877 Integration of magnetic tweezers and traction force microscopy for exploring the mechanobiology of keratinocyte cell-cell and cell-matrix anchoring junctions

Abstract

Elevated expression of osteopontin (OPN), a matricellular, adhesive glycoprotein, is associated with numerous cancer types. We have reported that ultraviolet B (UVB)-induced OPN is a critical driver for cutaneous squamous cell carcinoma (cSCC) development in the photocarcinogenesis model. In human skin cancer, UVB exposure is the major risk factor on the development of nonmelanoma skin cancer. Immunohistochemical analyses indicated that OPN protein is markedly increased in actinic keratoses, cSCC, and differentiated basal cell carcinoma (BCC), but not in solid basal cell epitheliomas when compared with no sun-exposed skin. This suggests OPN may play an important role in the maintenance of the pre-malignant and malignant keratinocytes in cSCC and differentiated BCC. Whether OPN splice variants, shown to have distinct functions, are expressed in normal and nonmelanoma skin cancer is not known. This study assessed whether 1) OPN splice variants differ in normal skin, cSCC, and BCC, and 2) their expression is altered in human adult keratinocytes (HaCaT) treated in vitro with UVB or 1, 25-dihydroxyvitamin D 3 (calcitriol), by real-time qPCR and Western blot analyses. Normal skin expressed minimal OPN-a, OPN-b and OPN-c mRNAs. In contrast, the transcripts of OPN variants were significantly (p<0.001) elevated in cSCC and BCC. In cSCC, each OPN isoform expression was at similar levels. In BCC, OPN-a was significantly (p<0.05) higher than OPN-c. Consistent with normal skin, HaCaT keratinocytes also expressed low levels of OPN-a and OPNb, however, OPN-c was not detected. UVB did not induce OPN expression, whereas calcitriol stimulated OPN-a and OPN-b only. Collectively, these results indicate elevated expression of the three OPN isoforms in nonmelanoma skin cancer compared to normal skin. Additionally, adult keratinocytes expressed only basal and induced level of OPN-a and OPN-b, suggesting that other cell types may be responsible for OPN-c expression.

Integration of Magnetic Tweezers and Traction Force Microscopy for the Exploration of Matrix Rheology and Keratinocyte Mechanobiology: Model Force- and Displacement-Controlled Experiments

“…The instrumentation and control schemes employed to operate our MT device are extensively detailed in a companion paper 27 an increase in hydroxyl moieties on the glass surface 29,30 . Next, the glass was silanized by covering with 97% 3-aminopropyltriethoxysilane (3-APTMS; Sigma Aldrich, St. Louis, MO) for 5 minutes, followed by multiple rinses with deionized (DI) water.…”

“…Real-time videos depicting continuum representations of the substrate displacement field, This finding is somewhat counterintuitive, based on the fact that for a constant nominal calibrated magnetic flux density setpoint, the magnetic actuation force, "# * ( ), increases as the superparamagnetic bead more closely approaches the needle tip 27 . However, based on the geometry of our MT setup, the vectoral component of "# * ( ) in the -plane of the collagen substrate remains roughly constant due to increases in the relative angle between ( ) and the -plane as the bead approaches the needle tip (see Fig.…”

“…Towards this end, we set out to demonstrate how integration of MT and DTM/TFM can be used to explore the mechanobiology of the human epidermis in the context of skin fragility disorders. As the design, operation, and calibration of our MT device is comprehensively reviewed in a companion paper 27 , herein we describe two model approaches to directly investigate force transmission in reconstituted collagen matrices and living human epidermal keratinocyte tissue constructs in vitro: force-control and displacement-control mode MT-DTM/TFM experiments. Explicit details are provided on the relevant physical approximations and computational methods used to analyze the large image-based data sets generated from each of these two types of MT-DTM/TFM experiment.…”