Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single cell dissemination, controlled by cadherinmediated cell-cell junctions. In clinical samples, E-cadherin expressing and deficient tumors both invade collectively and metastasize equally, implicating additional mechanisms controlling cell-cell cooperation and individualization. Using spatially defined organotypic culture, intravital microscopy of mammary tumors in mice and in silico modeling, we here identify cell density regulation by 3D tissue boundaries to physically control collective movement irrespective of the composition and stability of cell-cell junctions. Deregulation of adherens junctions, including E-cadherin and p120-catenin, resulted in a transition from coordinated to uncoordinated collective movement along extracellular boundaries, whereas singlecell escape depended on locally free tissue space. These data indicate that cadherins and ECM confinement cooperate to determine unjamming transitions towards step-wise epithelial fluidization and, ultimately, cell individualization.

Cancer invasion programs are adaptive by switching between metastatic collective and single-cell dissemination; however, current intravital microscopy models for epithelial cancer in mice fail to reliably recreate such invasion plasticity. Using microimplantation of breast cancer spheroids into the murine mammary fat pad and live-cell monitoring, we show microenvironmental conditions and cytoskeletal adaptation during collective to single-cell transition in vivo. E-cadherin-expressing 4T1 and E-cadherin-negative MMT tumors both initiated collective invasion along stromal structures, reflecting invasion patterns in 3D organotypic culture and human primary ductal and lobular carcinoma. Collectively invading cells developed weakly oscillatory actin dynamics, yet provided zones for single-cell transitions with accentuated, more chaotic actin fluctuations. This identifies collective invasion in vivo as a dynamic niche and efficient source for single-cell release.

Cancer fatalities result from metastatic dissemination and therapy resistance, both processes that depend on signals from the tumor microenvironment. To identify how invasion and resistance programs cooperate, we used intravital microscopy of orthotopic sarcoma and melanoma xenografts. We demonstrate that these tumors invade collectively and that, specifically, cells within the invasion zone acquire increased resistance to radiotherapy, rapidly normalize DNA damage, and preferentially survive. Using a candidate-based approach to identify effectors of invasion-associated resistance, we targeted β1 and αVβ3/β5 integrins, essential extracellular matrix receptors in mesenchymal tumors, which mediate cancer progression and resistance. Combining radiotherapy with β1 or αV integrin monotargeting in invading tumors led to relapse and metastasis in 40–60% of the cohort, in line with recently failed clinical trials individually targeting integrins. However, when combined, anti-β1/αV integrin dual targeting achieved relapse-free radiosensitization and prevented metastatic escape. Collectively, invading cancer cells thus withstand radiotherapy and DNA damage by β1/αVβ3/β5 integrin cross-talk, but efficient radiosensitization can be achieved by multiple integrin targeting.

Progression of epithelial cancers predominantly proceeds by collective invasion of cell groups with coordinated cell–cell junctions and multicellular cytoskeletal activity. Collectively invading breast cancer cells express the gap junction protein connexin-43 (Cx43), yet whether Cx43 regulates collective invasion remains unclear. We here show that Cx43 mediates gap-junctional coupling between collectively invading breast cancer cells and, via hemichannels, adenosine nucleotide/nucleoside release into the extracellular space. Using molecular interference and rescue strategies, we identify that Cx43 hemichannel function, but not intercellular communication, induces leader cell activity and collective migration through the engagement of the adenosine receptor 1 (ADORA1) and AKT signaling. Accordingly, pharmacological inhibition of ADORA1 or AKT signaling caused leader cell collapse and halted collective invasion. ADORA1 inhibition further reduced local invasion of orthotopic mammary tumors in vivo, and joint up-regulation of Cx43 and ADORA1 in breast cancer patients correlated with decreased relapse-free survival. This identifies autocrine purinergic signaling, through Cx43 hemichannels, as a critical pathway in leader cell function and collective invasion.