Figure 3. Observations of streaming, intravasation and dissemination of tumour cells in mammary tumours.

a | In vivo multiphoton microscopy of mammary primary tumours in mice (from REF. 5). MTLn3 rat breast adenocarcinoma cells, engineered to express a fusion protein comprised of enhanced green fluorescent protein (EGFP)–mammalian enabled homologue invasive splice variant (MENA^INV; green), move in a multicellular stream towards a blood vessel (red) over 30 minutes. Scale bar = 25 μm. The white arrow indicates the direction of cell movement. The asterisk indicates the location of the blood vessel. See also Supplementary information S2 (movie). b | Immunohistochemistry of a fixed and paraffin-embedded MTLn3 primary tumour with tumour cells overexpressing MENA^INV (T; pink) and F4/80-expressing macrophages (M; grey), imaged at x63 magnification (from REF. 5). Nuclear counterstain is shown in green. Scale bar = 20 μm. c | A tumour cell expressing EGFP (green) crossing the endothelium (red) of a blood vessel in a mammary tumour. Scale bar = 5 μm. Image courtesy of J. van Rheenen, J. Wyckoff and J.S.C., Albert Einstein College of Medicine. d | Photoconversion of dendra-expressing tumour cells from green to red allows the red tumour cells to be followed as they actively exit the primary tumour via blood vessels, with knowledge of their origin. e | Red photoconverted tumour cells arrive at the lung and remain there as either a disseminated non-dividing population (red) or as a dividing population (yellow). Scale bar = 25 μm. Parts d and e are reproduced, with permission, from REF. 128 © (2011) Macmillan Publishers Ltd. All rights reserved.