Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer

Development and Characterization of Nanoscale Gel-Core Liposomes Using a Short Self-Assembled Peptide Hydrogel: Implications for Drug Delivery

Targeted drug delivery systems, delivering drugs to specific locations, are an emerging research interest spanning the fields of nanotechnology, bionanotechnology, and precision medicine. Topical or transdermal delivery has many advantages over traditional injection and oral delivery. Not only can it reduce the risk of systemic exposure and overdose but also it is better tolerated by certain patients. However, skin penetration remains a challenge given the protection conferred by the outermost skin layer (stratum corneum), preventing foreign materials, such as pathogenic substances, from infiltrating the body. A promising alternative to overcome the stratum corneum delivery challenge combines the self-assembling properties of nanoscale systems, like liposomes, with peptide hydrogelators, creating an effective topical drug delivery system that couldcross the protective skin barrier. Presented herein is the successful production of gel-core liposomes using two short self-assembled peptide hydrogels. The subsequent composite gel-core liposomes were extensively characterized using a range of techniques including microscopy and SANS

A step further toward glyphosate-induced epidermal cell death: Involvement of mitochondrial and oxidative mechanisms.

International audienceA deregulation of programmed cell death mechanisms in human epidermis leads to skin pathologies. We previously showed that glyphosate, an extensively used herbicide, provoked cytotoxic effects on cultured human keratinocytes, affecting their antioxidant capacities and impairing morphological and functional cell characteristics. The aim of the present study, carried out on the human epidermal cell line HaCaT, was to examine the part of apoptosis plays in the cytotoxic effects of glyphosate and the intracellular mechanisms involved in the apoptotic events. We have conducted different incubation periods to reveal the specific events in glyphosate-induced cell death. We observed an increase in the number of early apoptotic cells at a low cytotoxicity level (15%), and then, a decrease, in favor of late apoptotic and necrotic cell rates for more severe cytotoxicity conditions. At the same time, we showed that the glyphosate-induced mitochondrial membrane potential disruption could be a cause of apoptosis in keratinocyte cultures

Cell imaging connected with protein mapping to investigate glyphosate induced toxicity on human keratinocytes

During the skin ageing which is accelerated by environmental factors, the generated reactive oxygen species make unstable the "proliferation/differentiation/apoptosis" coupling of epidermal cells. A biochemical study, previously realized on human keratinocyte cultures treated by a pesticide, glyphosate, allowed the validation of an in vitro model of skin ageing. In this work, we propose to study the loss of HaCaT cell integrity appearing after this chemical induced-oxidative stress. An original approach, combining a micro- to nanoscale cell characterization and analysis of fluctuating protein expression, is proposed: a) the exploration of cell growth and morphology through fluorescence and AFM imaging, b) a functional investigation using force spectroscopy for detection of oxidative stress biomarkers in membranes and c) the establishment of proteomic comparative profiles between safe and glyphosate-treated cells. Our results revealed up to now that a glyphosate treatment induced cell impairment in an acute dose-dependency. Indeed, we observed a breakage of plasma membrane and a release of cytosol, microtubules disorganization and fragmentation, and finally aggregation of chromatin. These observations characterized apoptotic key events. Our first force spectroscopy results revealed that a glyphosate coated tip interacted specifically with keratinocyte membrane structures. This nanobiotechnological tool will allow cell mapping establishment and screening original anti-oxidant molecules. The proteomic profiling would highlight cell oxidative stress cascade and identify corresponding major biomarkers in our skin ageing model. Original molecules with cytoprotective potentials will be tested and compared to well-known antioxidants, in order to correct pesticide induced cutaneous disorders. These data all together will contribute to better encircle the mechanisms of chemical and environmental hazards acting on the human skin

Technological innovation around protein and cell biochip for diagnosis: a translational research from nanoworld to patient

International audienceA great challenge in biosensors and diagnosis devices relies on the way to reconstitute relevant biological mechanisms on surface of the biochips and which analytical tools are convenient to provide accurate and rapid information on the structures and function of molecules attached to this surface. A better control in the realization of biochips can be obtained in combining different complementary approaches while always keeping in mind the biological key point. Researches in CLIPP are focused towards this objective. Conception, realization and characterization of protein and cell chips are presented. We detail different strategies of materials engineering1,2,3, chemical functionalizations and biomolecular graftings4, molecular and cellular characterization in physiological conditions5,6, which lead to the optimization of “biorecognitions events” at the surface of the chip. We present herein an original interdisciplinary approach, consisting to carry out in parallel a micro-scale analysis (SPR, fluorescence microscopy) and nano-scale characterizations (AFM, XPS, TOF-SIMS). Concerning protein interfaces, we demonstrated in particular that the molecular orientation in a protein monolayer can be determined based on the specific fragment ions from the protein in TOF-SIMS spectra7. These developments have also contributed to the establishment of a new biomolecular interaction analysis/mass spectrometry (BIA-MS) combination based on an entire “on-a-chip” procedure8. We report a low-cost approach combining Biacore 2000 analysis with homemade chips and MS and MS/MS identification directly onto the chips without elution step. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the “on-a-chip” processing to complete the BIA-MS approach in the discovery and analysis of protein complexes in biological fluids. Our interest is also focused on cell/surface interaction. The cell biochips we are developing consist either of circulating or adherent cells, that we characterized in terms of cell capture on biofunctionalized surface or growth with substrate dependency respectively. Parameters such as cell spreading, growth, morphology, and topography are particularly investigated and controlled by atomic force microscopy in physiological conditions6. With the aim to increase the throughput of analysis, we are currently working on cell and protein micro-arrays. Our expertise in cell and protein biochip preparation, and competences in micro- to nanoscale characterization in liquid conditions, represents precious assets enabling a relevant clinical proteomic research, thanks to deeply controlled steps of biosensor development and use

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

<div><p>The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.</p></div

Distinct Tpm isoforms differentially impact on the elastic modulus of the cell.

<p>Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from <i>n</i> = 3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01 compared to control cells. ***<i>P</i><0.001, ****<i>P</i><0.0001 compared to control.</p

Impact of the different Tpm isoforms on the organization of the actin cytoskeleton.

<p>(A) Representative immunofluorescence images of the Tpm-overexpressing B35 clones stained with 488-Atto phalloidin to visualise F-actin (green) and DAPI (blue) for the nucleus and the corresponding colored overlays defining F-actin in the cells. (B) A linear feature detection algorithm was employed to determine total F-actin length/per cell. Each clone was plated in 8 replicates, 6 random fields were imaged and a range of 320 to 590 cells were analysed from <i>n</i> = 3 independent experiments. Scale bar, 10 μm. ****<i>P</i><0.0001.</p

Total Tpm protein levels in the Tpm-overexpressing clones.

<p>The total Tpm protein expression levels were determined in each Tpm-overexpressing clone. The expression of each Tpm isoform, evaluated by western blotting, was determined with the use of Tpm recombinant proteins and expressed as μg of Tpm per μg of total cellular protein. The histogram depicts the sum of the most highly enriched Tpm isoforms (Tpm1.6, 1.7, 1.10, 1.11, 1.12, 2.1, 3.1, 4.2). <i>n</i> = 4 independent cell lysates, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001.</p

Effect of blebbistatin on the cell’s elastic properties.

<p>The elastic (Young) modulus for the Tpm1.12, Tpm4.2 and Tpm3.1-overexpressing clones was determined following DMSO or 50 μM of blebbistatin treatment for 30 mins. All data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 23–25 cells for each clone was measured from <i>n</i> = 3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01 compared to control cells.</p