Figure 2.
Clot formation in brain microvessels marks TC extravasation. (A) Representative images of intravascularly arrested TCs (green fluorescent protein, GFP) associated with a clot visualized with Rhodamine 6G dye. Scale bars, 10 µm. (B) Clot size was defined by the TC size:clot size ratio. Minor clot: clot volume:TC volume <0.5; Jimt1, n = 56 of 138; A2058, n = 132 of 156; H1, n = 94 of 127; extensive clot: clot volume:TC volume >0.5 and <1.0; Jimt1, n = 51 of 138; A2058, n = 12 of 156; H1, n = 29 of 127; excessive clot: clot volume:TC volume >1.0; Jimt1, n = 29 of 138; A2058, n = 5 of 156; H1, n = 0 of 127; no clot: Jimt1, n = 2 of 138; A2058, n = 7 of 156; H1, n = 4 of 127. (C) Representative image time course of a single H1 melanoma cell (green) associated with platelets (red) and an extensive clot: initial intravascular cell arrest, extravasation (day 5) into the perivascular position, outgrowth into a micrometastasis by vascular co-option (day 10), and finally successful macrometastasis after 1 month. Representative platelet accumulation on days 3 and 5 after TC injection (arrowheads). Distinct deep vessels are given as reference (asterisks). Scale bars: 50 µm, the dotted square represents the brain section of the previous imaging time point. (D-E) Quantification of the brain metastatic cascade, visualized in a flowchart. In all 3 cell lines, more than 100 individual arrested TCs were followed for more than 1 month; Jimt1, 138 metastases in 4 mice; A2058, 156 metastases in 4 mice; H1, 127 metastases in 3 mice. For each step of the metastatic cascade, the total number of surviving (white) vs dying (black) TCs is given, as well as the likelihood to reach the next step of the cascade (%). (D) Comparing TCs with (red) or without (blue) associated extensive clot formation during the intravascular phase. Jimt1 extravasation P = .013; A2058 extravasation P = .021; H1 extravasation P = .557 (χ2 test for all P values). (E) Comparing the fate of brain-arrested TCs with any surrounding clot vs those with no surrounding clot. Only TCs with any clot association successfully grow out into a macrometastasis. χ2 test analysis for successful extravasation or micro- or macrometastasis formation was not significant.

Clot formation in brain microvessels marks TC extravasation. (A) Representative images of intravascularly arrested TCs (green fluorescent protein, GFP) associated with a clot visualized with Rhodamine 6G dye. Scale bars, 10 µm. (B) Clot size was defined by the TC size:clot size ratio. Minor clot: clot volume:TC volume <0.5; Jimt1, n = 56 of 138; A2058, n = 132 of 156; H1, n = 94 of 127; extensive clot: clot volume:TC volume >0.5 and <1.0; Jimt1, n = 51 of 138; A2058, n = 12 of 156; H1, n = 29 of 127; excessive clot: clot volume:TC volume >1.0; Jimt1, n = 29 of 138; A2058, n = 5 of 156; H1, n = 0 of 127; no clot: Jimt1, n = 2 of 138; A2058, n = 7 of 156; H1, n = 4 of 127. (C) Representative image time course of a single H1 melanoma cell (green) associated with platelets (red) and an extensive clot: initial intravascular cell arrest, extravasation (day 5) into the perivascular position, outgrowth into a micrometastasis by vascular co-option (day 10), and finally successful macrometastasis after 1 month. Representative platelet accumulation on days 3 and 5 after TC injection (arrowheads). Distinct deep vessels are given as reference (asterisks). Scale bars: 50 µm, the dotted square represents the brain section of the previous imaging time point. (D-E) Quantification of the brain metastatic cascade, visualized in a flowchart. In all 3 cell lines, more than 100 individual arrested TCs were followed for more than 1 month; Jimt1, 138 metastases in 4 mice; A2058, 156 metastases in 4 mice; H1, 127 metastases in 3 mice. For each step of the metastatic cascade, the total number of surviving (white) vs dying (black) TCs is given, as well as the likelihood to reach the next step of the cascade (%). (D) Comparing TCs with (red) or without (blue) associated extensive clot formation during the intravascular phase. Jimt1 extravasation P = .013; A2058 extravasation P = .021; H1 extravasation P = .557 (χ2 test for all P values). (E) Comparing the fate of brain-arrested TCs with any surrounding clot vs those with no surrounding clot. Only TCs with any clot association successfully grow out into a macrometastasis. χ2 test analysis for successful extravasation or micro- or macrometastasis formation was not significant.

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