Pannexin1 drives multicellular aggregate compaction via a signaling cascade that remodels the actin cytoskeleton.

Pannexin 1 (Panx1) is a novel gap junction protein shown to have tumor-suppressive properties. To model its in vivo role in the intratumor biomechanical environment, we investigated whether Panx1 channels modulate the dynamic assembly of multicellular C6 glioma aggregates. Treatment with carbenoxolone and probenecid, which directly and specifically block Panx1 channels, respectively, showed that Panx1 is involved in accelerating aggregate assembly. Experiments further showed that exogenous ATP can reverse the inhibitive effects of carbenoxolone and that aggregate compaction is sensitive to the purinergic antagonist suramin. With a close examination of the F-actin microfilament network, these findings show that Panx1 channels act as conduits for ATP release that stimulate the P(2)X(7) purinergic receptor pathway, in turn up-regulating actomyosin function. Using a unique three-dimensional scaffold-free method to quantify multicellular interactions, this study shows that Panx1 is intimately involved in regulating intercellular biomechanical interactions pivotal in the progression of cancer.

Connexon-mediated cell adhesion drives microtissue self-assembly.

Microtissue self-assembly is thought to be driven primarily by cadherins, while connexons have been examined mainly in intercellular coupling. We investigated whether connexon 43 (Cx43)-mediated cell adhesion modulates self-assembly of human KGN granulosa cells, normal human fibroblasts (NHFs), and MCF-7 breast cancer cells seeded into nonadhesive agarose gels. We found that treatment with anti-Cx43 E2 (112 µg/ml), which suppresses Cx43 docking, significantly inhibited the kinetics of KGN and NHF self-assembly compared to the preimmune sera control (41.1 ± 4.5 and 24.5 ± 10.4% at 8 h, respectively). Likewise, gap junction inhibitor carbenoxolone also inhibited self-assembly of KGN, NHF, and MCF-7 cells in a dose-dependent manner that was specific to cell type. In contrast, Gap26 connexin mimetic peptide, which inhibits channel permeability but not docking, accelerated self-assembly of KGN and NHF microtissues. Experiments using selective enzymatic digestion of cell adhesion molecules and neutralizing N-cadherin antibodies further showed that self-assembly was comparably disrupted by inhibiting connexin- and cadherin-mediated adhesion. These findings demonstrate that connexon-mediated cell adhesion and intercellular communication differentially influence microtissue self-assembly, and that their contributions are comparable to those of cadherins.

Directed self-assembly of large scaffold-free multi-cellular honeycomb structures.

A significant challenge to the field of biofabrication is the rapid construction of large three-dimensional (3D) living tissues and organs. Multi-cellular spheroids have been used as building blocks. In this paper, we create large multi-cellular honeycomb building blocks using directed self-assembly, whereby cell-to-cell adhesion, in the context of the shape and obstacles of a micro-mold, drives the formation of a 3D structure. Computer-aided design, rapid prototyping and replica molding were used to fabricate honeycomb-shaped micro-molds. Nonadhesive hydrogels cast from these micro-molds were equilibrated in the cell culture medium and seeded with two types of mammalian cells. The cells settled into the honeycomb recess were unable to attach to the nonadhesive hydrogel and so cell-to-cell adhesion drove the self-assembly of a large multi-cellular honeycomb within 24 h. Distinct morphological changes occurred to the honeycomb and its cells indicating the presence of significant cell-mediated tension. Unlike the spheroid, whose size is constrained by a critical diffusion distance needed to maintain cell viability, the overall size of the honeycomb is not limited. The rapid production of the honeycomb building unit, with its multiple rings of high-density cells and open lumen spaces, offers interesting new possibilities for biofabrication strategies.