Visualizing extravasation dynamics of metastatic tumor cells

Abstract

Little is known about how metastatic cancer cells arrest in small capillaries and traverse the vascular wall during extravasation in vivo. Using real-time intravital imaging of human tumor cells transplanted into transparent zebrafish, we show here that extravasation of cancer cells is a highly dynamic process that involves the modulation of tumor cell adhesion to the endothelium and intravascular cell migration along the luminal surface of the vascular wall. Tumor cells do not damage or induce vascular leak at the site of extravasation, but rather induce local vessel remodeling characterized by clustering of endothelial cells and cell-cell junctions. Intravascular locomotion of tumor cells is independent of the direction of blood flow and requires beta1-integrin-mediated adhesion to the blood-vessel wall. Interestingly, the expression of the pro-metastatic gene Twist in tumor cells increases their intravascular migration and extravasation through the vessel wall. However, in this case, Twist expression causes the tumor cells to switch to a beta1-integrin-independent mode of extravasation that is associated with the formation of large dynamic rounded membrane protrusions. Our results demonstrate that extravasation of tumor cells is a highly dynamic process influenced by metastatic genes that target adhesion and intravascular migration of tumor cells, and induce endothelial remodeling.

Fig. 1.

Tumor cell extravasation correlates with metastatic potential. (A) Left panel shows whole-mount fluorescence image of a 2 d.p.f. Tg(fli1:EGFP) zebrafish embryo showing the perivascular tumor cell injection site (red arrow) and the ISV vessels in the tail region where cells typically arrest (box). Right panel is a multi-color confocal image of CFP- or RFP-expressing MDA tumor cells arrested in the ISV of Tg(fli1:EGFP) embryos and either inside or outside (extravasated) the vessel lumen. Image was taken 24 hours post injection (h.p.i.) with a 20× objective. (B) The percentage of extravasated tumor cells quantified 24 h.p.i. for several human tumor cell lines: MDA-MB-435 (MDAwt) MDA-MB-231, HT1080, SW480 and SW620. (C) The percentage of extravasated tumor cells quantified 24 h.p.i. for variants of MDA-MB-435 cell line used in the manuscript: MDAwt (wild type); MDA-MB-435 cells expressing the metastatic genes VEGFA (MDAVEGF) or Twist (MDAtwist). In addition, extravasation was measured for MDAwt cells depleted of the metastatic gene ITGB1 (MDAβ1KO) or these cells reconstituted with ITGB1 (MDAβ1KOR). Results are means ± s.e.m. Scale bars: 200 μm.

Fig. 2.

Tumor cells arrest in small vessels then actively migrate along the luminal surface of the vascular endothelium. (A) MDAwt cell expressing CFP and migrating within the ISV lumen for the indicated times (supplementary material Movie 1). Right panel shows 3D isosurface rendering of the tumor cell body for each time point (supplementary material Movie 2). (B) MDAtwist cell expressing RFP and migrating in the lumen of an ISV for the indicated times (supplementary material Movie 4). Right panel shows 3D isosurface rendering of the tumor cell body for each time point (supplementary material Movie 5). (C) Multi-color confocal images of MDAwt and MDAβ1KO cells labeled with CFP or RFP that have arrested in the ISV lumen. Lower panel shows a control 10 μm fluorescent Sepharose bead (yellow) arrested in the ISV. (D) Quantification of distance from tumor cell to vessel wall for MDAwt, MDAβ1KO cells, and Sepharose beads as shown in representative Fig. 2C above. Results are means ± s.e.m. Scale bars: 20 μm.

Fig. 3.

Twist promotes tumor cell extravasation by increasing tumor cell intravascular migration and membrane-protrusion dynamics dependent on ROCK kinase activity. (A) Comparison of membrane protrusion dynamics of MDAtwist and MDAwt cells. Middle panel, red and blue channels showing individual tumor cell shapes. Right panel shows schematic outlines of the blood vessel and MDAtwist tumor cell. (B) Intravascular shape index (sphericity, round=1.0) for individual MDAtwist and MDAwt cells for the indicated times. Each line in B represents relative change in sphericity over the indicated time period for individual MDAwt or MDAtwist tumor cells. (C) Average speed of shape index change for MDAwt and MDAtwist cells. (D) Effect of ROCK and MLCK inhibition on Twist-induced tumor cell extravasation. (E) β1 integrin expression on MDAwt and MDAtwist cells (MFI, mean fluorescence intensity units) as determined by FACS analysis. (F) Average numbers of MDAwt and MDAtwist cells attached to various extracellular matrixes (3 hour time point) per 40× field. Results are means ± s.e.m. Scale bars, 20 μm.

Fig. 4.

Tumor cells induce vessel remodeling in response to metastatic gene expression, but do not induce vascular leakage. (A) Single optical sections of MDAwt or MDAVEGF cells expressing CFP and arrested in ISVs with or without reduced VE-cadherin expression (VE-Cad morpholino), which serve as a positive control for vascular leakage (Montero-Balaguer et al., 2009). Vessel-wall integrity and leakage around arrested tumor cells (blue) was assessed by injection of Rhodamine-Dextran (MW=2×10⁶, red) into the circulation. (B) Representative images of vascular wall changes of ISVs with an arrested Sepharose bead (left), MDAwt cell (center) and MDAVEGF cell (right). (C) The thickness of the vessel wall was measured around arrested Sepharose beads or MDA cells expressing the indicated genes. (D) Left panel, representative image of MDAwt-RFP cell-induced movement of endothelial cell nuclei at the indicated times (supplementary material Movie 3). Right panel shows movement of endothelial cell nuclei in a normal vessel without an arrested tumor cell. Colored arrows indicate individual nuclei positions at the indicated times. (E) Nuclei speed was measured in ISVs with arrested Sepharose beads or MDA cells expressing the indicated genes. Each bar represents increase or decrease (percentage) above the average endothelial nuclei movement speed for a particular condition. (F) Representative image of endothelial cell nuclei clustering around a MDAwt cell expressing CFP (blue) and arrested in the ISV of a Tg(fli1:nEGFP) embryo in which GFP is expressed exclusively in the nucleus. (G) Quantification of endothelial cell nuclei clustering around ISV-arrested Sepharose beads or MDA cells expressing the indicted genes. (H) Confocal image of anti-ZO1 immunofluorescence staining (red) of cell-cell junctions (arrows) of an ISV with an arrested MDAwt cell (blue). Right panels show schematically EC cell-cell junctions and the tumor cell. Arrows indicate individual cell-cell junctions. Scale bars: 20 μm.