Fig. 3: Two-way demonstration of ICAM2 promotes the leptomeningeal metastasis in vitro and in vivo.

A IF staining of ICAM2 expression (green) was determined on scramble control (SCR) and two independent ICAM2 shRNA knock-down (sh#1 and sh#4) LeptoM3 cells (White arrow indicated that the membrane location of ICAM2). B The protein levels of ICAM2 and BCB adhesion ability were investigated in ICAM2 shRNA knock-down LeptoM3 cells compared with scramble control. After 10 min, Adhesive cell numbers were counted by the luciferase activity of cells. C The protein levels of ICAM2 and Trans-BCB ability were examined in ICAM2 shRNA knock-down LeptoM3 cells compared with scramble control. After 24 h, transferred cell numbers were counted by the luciferase activity of cells. After 7 weeks of IC injection, D Quantitative analysis of scramble control and ICAM2 shRNA knock-down LeptoM3 cells tracing by IVIS E Metastatic lesions in different organs were quantitatively analyzed using IVIS ex vivo. F The metastatic tumor cells in lumbar spine were detected by H&E staining (Black arrow indicated metastatic tumor cells). G The leptomeningeal metastatic ability of scramble control and ICAM2 shRNA knock-down LeptoM3 cells were evaluated by calculating the percentage of mice with leptomeningeal metastases. H IF staining of ICAM2 expression (green) was determined on vector control (vector) and ICAM2-overexpressing (ICAM2) MDA-MB-231 cells (White arrow indicated that the membrane location of ICAM2). I The protein levels of ICAM2 and BCB adhesion ability were investigated in ICAM2-overexpressing MDA-MB-231 cells compared with vector control. J The protein levels of ICAM2 and Trans-BCB migration ability were investigated in ICAM2-overexpressing MDA-MB-231 cells compared with vector control. After 7 weeks of IC injection, K Quantitative analysis of vector control and ICAM2-overexpressing MDA-MB231 cells tracing by IVIS. L Metastatic lesions in different organs were quantitatively analyzed using IVIS ex vivo. M The metastatic tumor cells in leptomeningeal space of spinal cord was determined by H&E staining (Black arrow indicated metastatic tumor cells). N The leptomeningeal metastatic ability of vector control and ICAM2-overexpressing MDA-MB-231 cells was evaluated by calculating the percentage of mice with leptomeningeal metastases. O The metastatic percentage of the different organ metastasis was calculated after 15 days of IC injection with 1 × 10⁴ ICAM2 overexpressing or vector control 4T1 cells in BALB/c mice. P Metastatic lesions in different organs were quantitatively analyzed using IVIS ex vivo. Q The leptomeningeal metastatic ability of vector control and ICAM2-overexpressing 4T1 cells was evaluated by calculating the percentage of mice with leptomeningeal metastases. (*P < 0.05; ***P < 0.001).