Anti pd l1

Antibodies used in this study were anti-DNMT1 (Abcam), anti-5mC (Cell Signaling), anti-GAPDH, anti-ERK, anti-phospho-ERK (Cell signaling), anti-SMA (Sigma), anti-CD8 (Biolegend, BioXcell, or eBiosciences), anti-CD11c, anti-CD45, anti-F480, anti-MECA-79, anti-Vcam1, and anti-CD4 (all from BD Biosciences), anti-granzyme B (Cell Signaling), anti-mouse IgG (BioXcell), anti-PD-1 (CD279, BioXcell), and anti-PD-L1 (Genentech, MTA program). MGECs were isolated and cultured as described previously by our lab^{60 ,61 (link)}. Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza and cultured in EBM-2 supplemented with the EGM-2 bullet kit. Culture dishes were precoated with 1% gelatin. For the labeling of CD8⁺ T-cells, CellTracker^TM green dye was used (Molecular Probes). The culture of EO771 and PyMT cells was described previously^{62 (link),63 (link)}. Other reagents include a JAK2 inhibitor (AG490), NFκΒ inhibitor (JSH23, Sigma), TNFα (Peprotech), IFNγ (EMD Millipore), GSK3484862 (Med Chem Express), and 5-Azacytidine (Sigma).

UMBO was delivered to both UMBO and UMBO plus Anti-PD-L1 groups. Anti-PD-L1 (6E11 Genentech) was administered intraperitoneally in two independent experiments (n = 8) at a dose of 200 µg sixty minutes before UMBO application. Mice were maintained under anesthesia with isoflurane (2%, 2 L/min O₂). For each UMBO application, 10 mL/kg SonoVue^® was injected through the intravenous route less than 10 s before the start of the ultrasound application. For each session, UMBO was validated using an additional control mouse. Each control mouse was injected intravenously with a solution of 2.7% Evans blue (Sigma, E2129) in phosphate buffer saline (PBS) at a dose of 4 mL/kg ten minutes post-sonication. All mice received 10 mL/kg warm saline injection in each treatment protocol before anesthesia to prevent any possible hypovolemia or hypothermia effect. I.P injection of Anti-PD-L1 injection was given 60 min before sonication to ensure Anti-PD-L1 absorption (Supplementary Material, Figure S1B). UMBO test mice were sacrificed 15 min following Evans’ blue injection, and their brain was harvested. The passage of Evans blue was assessed both visually and by ZEISS Axio-Scan fluorescence imaging of cryo-sectioned brains.