(F-240) Investigating the Effects of Collagen Receptor DDR2 on Cell Mechanosensing

Introduction: Cancer progression and metastasis are caused by changes in extracellular matrix (ECM) components and mechanical properties. The mechanosensitivity of cancer cells enables them to sense the alterations in ECM stiffness and adapt their cellular behavior, including cell stiffness, migration, and growth (1). The cell-ECM adhesions are a vital component in sensing ECM stiffness. Discoidin domain receptor 2 (DDR2) is a tyrosine receptor kinase that is activated by binding to collagen and promotes cell adhesion to collagen in the ECM (2)(3). DDR2 is essential to the normal function of organs in the human body, but disruptive expression can cause carcinogenesis, resulting in the presence of DDR2 in many cancer cells (4). We hypothesize that DDR2 plays a role in cell mechanosensing and aim to analyze the effects of down-regulating DDR2 expression on the ability of cells to adapt to substrate stiffness changes. 

Materials and Methods: (a) Cell line: SH-SY5Y neuroblastoma cells were transfected with a Tet-shDDR2 to construct a cell line with inducible DDR2 downregulation. Doxycycline was used to induce DDR2 downregulation in cells. Treated cells, shiDDR2 (-) are kept in culture with doxycycline and untreated cells, shiDDR2 (+) are grown in the absence of doxycycline. (b) Traction Force Microscopy (TFM): TFM was utilized to determine the traction force and maximum traction stress exerted by cells.  The cells were plated on polyacrylamide gels (PAA) of the following stiffnesses to mimic varying interstitial stiffnesses: 0.05, 0.8, 2, 7.5, 13, and 20 kPa. Fluorescent beads of 0.1 um in diameter were embedded within a 5 um layer below the surfaces of PAA gels. All gels were coated with collagen (0.1 mg/mL). Phase contrast images of the cells and fluorescent images of the beads underneath were taken of single cells attached to the gels. To detach the cells from the gel surface, 1% trypsin was added. Phase contrast and fluorescent images were retaken in the same locations. A MATLAB script was then applied to measure the displacements of the fluorescent beads before and after trypsinization. The traction stress was computed from the bead displacements using the finite element analysis software, ANSYS. Cell area and aspect ratio are determined using freehand tool in ImageJ.

Results, Conclusions, and Discussions:

Cells with undisturbed DDR2 levels demonstrated greater cell area as substrate stiffness increased, while shiDDR2 (-) cells showed an inability to react to changes in substrate stiffness, as cell area did not change with varying gel stiffness. shiDDR2 (-) cells demonstrated a higher total force and maximum traction stress when compared to shiDDR2 (+) cells. 

While the control cells demonstrated the ability to increase cell area as gel stiffness increased, shiDDR2 (-) cells were unable to change their area in relation to gel stiffness. This indicates that the downregulation of DDR2 decreased the cells’ mechanosensitivity, resulting in a diminished ability to adapt to their substrate stiffness. Cells rely on traction force to sense substrate stiffness.  However, our data shows the shiDDR2 (-) cell line exerts higher traction force and maximum traction stress on substrates when compared to shiDDR2 (-) cells. This phenomenon may be due to the overexpression of integrins to aid in cell adhesion upon the down-regulation of DDR2. Future directions include testing on fibronectin-coated gels of all stiffnesses to analyze how different components in the cells’ environment affect their mechanosensing properties in comparison to collagen. This will aid in further understanding the full impact that DDR2 has on cancer cells throughout the human body.



Acknowledgments: This work was supported by NSFREU grant EEC2150076.

References: (1) Terra M. Kruger, Kendra J. Bell, Thiranjeewa I. Lansakara, Alexei V. Tivanski, Jonathan A. Doorn, and Lewis L. Stevens ACS Chemical Neuroscience 2019 10 (3), 1284-1293.(2) Bayer, S. V. H. (2019). The role of tumor stromal discoidin domain receptor 2 (DDR2) in breast cancer metastasis (Order No. 13809588). (3) Xu H, Bihan D, Chang F, Huang PH, Farndale RW, Leitinger B (2012) Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation. PLoS ONE 7(12): e52209. (4) Gao Y, Zhou J, Li J. Discoidin domain receptors orchestrate cancer progression: A focus on cancer therapies. Cancer Sci. 2021 Mar;112(3):962-969.