Investigating the Role of P-cadherin in Acini Formation and HGF-Induced Tubulogenesis

Introduction: Collective cell migration (CCM), where cells move as a coordinated and cohesive group, is an essential process to establishing and maintaining biological processes, one such being epithelial morphogenesis (Olson & Nechiporuk, 2018). Cadherins (i.e., P-, E-, and N-cadherin), located along the transmembrane, are principal adhesion proteins responsible for mediating cell-cell contact and stability (Maître & Heisenberg, 2013). Acting as a link between the transmembrane and the actomyosin cytoskeleton, cadherins are essential to transmitting mechanical forces not only to neighboring cells, but also to the extracellular matrix (ECM) in response to internal and external cues (Pannekoek et al., 2019). The mechanosensitive nature of cadherin complexes is vital to regulating directionality, organization, and polarity during CCM (Maître & Heisenberg, 2013; Pannekoek et al., 2019). While epithelial cells co-express both E-cadherin and P-cadherin, most literature studies have focused on understanding E-cadherin function yet work on P-cadherin has not been as well-investigated. In 3D, epithelial cells form into acini, a spherical structure containing a single hollow lumen in the center (Narayanan et al., 2020). Dysregulated lumen formation has been shown to be linked to disease or developmental disorders like cancer and inherited renal diseases (Buckley & St Johnston, 2022). The acini and lumen development process are vital to tissue morphogenesis and homeostasis. The overarching objective of this proposal is to investigate how P-cadherin contributes to CCM in 3D epithelial morphogenesis. Our hypothesis is that P-cadherin has a critical role in maintaining lumen formation and mediating cell migration of epithelial cells in 3D.

Materials and

Methods: In this study, our lab used Madin-Darby canine kidney (MDCK) cell acini as our 3D in vitro system to model epithelial morphogenesis and hepatocyte growth factor (HGF)-induced branching to understand how P-cadherin affects collective migration in 3D. To probe whether lumen development and cell migration are dependent on P-cadherin, we generated a MDCK P-cadherin knockout cell line (MDCK PCAD-/-) using CRISPRi. MDCK E-cadherin knockout cells (MDCK ECAD-/-) were gifted by Sanjeevi Sivasankar at UC Davis. MDCK acini (WT, PCAD-/-, and ECAD-/-) were cultured in hydrogels consisting of type I collagen and fibrin for a total of 7 days. Acini were fixed at days 3 and 7 to compare early and mature stages of lumen formation. Acini were also exposed to media containing HGF after day 3 for 4 additional days to induce tubulogenesis. Samples were fixed and immunofluorescence staining was performed to identify the nuclei (DAPI), actin cytoskeleton (phalloidin), tight junction (ZO-1), and lumen (GP135, podocalyxin).

Results, Conclusions, and Discussions: Our findings demonstrate that different aspects of lumen formation are regulated by P-cadherin and E-cadherin. MDCK WT acini form single, hollow lumens at early stages of acini development (Fig. 1A). After 7 days of culture, MDCK PCAD-/- acini did not have hollow lumen, indicating a role between P-cadherin and lumen hollowing during acini development (Fig. 1B). MDCK ECAD-/- acini were unable to form single, hollow lumens, but displayed multiple small lumens instead (Fig. 1C). We concluded that P-cadherin affects the ability for acini to form a hollow lumen while E-cadherin affects the ability for acini to form a single lumen. Under conditions with HGF, we found that MDCK PCAD-/- acini are not responsive to HGF while both MDCK WT and ECAD-/- acini form protrusions and tubules (Fig. 2A-C). In conclusion, our studies demonstrate that P-cadherin and E-cadherin individually regulate different aspects of lumen formation. Further, we reveal that HGF-induced protrusions is a P-cadherin-dependent response. Overall, our studies demonstrate a significant role for P-cadherin in 3D epithelial cell migration that is independent of E-cadherin. In future work, we will perform live-cell imaging to observe how acini and lumen formation occur differently between cell types as well as characterize differences in branching in response to HGF.

Acknowledgements (Optional): Thanks to the VCU Nanomaterials Characterization Core for use of facilities.