Fig. 2.

Direct observation of polyclonal collective invasion, polyclonal disseminated tumor emboli, and polyclonal CTC clusters. (A) Representative micrograph of polyclonal collective invasion arising from mosaic ROSA^mT/mG;MMTV-PyMT transplanted tumors stained with phalloidin (F-actin) and DAPI (n = 75 units, 5 tumors). (B) Schema of two potential outcomes for disseminated tumor cell clusters at the tumor stromal interface. (C) Representative micrographs of a polyclonal disseminated tumor cluster (yellow arrow) in the x-y plane with successive images along the z axis (Left panels) and reconstructed 3D image (Right) (n = 25 units, 5 tumors). (D) Representative micrograph of a polyclonal disseminated tumor embolus contained within a vessel. The transplanted tumor is composed of mTomato⁺ and CFP⁺ tumor cells. Injection with VE-Cadherin and CD31 fluorescently labeled antibodies marked functional vasculature. (E) Representative micrographs demonstrating E-cadherin⁺ polyclonal collective invasion, dissemination, intravascular embolus (from left to right). Yellow hash marks: vessel lumen. (F) Representative micrographs of CTC clusters composed of mTomato⁺ and CFP⁺ tumor cells and stained for K14 and DAPI (n = 1 multicolored cluster, n = 13 mTomato⁺ clusters, n = 2 CFP⁺ cluster). (G) The number of events for each CTC cluster size is presented as a histogram (n = 134 events, 3 transplanted mice). (H) The median percentage of cells that are K14⁺ in CTCs of different cluster sizes are presented as a boxplot (n = 17 clusters). [Scale bars, 2 mm (A, Left), 40 μm (A, Right), 20 μm (C–E), and 10 μm (F).]