Cell-Cell Mechanical Cues Modulating ECM Responsivity

While much of the focus in this review has been on cell-ECM interactions that occur in the cellular niche, there are also non-matrix adhesive interactions present in cellular niches that can control cellular behaviors – chief among these are cell-cell adhesive interactions. These cell-cell contacts are the main source of mechanical interaction at early stages of development, and eventually yield to cell-ECM interactions in a tissue-specific fashion as cells begin to secrete matrix components (Kalson, Lu et al. 2015). These cell-cell interactions are mainly governed by the cadherin family of transmembrane adhesive proteins, though these interactions can also be mediated by a variety of less predominant proteins, including nectins and IgG-family CAMs. The main cell-cell adhesive interaction arises from the homotypic interactions of cadherins from one cell to cadherins on a neighboring cell. The extracellular domains of cadherins consist of multiple repeat domains that are essential for adhesive interactivity, followed by a transmembrane region, and then an intercellular domain that functions as both a signaling hub and a linkage to the actin cytoskeleton. As such, cadherins play an essential role in development and disease. Cell-cell interaction through cadherins can result in robust changes in cellular phenotype, due to the dual functional role of these proteins in signaling and adhesivity. While there are many sub-types of cadherin, the three most commonly expressed and studied types are E-cadherin, N-cadherin, and VE-cadherin. Each of these cadherin sub- types have the same general adhesive mechanisms, but vary in the results and mediators of their downstream signaling (Sayegh, Kapus et al. 2007). Importantly for stem cell differentiation, cadherins can signal through the Hippo pathway and become a negative regulator of YAP/TAZ localization in dense cell culture conditions (Gumbiner and Kim 2014), altering proliferation, migration, and differentiation (Dupont, Morsut et al. 2011).

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GTPase signaling pathways (Sayegh, Kapus et al. 2007, Heuberger and Birchmeier 2010). On a mechanical level, cadherin also functions to enable complex multi-cellular behaviors such as collective migration and wound closure (Cai, Chen et al. 2014).

Since cadherin plays a large role in tuning the mechanical state of the cell through signaling and adhesion, it is important to consider crosstalk between cell-cell signaling and cell-ECM signaling when designing biomaterials (Weber, Bjerke et al. 2011, Mui, Bae et al. 2015, Mui, Chen et al. 2016). It has long been known that dense cell culture and the increased cell-cell interaction because of this cellular density can have profound impacts on cellular behaviors such as differentiation (McBeath, Pirone et al. 2004), though decoupling cell-cell interactivity from reduced cell-ECM adhesive cues remains difficult. One approach to induce adherens junction formation while controlling ECM interaction was to micropattern adhesive islands while forcing some degree of overlap between the two islands, thereby inducing cell-cell interaction while restricting cell-ECM area (Liu, Tan et al. 2010, Mui, Bae et al. 2015). However, experimental issues still remain with respect to varying cell-cell contact length in these systems, as adherens junctions can form on the diagonal axes and are not always consistent in adhesive area. One of the most widely adopted tools for studying cadherin mechanobiology is the Fc-tagged recombinant cadherin extracellular domain, which can be conjugated to biomaterials systems (Lambert, Padilla et al. 2000, Nagaoka, Koshimizu et al. 2006, Yue, Murakami et al. 2010, Fichtner, Lorenz et al. 2014, Vega, Lee et al. 2016). The advantage of this platform is that the native molecule conformation can be used, and cells can form reinforced adherens junction-like structures on these substrates (Tsai and Kam 2009, Chopra, Tabdanov et al. 2011). Yet, due to the size and chemistry of these molecules, adaptability and “off-target” interactivity

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of these molecules becomes an issue, where at certain densities it is likely that the Fc- cadherin molecules form trans-dimers and other structures.

While short peptide mimics of adhesive domains in ECM proteins have been widely incorporated into biomaterials design, there has only recently been a focus on the incorporation of short peptide mimics for cell-cell adhesive domains (Williams, Williams et al. 2000, Williams, Williams et al. 2002). This is in part due to the complexity of binding and signaling interactions within cadherins, as there are multiple states of binding for a cadherin-cadherin binding pair (X-dimer, strand-swapped dimer) as well as a more complex set of reinforcement adhesive sites that are crucial for cis- and trans- cadherin mechanical interactions (Hoffman and Yap 2015, Priest, Shafraz et al. 2017). However, one key short peptide sequence has been discovered in E-cadherin and N-cadherin, the HAV adhesive motif. This motif appears to mediate a large degree of cadherin adhesivity (Williams+, JCB, 2000). Originally developed for use as a cancer therapeutic agent, the HAV motif is located in the EC1 domain of type-1 cadherin molecules, and, when added as a soluble antagonist, has been shown to mediate up to 90% of cadherin adhesivity (Figure 2-6)(Williams+ JBC 2000). Similar to flanking RGD motifs and tertiary structure in integrin specificity (Hersel, Dahmen et al. 2003), the flanking resides to the HAV peptide have also been shown to provide for specificity between E-Cadherin and N-Cadherin; where HAVS mediates the E-Cadherin interactions and HAVDI mediates the N-Cadherin interaction (Williams, Williams et al. 2000, Williams, Williams et al. 2002).

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Figure 2-6 – The HAVDI domain on N-Cadherin mediates the strand-swapped dimer formation (inset). Adapted from (Brasch et al. 2012, Gumbiner 2005).

Despite these advances, there is still much unknown about the function of the HAV motif. This motif resides in the hydrophobic pocket of the EC1 domain of type-1 cadherins (Nardone, Lucarelli et al. 2016), which is responsible for mediating the stable strand- swapped dimer formation. While there exist other adhesive domains in N-Cadherin, there is an extensive set of literature that demonstrates the critical role of the HAV adhesive domain within the EC1 repeat of type-1 cadherins in cadherin specific responses in development, including data from both in vivo and in vitro studies (Halbleib and Nelson 2006, Sayegh, Kapus et al. 2007). Structural studies have revealed that the cadherin- HAV interaction is the key mechanical interaction for the stable mechanical linkage

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(strand-swapped) between two single cadherin molecules, and it is likely that the HAV motif alone does not allow for all of the more complex binding interactions that happen during adhesions junction formation, such as EC4-EC4 linkage and trans-cadherin binding (Hoffman and Yap 2015). Other work has illustrated other minimal peptides from cadherin that can have downstream effects on cellular behavior, such as IKVAV and INSPG (Williams, Williams et al. 2001), though these seem to play a less crucial role in signaling compared to the HAV domain (Schense, Bloch et al. 2000). Similarly, previous work has shown that the first two extracellular domains of cadherin (in which the HAV motif resides) allow for weak adhesive interactions when compared to the full ectodomain of cadherin (Fichtner, Lorenz et al. 2014, Vega, Kwon et al. 2016). Indeed, our unpublished data supports the fact that mesenchymal cells cannot robustly attach and spread on to substrates containing HAVDI, whereas cells can reliably attach onto fc-N-cadherin coated substrates of any stiffness. This suggest that cells require other adhesive domains for robust attachment and adherens junction formation, and that the HAVDI peptide mimics early cadherin-cadherin binding without cis- or trans- reinforcement.

That is not to say that dense adherens junctions are the only structures of relevance for cadherin signaling. In this context, it is important to consider microenvironmental polarity and dynamics, as most work on cadherin mechanobiology has been done in the context of reinforced E-cadherin/VE-cadherin adherens junctions that are typical of most cellular niches in stable patent environments such as the vessel lumen (Mui, Bae et al. 2015). However, not all cellular niches are permissive to adherens junction formation, such as the mesenchymal cell niche. In this niche, N-cadherin is the dominant cadherin, and cell- cell contacts show almost no order. This is compounded with the high degree of cell

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motility, with cells rarely staying in one place long enough to allow reinforced adherens junctions to form beyond a specific point in development. As such, many of the engineered systems that engender adhesive junction formation are not directly relevant to this niche. In these environments, HAVDI presentation is likely a closer mimic to the cell-cell interactions these cells would experience, where transient cadherin adhesion likely plays a role in signaling in lieu of mechanical reinforcement of an adherens junction.

Further development on HAV peptide structure is likely needed to recapitulate full adherens junction formation. There are two possible explanations as to why there are not robust adherens junctions with the HAV peptide. It could be either structural or could involve incorporation of multiple synergistic binding interactions. Perhaps the length scale and flexibility of the HAV peptide is important, as more stable HAVDI bonds might be formed if a flexible linker was incorporated in peptide design in order to more robustly bind in the W2 pocket of the cellular cadherin. Or, similar to the synergistic binding effects of RGD with the synergy sequence PHSRN (Aota, Nomizu et al. 1994, Redick, Settles et al. 2000), more complex structure and presentation schema of additional domains, such as the residues found in EC4, might be needed to allow for tight adherens junction formation. Regardless, the HAV motif remains an exciting tool for use as a therapeutic modification to biomaterials to better tune cell-material interactions and the subsequent interpretation of substrate stiffness (Cosgrove, Mui et al. 2016) or engender other cellular responses (Bian, Guvendiren et al. 2013, Lim, Mosley et al. 2016, Lim, Khan et al. 2017). While the HAV motif lacks the complexity of full adherens junction, future work to study stem cell interactions with materials as well as future development of this residue could potentially increase the usefulness of this interaction in directing a wider array of cellular behaviors.

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Many questions about cadherin adhesivity remain, and our knowledge of cadherin binding still lags behind our knowledge of integrin binding. However, new technologies are quickly providing mechanistic insights into cadherin adhesivity (Manibog, Li et al. 2014, Manibog, Sankar et al. 2016), organization (Bertocchi, Wang et al. 2017) and signaling (Yao, Qiu et al. 2014), and this knowledge will hopefully allow for the more precise design and control of cell-cell interactions to create more native-like biomaterials.