Browsing by Author "Thompson, David"

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    Probing cellular mechano-sensitivity using biomembrane-mimicking cell substrates of adjustable stiffness
    (2015-12) Lin, Yu-Hung; Naumann, Christoph A.; Das, Chittaranjan; Thompson, David; Long, Eric C.
    It is increasingly recognized that mechanical properties of substrates play a pivotal role in the regulation of cellular fate and function. However, the underlying mechanisms of cellular mechanosensing still remain a topic of open debate. Traditionally, advancements in this field have been made using polymeric substrates of adjustable stiffness with immobilized linkers. While such substrates are well suited to examine cell adhesion and migration in an extracellular matrix environment, they are limited in their ability to replicate the rich dynamics found at cell-cell interfaces. To address this challenge, we recently introduced a linker-functionalized polymer-tethered multi-bilayer stack, in which substrate stiffness can be altered by the degree of bilayer stacking, thus allowing the analysis of cellular mechanosensitivity. Here, we apply this novel biomembrane-mimicking cell substrate design to explore the mechanosensitivity of C2C12 myoblasts in the presence of cell-cell-mimicking N-cadherin linkers. Experiments are presented, which demonstrate a relationship between the degree of bilayer stacking and mechanoresponse of plated cells, such as morphology, cytoskeletal organization, cellular traction forces, and migration speed. Furthermore, we illustrate the dynamic assembly of bilayer-bound N-cadherin linkers underneath cellular adherens junctions. In addition, properties of individual and clustered N-cadherins are examined in the polymer-tethered bilayer system in the absence of plated cells. Alternatively, substrate stiffness can be adjusted by the concentration of lipopolymers in a single polymer-tethered lipid bilayer. On the basis of this alternative cell substrate concept, we also discuss recent results on a linker-functionalized single polymer-tethered bilayer substrate with a lateral gradient in lipopolymer concentration (substrate viscoelasticity). Specifically, we show that the lipopolymer gradient has a notable impact on spreading, cytoskeletal organization, and motility of 3T3 fibroblasts. Two cases are discussed: 1. polymer-tethered bilayers with a sharp boundary between low and high lipopolymer concentration regions and 2. polymer-tethered bilayers with a gradual gradient in lipopolymer concentration.
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    Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma
    (Impact Journals, 2022-09-06) Aayush, Aayush; Darji, Saloni; Dhawan, Deepika; Enstrom, Alexander; Broman, Meaghan M.; Idrees, Muhammad T.; Kaimakliotis, Hristos; Ratliff, Timothy; Knapp, Deborah; Thompson, David; Pathology and Laboratory Medicine, School of Medicine
    Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.