Poster U17 - Aligned matrix architecture promotes directional protrusions through P-cadherin mediated matrix deformations in beginning stages of tubulogenesis

Introduction: Collective cell migration, where cells migrate as a coordinated cluster, regulates many biological phenomena such as morphogenesis, wound healing, and cancer metastasis. Our prior published studies revealed P-cadherin (CDH3) regulates collective cell migration in breast cancer (Hwang et al., 2019, 2023). We also demonstrated that matrix architecture promotes directional collective migration: tumor cell clusters extend protrusions in the direction of aligned parallel collagen fibers, suggesting the aligned collagen fibers delineates a path for migration (Lichtenberg et al., 2024). However, the mechanisms by which cells sense the aligned collagen fibers is not well understood. It has been established that forces from collectively migrating cells coupled with tension on their matrix is necessary for migration to occur (Gjorevski et al., 2015). The objective of this study is to elucidate whether CDH3 effects directional epithelial cell cluster response to aligned collagen fibers, and whether cell-matrix forces govern this phenomenon. To accomplish our objective, we measured matrix deformation of wild-type (WT) and P-cadherin knockout (CDH3-/-) clusters cultured in aligned and randomly oriented ECM using a microfluidic platform. We hypothesize clusters in aligned devices will exert increased deformations on their ECM, but in clusters where CDH3 is knocked out there will be no difference in ECM deformation upon mechanical or chemical stimulation. These findings can contribute significantly to the field of development, specifically tissues generated in epithelial morphogenesis.

Materials and

Methods: In our study, we used an established model of epithelial morphogenesis: luminal clusters, also called acini, are created using MDCK cells in a Matrigel assay. After establishing clusters, they are encapsulated in 3D collagen 1 gels (2mg/mL) with 1% w/v fluorescent microbeads and loaded into either microfluidic devices (random or aligned) or 8-well chamber slides (Fig. 1A). Clusters are cultured for 24 hours at 37°C and tubulogenesis is induced by delivering hepatocyte growth factor (HGF) (Leroy & Mostov, 2007). Live-cell imaging is performed so we can capture 3D frames of bead and cell movement over time. To quantify ECM deformation, we developed an assay with a complementary MATLAB code, CellXMD, to measure ECM deformation rates by the cells. Images are cropped to analyzed 1 cluster at a time using ImageJ, converted to tiff files, and analyzed using CellXMD MATLAB code, where movement of each bead is tracked in each time frame for the entire imaging period (Fig. 1B).

Results, Conclusions, and Discussions: First, we cultured MDCK clusters in chamber slides and treated with HGF to induce the onset of protrusions, in the beginning stages of tubulogenesis. We measured larger matrix deformations and more protrusions with HGF treatment compared to no treatment controls (Fig. 2A,B,D). We also cultured MDCK CDH3-/- clusters in chamber slides and treated with HGF; in CDH3-/- clusters, we did not measure significant differences in phenotype or matrix deformation after HGF treatment (Fig. 2A,C). These qualitative observations support our hypothesis that matrix deformation is regulated by CDH3 and cell generated protrusions.
When the MDCK cell clusters were loaded into microfluidic devices that generate aligned and randomly oriented collagen matrices (Lichtenberg et al., under review), we also measured higher matrix deformation after treatment with HGF of MDCK WT cell clusters (Fig. 3A,B). In aligned devices, MDCK WT clusters sent out protrusions in the direction of the matrix fibers (Fig. 3A,D), and each protrusion exerted higher matrix deformation (Fig. 3B) compared to randomly oriented collagen fibers (Fig. 3A,B,D). MDCK CDH3-/- clusters showed no significance in ECM deformation with or without HGF treatment in either device (Fig. 3A,C). Together our results demonstrate matrix alignment can promote directional cell protrusions that could direct tubulogenesis. Further, we demonstrate that directional protrusions along aligned collagen, coincide with higher matrix deformations compared to radially protrusive clusters in random fiber matrices, and the ability for cell clusters to successfully generate these protrusions is regulated by CDH3 (Hwang et al., 2019, 2023). These results further validate the significance of matrix architecture in influencing cell-matrix interactions. Future directions include the investigation of other extracellular mechanical cues of migration such as stiffness or fluid flow and understanding modes of migration of these epithelial luminal cell clusters.

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