer. Haptotaxis has not been well-studied due to the lack of biomimetic, biocompatible models, where, for example, microcontact printing and microfluidics approaches are primarily limited to 2D surfaces and cannot produce the 3D submicron features to which cells respond. Here we used multiphoton excited (MPE) phototochemistry to fabricate nano/microstructured gradients of laminin (LN) as 2.5D models of the ovarian basal lamina to study the haptotaxis dynamics of a series of ovarian cancer cells. Using these models, we found that increased LN concentration increased migration speed and also alignment of the overall cell morphology and their cytoskeleton along the linear axis of the gradients. Both these metrics were enhanced on LN compared to BSA gradients of the same design, demonstrating the importance of both topographic and ECM cues on the adhesion/migration dynamics. Using two different gradient designs, we addressed the question of the roles of local concentration and slope and found that the specific haptotactic response depends on the cell phenotype and not simply the gradient design. Moreover, small changes in concentration strongly affected the migration properties. This work is a necessary step in studying haptotaxis in more complete 3D models of the tumor microenvironment for ovarian and other cancers

Ovarian Cancer Cell Adhesion/Migration Dynamics on Micro-Structured Laminin Gradients Fabricated by Multiphoton Excited Photochemistry

Haptotaxis, i.e., cell migration in response to adhesive gradients, has been previously implicated in cancer metastasis. A better understanding of cell migration dynamics and their regulation could ultimately lead to new drug targets, especially for cancers with poor prognoses, such as ovarian cancer. Haptotaxis has not been well-studied due to the lack of biomimetic, biocompatible models, where, for example, microcontact printing and microfluidics approaches are primarily limited to 2D surfaces and cannot produce the 3D submicron features to which cells respond. Here we used multiphoton excited (MPE) phototochemistry to fabricate nano/microstructured gradients of laminin (LN) as 2.5D models of the ovarian basal lamina to study the haptotaxis dynamics of a series of ovarian cancer cells. Using these models, we found that increased LN concentration increased migration speed and also alignment of the overall cell morphology and their cytoskeleton along the linear axis of the gradients. Both these metrics were enhanced on LN compared to BSA gradients of the same design, demonstrating the importance of both topographic and ECM cues on the adhesion/migration dynamics. Using two different gradient designs, we addressed the question of the roles of local concentration and slope and found that the specific haptotactic response depends on the cell phenotype and not simply the gradient design. Moreover, small changes in concentration strongly affected the migration properties. This work is a necessary step in studying haptotaxis in more complete 3D models of the tumor microenvironment for ovarian and other cancers

Tuning the Electronic Structure of Graphite Oxide through Ammonia Treatment for Photocatalytic Generation of H₂ and O₂ from Water Splitting

Graphite oxide (GO) synthesized from the oxidation of graphite powders exhibits p-type conductivity and is active in photocatalytic H₂ evolution from water decomposition. The p-type conductivity hinders hole transfer for water oxidation and suppresses O₂ evolution. Treating GO with NH₃ gas at room temperature tunes the electronic structure by introducing amino and amide groups to its surface. The ammonia-modified GO (NGO) exhibits n-type conductivity in photoelectrochemical analysis and has a narrower optical band gap than GO. Electrochemical analysis attributes the band gap reduction to a negative shift of the valence band. An NGO-film electrode exhibits a substantially higher incident photo-to-current efficiency in the visible light region than a GO electrode. Photoluminescence analyses demonstrate the above-edge emission characteristic of GO and NGO. NH₃ treatment enhances the emission by removing nonirradiative epoxy and carboxyl sites on the GO. In half-reaction tests of water decomposition, NGO effectively catalyzes O₂ evolution in an aqueous AgNO₃ solution under mercury-lamp irradiation, whereas GO is inactive. NGO also effectively catalyzes H₂ evolution in an aqueous methanol solution but shows less activity than GO. Under illumination with visible light (λ > 420 nm), NGO simultaneously catalyzes H₂ and O₂ evolutions, but with a H₂/O₂ molar ratio below 2. The n-type conductivity of NGO may hinder electron transfer and form peroxide species instead of H₂ molecules. This study demonstrates that the functionality engineering of GO is a promising technique to synthesize an industrially scalable photocatalyst for overall water splitting