Metastasis is the leading cause of cancer-related deaths; metastatic lesions develop from disseminated cancer cells (DCCs) that can remain dormant1. Metastasis-initiating cells are thought to originate from a subpopulation present in progressed, invasive tumours2. However, DCCs detected in patients before the manifestation of breast-cancer metastasis contain fewer genetic abnormalities than primary tumours or than DCCs from patients with metastases3,4,5. These findings, and those in pancreatic cancer6 and melanoma7 models, indicate that dissemination might occur during the early stages of tumour evolution3,8,9. However, the mechanisms that might allow early disseminated cancer cells (eDCCs) to complete all steps of metastasis are unknown8. Here we show that, in early lesions in mice and before any apparent primary tumour masses are detected, there is a sub-population of Her2+p-p38lop-Atf2loTwist1hiE-cadlo early cancer cells that is invasive and can spread to target organs. Intra-vital imaging and organoid studies of early lesions showed that Her2+ eDCC precursors invaded locally, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent epithelial–mesenchymal transition (EMT)-like dissemination program but without complete loss of the epithelial phenotype, which was reversed by Her2 or Wnt inhibition. Notably, although the majority of eDCCs were Twist1hiE-cadlo and dormant, they eventually initiated metastasis. Our work identifies a mechanism for early dissemination in which Her2 aberrantly activates a program similar to mammary ductal branching that generates eDCCs that are capable of forming metastasis after a dormancy phase.
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We thank R. Parsons and P. Polulikakos for PI3K and AKT inhibitors, S. Aaronson and H.-C. Wen for WNT3A, SFRP1 and DKK1 reagents and expertise. Grant support:. HHMI (R.J.D.). SWCRF (J.A.A.-G. and E.F.F.), CA109182, CA196521 (J.A.A.-G.), CA163131 (J.A.A-G and J.C.), CA100324 (J.C), F31CA183185 (K.H.), BC132674 (J.A.A.-G and J.C.), BC112380 (M.S.S.). NIH 1S10RR024745. Microscopy CoRE at ISMMS. DFG KL 1233/10-1 and the ERC (322602) (C.A.K.).
Extended data figures
Extended data tables
Two independent MMTV-Her2 organoids cultured for 4 days in Matrigel were imaged for 2 hours and frames were collected every 20 minutes. Top row shows the differential interference contrast (DIC) images of the two organoids with cell movement and microinvading cells toward the bottom of the field of view. The bottom row shows a variance filter from Image J software that highlights the cell edges and allows having a bright view of the microinvading cells from the organoids into the Matrigel.
Intra-vital imaging of mammary glands in MMTV-Her2-CFP mice was performed using a specially designed mammary gland imaging window (see Experimental Procedures for details). CFP+/Her2+ cells can be seen in the cyan/light cyan signal and vasculature was delimited using i.v. delivered dextran-rhodamine (red). Note the apparent normal duct architecture at this time, which remained organized with some intra-ductal movement observed mainly in luminal spaces. Still image of this video is shown in Figure 2a left panel.
Mammary gland imaging windows were used as in Video S2 in MMTV-Her2-CFP mice at age 15 weeks when early lesions are present (Extended Data 1a-c). Motile single CFP+ cells were found micro-invading from lesions and are outlined in purple and green in the video. CFP+/Her2+ cells can be seen in cyan and vasculature was stained with dextran-rhodamine (Red). Note that here a greater digital zoom was used to show in detail the motile cells micro-invading from the ductal structure that occupies the lower right quadrant of the video. Still image of video is shown in Figure 2a middle panel.
Mammary gland imaging windows were used to image ducts of MMTV-Her2-CFP mice at age 18 weeks when early stage lesions are present. Single cells were found microinvading from the early stage ducts (upper right) as outlined in purple in the video. One single cell can be traced through the stroma between ducts. CFP+/Her2+ cells can be seen in cyan and vasculature was stained with dextran-rhodamine (red), which also revealed macrophages that capture the labeled dextran. Still images of video are shown in Figure 2a right panel.
Intra-vital imaging of multiple micro-invasion events in MMTV-Her2-CFP mice treated for two weeks with SB203580
Imaging of MMTV-Her2-CFP mammary glands following treatment for two weeks with SB203580 (10mg/kg, i.p every 48hr). This video shows an early lesion and CFP+/Her2+ cells in cyan and vasculature stained with dextran-rhodamine (red). Note the luminal filling of ducts and increased movement of cells along the ductal walls into the stroma and into a low contrast vessel between the two lobules of the ductal structure. Still images are in Figure 2b.
Digital magnification of the region of Video S5 highlighted in a dotted line square inFigure 2b (inset b’)
Note the amoeboid like movement of these CFP+/Her2+ cells micro-invading cells breaking the lesion boundaries into the stroma. Note also that the motility seem to occur at the single cell level and not collectively. Still image of video is shown in Figure 2b’.
3D rendering of the micro-invasion events in MMTV-Her2-CFP mice treated with the p38a/b inhibitor SB203580
3D reconstruction of Video S6 revealed that CFP+/Her2+ cells were entering the lumen of the blood vessel (RED) as revealed by the yellow overlap signal and suggesting active intravasating capacity as supported by the video S8 and the detection of CTCs and DTCs in Extended data 4h, i, j. Entry into the vessel lumen is marked when CFP+ and Dextran-Rhodamine (Red) signals overlap shown in yellow. Still images of video are shown in Figure 2c.
Digital Magnification of an early lesion reveals a CFP+ cells entering the vasculature from the stroma in SB203580 treated mice. Left panel shows original imaging with a delineated vessel (yellow line) and the intravasating cell (purple line). Right panel shows a digital segmentation to isolate the vasculature (RED) and the intravasating eDCC precursor (CYAN) that turns yellow when the optical signals overlap. Still images of this sequence are shown in Figure 2d.
About this article
Nature Cell Biology (2018)