To gain a greater understanding of the factors that drive spatial organization in multicellular aggregates of cancer cells, we investigate the segregation patterns of 6 breast cell lines (MDA-MB-231, MDA-MB-468, MDA-MB-436, MDA-MB-157, ZR-75-1, and MCF-10A) of varying degree of mesenchymal character during formation of mixed aggregates. We consider cell sorting in the context of available adhesion proteins and cellular contractility, biophysical properties that are typically considered in models of cell sorting. We characterize the mechanisms of spheroid formation as being primarily cadherin- or integrin-driven. The primary compaction mediator for a given cell type plays an important role in compaction speed, which in turn is the major factor dictating preference for interior or exterior position within mixed aggregates. In particular, cadherin-deficient, invasion-competent cells tend to position towards the outside of aggregates, facilitating access to extracellular matrix. We show that reducing actomyosin contractility has a differential effect on spheroid formation depending on the compaction mechanism. Inhibition of contractility has a significant stabilizing effect on cell-cell adhesions in integrin-driven aggregation and a mildly destabilizing effect in cadherin-based aggregation. This differential response is exploited as a spheroid formation method and as a method through which to statically control aggregate organization and dynamically rearrange cells in pre-formed aggregates. Sequestration of invasive cells in the interior of spheroids provides a physical barrier that reduces invasion in three-dimensional culture, revealing a potential strategy for containment of invasive cell types. [Media: see text] [Media: see text] [Media: see text]

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease-and adhesion-independent manner. Here, we delineate a contractilitydependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional β1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

We demonstrate simultaneous spatial and spectral subtraction-based super-resolution microscopy based on photobleaching, photoblinking, or photoactivation in multichromophoric systems to detail the characteristics of individual emitters in small aggregates isolated and immobilized in polymeric films. In particular, we investigate the prototypical multichromophoric electron-donating system poly­[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) and electron-accepting N,N′-dipentyl-3,4,9,10-perylenedicarboximide (pPDI) molecules prepared as small aggregates via tandem analysis of spatial and spectral information of individual emitters within a diffraction-limited spot. In MEH-PPV aggregates, this approach reveals evidence of both chain-limited and domain-limited exciton migration as well as preferential photodegradation of regions with a high number of intermolecular contacts. In small aggregates of pPDI, evidence of individual subunits with distinct spectral characteristics and electronic coupling properties is found.

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