Additional file 1 of Drug-loaded microbubble delivery system to enhance PD-L1 blockade immunotherapy with remodeling immune microenvironment
Creators: name: Zheng, Jun
name: Huang, Ju
name: Zhang, Liang
name: Wang, Mengna
name: Xu, Lihong
name: Dou, Xiaoyun
name: Leng, Xiaojing
name: Fang, Mingxiao
name: Sun, Yang
name: Wang, Zhigang
Publisher: name: figshare
Year: 2023
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Supporting Figure S1. Characterization of RD@MBs. (a) Light microscopy image of RD@MBs (scale bar = 10 μm). (b-c) The standard curves of DTX and R837 measured by LC-MS. Supporting Figure S2. Biosafety of RD@MBs. (a) Cell viabilities of 4T1 cells treated with different concentrations of RD@MBs (without US irradiation) (n = 3). (b) Heatmap of the blood biochemical and blood routine analysis (n = 5). (c) H&amp;E images of major organs (heart, liver, spleen, lung and kidney) treated with different concentrations of RD@MBs for in vivo biosafety evaluation. Data are expressed as mean ± SD. Supporting Figure S3. UTMD drug delivery system. (a-b) In vitro US imaging and the corresponding echo intensities of RD@MBs at various concentrations (20, 40, 60, 80, 100 µg/mL) (n = 3). (c-d) In vivo US imaging and corresponding echo intensities of tumor regions at different time points (n = 3). Supporting Figure S4. UTMD drug delivery system. (a-b) FCM results of drug release in tumor sites and the corresponding quantitative analysis of MFI. Supporting Figure S5. Cell viabilities of 4T1 cells treated with different concentrations of R@MBs, D@MBs and RD@MBs (n = 3). Data are expressed as mean ± SD. Statistical significances were calculated via Student’s t test, *p &lt; 0.05, **p &lt; 0.01 and ***p &lt; 0.001. Supporting Figure S6. (a-b) Primary and distant tumor TGI after different treatments in 4T1 orthotopic tumor bearing mice (n = 5). (c-d) Representative digital photos of tumor nodules in the 4T1 orthotopic tumor bearing mice lungs and corresponding quantification of the numbers of lung nodules (n = 5). (e) H&amp;E and TUNEL staining images of primary tumor in 4T1 orthotopic tumor bearing mice. PCNA staining images of distant tumor in 4T1 orthotopic tumor bearing mice. (Scale bar = 200 μm). (f) Body weight of the 4T1 orthotopic tumor bearing mice after different treatments (n = 5). (g-h) Primary and distant tumor TGI after different treatments in CT26 subcutaneous tumor bearing mice (n = 5). (i) Body weight of the CT26 subcutaneous tumor bearing mice after different treatments (n = 5). Data are expressed as mean ± SD. Statistical significances were calculated via Student’s t test, *p &lt; 0.05, **p &lt; 0.01 and ***p &lt; 0.001. Supporting Figure S7. Immunofluorescence staining images for expression of CD86 and CD206 in 4T1 tumor tissues after different treatments. Blue, DAPI-labeled nucleus; green, anti-CD86 antibody-labelled M1-TAMs; red, anti-CD206 antibody-labeled M2-TAMs. (Scale bar = 200 μm). Supporting Figure S8. Immunofluorescence staining images for expression of MMP-9 and VEGF in 4T1 tumor tissues after different treatments. Blue, DAPI-labeled nucleus; green, anti-MMP-9 antibody-labelled MMP-9; red, anti-VEGF antibody-labeled VEGF (scale bar = 200 μm).Supporting Figure S9. (a) FCM results of Tregs (CD3+CD4+Foxp3+) in spleen. (b) FCM results of Tregs (CD3+CD4+Foxp3+) in tumor. Supporting Figure S10. Immunofluorescence staining images for infiltration of CTLs in primary tumor of 4T1 orthotopic tumor bearing mice after different treatments. Blue, DAPI-labeled nucleus; green, anti-CD8 antibody-labelled CTLs (scale bar = 100 μm).