(B-69) Quantification of CDH3 mediated 3D cell forces using bead tracking technology

Introduction:Collective cell migration is an important biological phenomenon seen in morphogenesis, wound healing, and cancer metastasis. In clusters, cells exist as heterogeneous populations, with different cadherins to adhere to one another. In breast cancer metastasis, single cell sequencing analysis of migrating, primary tumor clusters demonstrate there exists unique cell clusters containing the classical cadherins: P-cadherin (CDH3), E-cadherin (CDH1), and N-cadherin (CDH2) expression [1]. While it is known that cadherin complexes play a role in mediating cell traction forces, it is unclear how differences in cadherins affect the magnitude of forces. The objective of this study is to measure, quantify, and compare the 3D forces exerted by epithelial cells whereby we selectively knockout different classical cadherins. Specifically, in this study, we focus on investigating the contributions of P-cadherin mediated forces.
Materials and Methods:

First, we generated novel, stable knockout cell lines using Madin-Darby canine kidney (MDCK) epithelial cells where we selectively knocked-out CDH3 (validated via immunostaining and western blot; Fig 1A and B). We compare all of our findings to MDCK wild-type cells (CDH3+/+ and CDH1+/+) and MDCK CDH1-/-. Next, we generated a custom-MATLAB code to quantify forces exerted on the matrix using fluorescent microbead tracking. MDCK wild-type, CDH3-/-, and CDH1-/- cells were encapsulated in separate 3D collagen 1 gels (1.5mg/mL) along with 5% w/v fluorescent microbeads. After overnight adhesion at 37°C, we performed live-cell imaging with heated stage and CO2/O2 control (Nikon TIE2; images were taken every 10 minutes for 2 hours). Afterwards, images were cropped to analyze 1 cell at a time using ImageJ, converted to tiff files, and analyzed through our custom MATLAB code, where we tracked movement of each bead in each time frame for the entire imaging period. Bead movement was converted into a force using the gel stiffness, via Hooke’s Law, and the total force magnitude and net force were computed for each representative cell.

Results, Conclusions, and Discussions:

In the absence of CDH3 there is an approximate 25%-decrease fold change in the amount of 3D cell forces generated compared to MDCK wild-type cells, and without CDH1 there is 85%-decrease fold change, compared to MDCK wild-type cells (Fig. 1C). These findings suggest CDH1 has a larger role in contributing to forces exerted on the surrounding matrix compared to CDH3. Our results are supported by previous 2D studies that have correlated increased CDH1 expression to increased pulling force at cell-matrix focal adhesions [2]. Another study shows recruitment of CDH1 through magnetic beads with mechanical stimulation by tweezers [3]. In future studies we will form 3D acini using MDCK WT, CDH3-/-, and CDH1-/- cell lines to understand if and how forces exerted on the surrounding matrix are influenced by cellular configuration of the MDCK cells.

References:

[1] Hwang PY, Mathur J, Cao Y, Almeida J, Ye J, Morikis V, Cornish D, Clarke M, Stewart SA, Pathak A, and Longmore GD. (2023). A Cdh3-β-catenin-laminin signaling axis in a subset of breast tumor cells control leader cell polarization and directional collective migration. Developmental Cell. 58: 34-50.

[2] Jasaitis A, Estevez M, Heysch J, Ladoux B, and Dufour S. (2012). E-cadherin-dependent stimulation of traction force at focal adhesions via the Src and PI3K signaling pathways. Biophysical Journal. 103(2): 175-184.

[3] Labernadie A, Takuya K, Brugues A, Serra-Picamal X, Derzsi S, Arwert E, Weston A, Gonzalez-Tarrago V, Elosegui-Artola A, Albertazzie L, Alcaraz J, Roc-Cusachs, Sahai E, and Trepat X. (2017). A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nature Cell Biology. 19(3): 224-237.