Fig 2: (A) NIR fluorescence imaging of the biodistribution of Cy5.5-labeled peptide conjugates in CT26 tumor-bearing BALB/c mice in vivo; (B) Quantitative fluorescence analyses of (A) at the corresponding time; (C) Immunofluorescence CLSM images of CRT induction by F-pY-T in the CT26 tumor-bearing BALB/c mice in vivo. The scale bars represent 50 μm; (D) Schematic illustration of F-pY-T and anti-PD-L1 blockade-mediated combination therapy; (E) Single-component tumor growth curves (n = 8); (F) Tumor weight in the different groups. Two-tailed t-test, ∗∗∗P＜0.001, ∗∗P < 0.01, ∗P < 0.05. n = 8; (G) Optical image of tumors (1: anti-PD-L1, 2: F-pY-T, and 3: F-pY-T plus anti-PD-L1).

Fig 3: (A) CLSM images of ecto-CRT levels on CT26 cells treated with different compounds for 24 h. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI: blue fluorescence). The scale bars represent 25 μm; (B) Flow cytometric analysis of ICD induction in CT26 cells in vitro with or without treatment with different compounds; Quantitative analyses of (C) ATP and (D) HMGB1 in the cell supernatants. One-way ANOVA, mean ± SD, ∗∗∗P＜0.001, ∗∗P < 0.01. n = 3; (E) Immunofluorescence CLSM images of CT26 cells treated with or without F-pY-T. The scale bar represents 25 μm; (F) Schematic illustration of the ICD induction profile of F-pY-T.

Fig 4: Antitumor immunity induced by the sHDLs in vivo. (A) Flow cytometry qualification of CRT levels on tumor cell surface. (B) ELISA qualification of HMGB1 in the tumors. (C) Western-blot analysis of pTBK1 and pIRF3 levels in the tumors. (D) Flow cytometry qualification of the percentage of mature BMDCs (CD80+CD86+) in the DLNs collected 3 days after the administration of different sHDLs (n = 5). (E) ELISA qualification of TNF-α, IL12p40, and IFN-γ secretion in the DLNs collected 3 days after the administration of different sHDLs (n = 3). In an additional experiment, the tumors were collected 7 days after the last treatment of a three-treatment regimen (n = 5). (F) Intratumoral densities of mature DCs. (G) ELISA qualification of IFN-α and IFN-β in the tumors. Intratumoral densities of CD3+ T lymphocytes (H), CD8+ T cells (I), and CD8+IFN-γ+ T cells (J). (K) CD8+ T cell-to-Treg (CD4+Foxp3+ T cells) ratio. (L) M1-to-M2 ratio. (M) The expression levels of PDL1 on tumor cells after different treatments. Statistical significance was calculated using a two-sided one-way ANOVA test. The data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Fig 5: Roxadustat increased lipid peroxidation and calreticulin exposure.A Representative fluorescent images of lipid peroxidation in GL261 or U87 cells after incubation with Erastin (10 μM) or RXD (100 μM towards U87, 200 μM towards GL261) with or without GSH (500 μM) for 48 h. Scale bar: 100 μm. B Representative flow cytometry analysis and statistical histogram of LPO-positive cells in GL261 or U87 cells with the different treatments. C Representative fluorescent images of CRT in GL261 or U87 cells after the different treatments. Scale bar: 100 μm. D Representative flow cytometry analysis and statistical histogram of CRT-positive cells in GL261 or U87 cells after the different treatments. *P < 0.05, **P < 0.01, ***P < 0.001.