American Association for Cancer Research
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Supplementary Figures 1 through 12 from Targeting CD47 and Autophagy Elicited Enhanced Antitumor Effects in Non–Small Cell Lung Cancer

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journal contribution
posted on 2023-04-03, 23:05 authored by Xuyao Zhang, Jiajun Fan, Shaofei Wang, Yubin Li, Yichen Wang, Song Li, Jingyun Luan, Ziyu Wang, Ping Song, Qicheng Chen, Wenzhi Tian, Dianwen Ju

Supplementary Figure S1. The designed amino acid sequence of SIRPalphaD1-Fc. Supplementary Figure S2. Macrophage-mediated cytotoxicity in NSCLC cells after treatment with SIRPalphaD1-Fc. Supplementary Figure S3. SIRPalphaD1-Fc induced accumulation of autophagosomes and triggered autophagic flux in NSCLC cells. Supplementary Figure S4. Inhibiting autophagy enhanced macrophage-mediated phagocytosis. Supplementary Figure S5. Rapamycin exhibited negligible effect on the cytotoxicity mediated by macrophage against NSCLC cells. Supplementary Figure S6. Inactivation of Akt/mTOR signaling pathway after NSCLC cells treated with SIRPalphaD1-Fc. Supplementary Figure S7. Inactivation of Akt/mTOR signaling pathway in vivo after SIRPalphaD1-Fc treatment. Supplementary Figure S8. Representative fluorescence images of A549 cells co-stained with Cyto-ID(c) green dye and MitoSox(tm) red dye after exposed to SIRPalphaD1-Fc for the indicated time. Supplementary Figure S9. Photos of NSCLC xenograft tumors after treatment with SIRPalphaD1-Fc and/or chloroquine. Supplementary Figure S10. Analysis of SIRPalphaD1-Fc-treated A549 xenograft tumor tissues by TEM, H&E and immunohistochemistry staining. Supplementary Figure S11. Representative fluorescence images of ROS formation in NCI-H1975 xenograft tumors. Supplementary Figure S12. Simultaneously targeting CD47 and autophagy activated apoptosis-related pathways in vivo.



CD47-specific antibodies and fusion proteins that block CD47–SIRPα signaling are employed as antitumor agents for several cancers. Here, we investigated the synergistic antitumor effect of simultaneously targeting CD47 and autophagy in non–small cell lung cancer (NSCLC). SIRPαD1-Fc, a novel CD47-targeting fusion protein, was generated and was found to increase the phagocytic and cytotoxic activities of macrophages against NSCLC cells. During this process, autophagy was markedly triggered, which was characterized by the three main stages of autophagic flux, including formation and accumulation of autophagosomes, fusion of autophagosomes with lysosomes, and degradation of autophagosomes in lysosomes. Meanwhile, reactive oxygen species and inactivation of mTOR were shown to be involved in autophagy initiation in SIRPαD1-Fc–treated cells, indicating a probable mechanism for autophagy activation after targeting CD47 by SIRPαD1-Fc. Inhibition of autophagy enhanced macrophage-mediated phagocytosis and cytotoxicity against SIRPαD1-Fc–treated NSCLC cells. In addition, simultaneously targeting both CD47 and autophagy in NSCLC xenograft models elicited enhanced antitumor effects, with recruitment of macrophages, activated caspase-3, and overproduction of ROS at the tumor site. Our data elucidated the cytoprotective role of autophagy in CD47-targeted therapy and highlighted the potential approach for NSCLC treatment by simultaneously targeting CD47 and autophagy. Cancer Immunol Res; 5(5); 363–75. ©2017 AACR.See related Spotlight by Kaufman, p. 355.