Poster V7 - Collective Transitions from Orbiting to Matrix Invasion in 3D Multicellular Spheroids

Introduction: Coordinated migration dynamics where groups of cells swirl or travel in parallel as multicellular strands are widely observed in embryonic development and wound healing, but remain poorly understood in the context of cell-cell and cell-matrix adhesions. Indeed, there exists a dynamic reciprocity where matrix architecture shapes cell migration, but in turn, cell migration also shapes matrix architecture. Here, we show that multicellular spheroids in 3D matrix exhibit sustained circumferential orbiting motions that gradually transition towards radial invasion by multicellular stands, governed by the dynamic remodeling of the boundary between spheroid and matrix. We elucidate the fundamental principles underlying such morphogenesis using 3D traction force microscopy and systematic perturbations of cell-cell and cell-matrix adhesions.

Materials and

Methods: Multicellular spheroids were prepared as hanging drops using mammary epithelial cells (MCF-10A) stably transfected with a red fluorescent nuclear marker (mCherry-H2B). The clusters were then embedded in collagen I mixed with fluorescent beads to facilitate visualization. Live cell confocal microscopy was utilized to concurrently track collective cell migration and matrix deformation following the mCherry-H2B and bead signals, respectively. Relative angular positions of peripheral nuclei over time were captured using kymograph analysis. Cell migration and matrix deformation were quantified using optical flow algorithms. Physical and biochemical perturbations were applied to decouple the contributing factors of the cell-ECM and cell-cell interactions.

Results, Conclusions, and Discussions: Kymograph analysis and optical flow algorithm reveal that there are distinct phases in progression of collective migration over time. Upon embedding, the multicellular mammary gland cell spheroids exhibit minimal migration. However, the non-oriented, oscillatory motions soon turn into highly coordinated circumferential orbiting, either clockwise or counterclockwise. Throughout the orbiting phase, at least 75% peripheral cells are engaged in the coordinated orbiting, with this proportion peaking near 90%, as determined by the optical flow algorithm. The orbiting phases persist until the spheroids develop protrusions which evolve into invasive strands oriented radially (Figure. 1).
Intriguingly, alterations in the global morphology and the tractions exerted against the surrounding matrix are observed during the orbiting phases. Initially, most spheroids deviate slightly from perfect sphericity; however, during the orbiting phase, they gradually become more spherical, adjusting their bulge boundaries in and flat boundaries out. Specifically, areas where the spheroid boundary retreats during the round compactions correlate with the hotspots of radial tractions, and these spots are predictive of subsequent strand invasion.
Compressive stress conditions impede the coordinated circumferential orbiting by inhbiting the round compaction and matrix remodeling. Furthermore, including cells with weaker cell-cell interaction into the spheroid also prevented collective orbiting motion, underscoring the significance of cell-cell interactions. These mechanical and biochemical perturbations provide mechanistic insight into the transition between the collective orbiting and invasion phases. We envision these governing principles will be broadly relevant to understanding organoid morphogenesis for embryonic development and disease progression.

Acknowledgements (Optional): We acknowledge funding from NIGMS (R01GM140108) and the Brown University Hibbitt Postdoctoral Fellowship.