Figure 1.
Ultrastructural and intravital imaging of clot components during BM. (A) EM images of intravascularly arrested Jimt1 (breast cancer) TCs (green) associated with a clot (blue) and platelet aggregates (purple). The vessel wall is depicted in red. Platelets (dotted outlines in high-magnification images) are embedded in the clot. Scale bars in colorized overview panels, 5 µm; scale bars in high-magnification panels: 1 µm. (B) Quantification of arrested TCs on days 5 to 7 p.i.i. with associated (platelet) clot in EM imaging (32 metastatic Jimt1 breast cancer cells from 9 mice). Error bars: 95% CI. (C) Experimental design for studying the impact of the coagulation system on the steps of BM formation in mice. TCs are injected intracardially and followed immediately by in vivo MPLSM through a chronic cranial window. The superior sagittal sinus (white arrow) serves as an anatomical orientation. Microscopy images of the same brain regions over 1 month are used to follow single arrested TCs as they process through the brain metastatic cascade: initial intravascular cell arrest (days 0-5), early extravasation between days 2 and 7, single cell in the perivascular niche (days 2-10), followed by outgrowth into a micrometastasis (3-50 cells) and finally macrometastasis (>50 cells) on day 28. Magnification micrograph: coregistration of circulating platelets (red) and FITC-labeled anti-VWF antibody (green) bound to the typically large VWF fibers (arrowhead) on day 7 p.i.i. next to an intra- and extravascular TC (blue). Scale bars, 10 µm. (D) Representative in vivo MPLSM images over time of the distinct steps of the brain metastatic cascade of all 3 cell line models, which were investigated with MPLSM (without clot imaging). At persistent cell arrest, TCs fill out the whole lumen of the brain capillary with a cell-vessel diameter ratio of 1 (H-bars). Distinct deep vessels are given as reference points (white asterisks) for orientation. Vascular remodeling occurs around distinct arrested/extravasating TCs. Scale bars, 50 µm. (E) Mean diameter of brain capillaries where TCs arrest. Diameter was measured as indicated (H-bar) where intravascularly arrested TCs were in vicinity (105 cells in 6 mice; boxplot; median, 8 µm). Scale bar, 10 µm. TRITC, tetramethylrhodamine-isothiocyanate.

Ultrastructural and intravital imaging of clot components during BM. (A) EM images of intravascularly arrested Jimt1 (breast cancer) TCs (green) associated with a clot (blue) and platelet aggregates (purple). The vessel wall is depicted in red. Platelets (dotted outlines in high-magnification images) are embedded in the clot. Scale bars in colorized overview panels, 5 µm; scale bars in high-magnification panels: 1 µm. (B) Quantification of arrested TCs on days 5 to 7 p.i.i. with associated (platelet) clot in EM imaging (32 metastatic Jimt1 breast cancer cells from 9 mice). Error bars: 95% CI. (C) Experimental design for studying the impact of the coagulation system on the steps of BM formation in mice. TCs are injected intracardially and followed immediately by in vivo MPLSM through a chronic cranial window. The superior sagittal sinus (white arrow) serves as an anatomical orientation. Microscopy images of the same brain regions over 1 month are used to follow single arrested TCs as they process through the brain metastatic cascade: initial intravascular cell arrest (days 0-5), early extravasation between days 2 and 7, single cell in the perivascular niche (days 2-10), followed by outgrowth into a micrometastasis (3-50 cells) and finally macrometastasis (>50 cells) on day 28. Magnification micrograph: coregistration of circulating platelets (red) and FITC-labeled anti-VWF antibody (green) bound to the typically large VWF fibers (arrowhead) on day 7 p.i.i. next to an intra- and extravascular TC (blue). Scale bars, 10 µm. (D) Representative in vivo MPLSM images over time of the distinct steps of the brain metastatic cascade of all 3 cell line models, which were investigated with MPLSM (without clot imaging). At persistent cell arrest, TCs fill out the whole lumen of the brain capillary with a cell-vessel diameter ratio of 1 (H-bars). Distinct deep vessels are given as reference points (white asterisks) for orientation. Vascular remodeling occurs around distinct arrested/extravasating TCs. Scale bars, 50 µm. (E) Mean diameter of brain capillaries where TCs arrest. Diameter was measured as indicated (H-bar) where intravascularly arrested TCs were in vicinity (105 cells in 6 mice; boxplot; median, 8 µm). Scale bar, 10 µm. TRITC, tetramethylrhodamine-isothiocyanate.

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