Figure 5.
Effects of antithrombotic drugs on BM formation. (A) Experimental design. APT via anticoagulation with tinzaparin (tinza) or dual platelet inhibition with ASA/clopidogrel (ASAC) was started 2 days before heart injection and continued with daily application until day 10 after heart injection. (B) Flowchart of the brain metastatic cascade over 1 month to compare the fate of brain-arrested TCs with anticoagulative or antithrombotic treatment compared with a control (ctr) group. 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 (%). Jimt1, 138 metastases in 4 control mice vs 212 metastases in 3 tinzaparin-treated mice vs 204 metastases in 3 ASAC-treated mice; A2058, 156 metastases in 4 control mice vs 130 metastases in 3 tinzaparin-treated mice; H1, 127 metastases in 3 control mice vs 162 metastases in 3 tinzaparin-treated mice. χ2 test analysis for successful extravasation or micro- or macrometastasis formation was not significant. (C) Representative images of hematoxylin and eosin (H&E)–stained brain sections for quantification of overall tumor load at the ultimate end point on day 28. Metastases were counted in whole brain slides (white arrows). The higher magnifications show the morphology of 1 single brain metastasis and the colored outline used for area calculations: vascular co-optive growing metastases present with extensive perivascular protrusions and show a diffuse infiltration of TCs into the surrounding parenchyma, which were then counted as parts of 1 major metastasis. Scale bars for overview, 1 mm; for zoomed-in view, 200 µm. (D) Quantification of mean metastasis size and number in control vs tinzaparin-treated mice with breast cancer metastases. Jimt1, 1222 metastases in 4 control mice vs 997 metastases in 3 tinzaparin-treated mice (metastasis area, P = .022; metastasis number, P = .009. Error bars: 95% CI. (E-F) Quantification of metastasis number and overall tumor burden in control vs antithrombotic-treated mice after breast cancer cell injection. Dabigatran, 12 mice vs 11 control mice (metastasis number, P = .020; tumor load, P = .011); ASAC, 10 mice vs 11 control mice (metastasis number, P = .539; tumor load, P = .675); tirofiban, 7 mice vs 11 control mice (metastasis number, P = .119; tumor load, P = .230); anti-GPIb platelet depletion, 7 mice vs 11 isotype-control mice (metastasis number, P = .141; tumor load, P = .529. Boxplot. (G) Representative images of high-resolution MRI scans of mouse brains. BMs (arrowheads) are hyperintense in T1_RARE weighted contrast-enhanced images. Scale bar: 10 mm. p.o., orally; s.c., subcutaneously. All P values from Student t test. *P < .05.

Effects of antithrombotic drugs on BM formation. (A) Experimental design. APT via anticoagulation with tinzaparin (tinza) or dual platelet inhibition with ASA/clopidogrel (ASAC) was started 2 days before heart injection and continued with daily application until day 10 after heart injection. (B) Flowchart of the brain metastatic cascade over 1 month to compare the fate of brain-arrested TCs with anticoagulative or antithrombotic treatment compared with a control (ctr) group. 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 (%). Jimt1, 138 metastases in 4 control mice vs 212 metastases in 3 tinzaparin-treated mice vs 204 metastases in 3 ASAC-treated mice; A2058, 156 metastases in 4 control mice vs 130 metastases in 3 tinzaparin-treated mice; H1, 127 metastases in 3 control mice vs 162 metastases in 3 tinzaparin-treated mice. χ2 test analysis for successful extravasation or micro- or macrometastasis formation was not significant. (C) Representative images of hematoxylin and eosin (H&E)–stained brain sections for quantification of overall tumor load at the ultimate end point on day 28. Metastases were counted in whole brain slides (white arrows). The higher magnifications show the morphology of 1 single brain metastasis and the colored outline used for area calculations: vascular co-optive growing metastases present with extensive perivascular protrusions and show a diffuse infiltration of TCs into the surrounding parenchyma, which were then counted as parts of 1 major metastasis. Scale bars for overview, 1 mm; for zoomed-in view, 200 µm. (D) Quantification of mean metastasis size and number in control vs tinzaparin-treated mice with breast cancer metastases. Jimt1, 1222 metastases in 4 control mice vs 997 metastases in 3 tinzaparin-treated mice (metastasis area, P = .022; metastasis number, P = .009. Error bars: 95% CI. (E-F) Quantification of metastasis number and overall tumor burden in control vs antithrombotic-treated mice after breast cancer cell injection. Dabigatran, 12 mice vs 11 control mice (metastasis number, P = .020; tumor load, P = .011); ASAC, 10 mice vs 11 control mice (metastasis number, P = .539; tumor load, P = .675); tirofiban, 7 mice vs 11 control mice (metastasis number, P = .119; tumor load, P = .230); anti-GPIb platelet depletion, 7 mice vs 11 isotype-control mice (metastasis number, P = .141; tumor load, P = .529. Boxplot. (G) Representative images of high-resolution MRI scans of mouse brains. BMs (arrowheads) are hyperintense in T1_RARE weighted contrast-enhanced images. Scale bar: 10 mm. p.o., orally; s.c., subcutaneously. All P values from Student t test. *P < .05.

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