A NRF2 Regulated and the Immunosuppressive Microenvironment Reversed Nanoplatform for Cholangiocarcinoma Photodynamic‐Gas Therapy

Abstract Photodynamic therapy (PDT) is a minimally invasive and controllable local cancer treatment for cholangiocarcinoma (CCA). However, the efficacy of PDT is hindered by intratumoral hypoxia and the presence of an antioxidant microenvironment. To address these limitations, combining PDT with gas therapy may be a promising strategy to enhance tumor oxygenation. Moreover, the augmentation of oxidative damage induced by PDT and gas therapy can be achieved by inhibiting NRF2, a core regulatory molecule involved in the antioxidant response. In this study, an integrated nanotherapeutic platform called CMArg@Lip, incorporating PDT and gas therapies using ROS‐responsive liposomes encapsulating the photosensitizer Ce6, the NO gas‐generating agent L‐arginine, and the NRF2 inhibitor ML385, is successfully developed. The utilization of CMArg@Lip effectively deals with challenges posed by tumor hypoxia and antioxidant microenvironment, resulting in elevated levels of oxidative damage and subsequent induction of ferroptosis in CCA. Additionally, these findings suggest that CMArg@Lip exhibits notable immunomodulatory effects, including the promotion of immunogenic cell death and facilitation of dendritic cell maturation. Furthermore, it contributes to the anti‐tumor function of cytotoxic T lymphocytes through the downregulation of PD‐L1 expression in tumor cells and the activation of the STING signaling pathway in myeloid‐derived suppressor cells, thereby reprogramming the immunosuppressive microenvironment via various mechanisms.


Figure S1 .
Figure S1.Expression of antioxidant genes in CCA.(A) Pan cancer analysis of NRF2 expression by TCGA database.(B) NRF2 mRNA levels in cancer and paracancer tissues (n=15).(C) IHC images of NRF2 in cancer and paracancerous tissues.Scale bar: 50 μm.(D) GSEA enrichment analysis of antioxidant gene sets between low and high NRF2 level by

Figure S4 .
Figure S4.NO generation of Arg@Lip in an environment with different concentrations of H2O2 and different pH.(A) pH 5.5, (B) pH 6.8, (C) pH 7.4.

Figure S5 .
Figure S5.The mean fluorescence intensity of the fluorescence images in Figure 2C.

Figure S6 .
Figure S6.Flow cytometry analysis of the production of ROS, NO and ONOO -in various groups.(A) ROS was detected by DCFH-DA after different treatments.(B) NO was detected by DAF-FM DA after different treatments.(C) ONOO -was detected by DAX-J2 PON Green after different treatments.

Figure S7 .
Figure S7.Detection of intracellular antioxidant system after different treatments.(A) and (B) WB and qPCR analysis of NRF2, NQO1, GCLC and GPX4.(C) The relative level of intracellular GSH was detected at various times after different treatments.

Figure S8 .
Figure S8.Cell viability and cell death after different treatments.(A) Detection of cell vitality after different liposomes (dose: 20 μg/ml Ce6) with or without light (650 nm, 100 mW/cm 2 , 15 min) by CCK8.(B) and (C) Flow cytometry typical images and statistical analysis of cell death (7-AAD + ) under different conditions.

Figure S9 .
Figure S9.GSEA analysis of the GSE132305 dataset.(A) GSEA enrichment analysis of apoptosis gene sets between tumor and normal tissues.(B) GSEA enrichment analysis of ferroptosis gene sets between tumor and normal tissues.(C) Heat map of apoptosis genes (normal vs tumor).(D) Heat map of ferroptosis genes (normal vs tumor).

Figure S12 .
Figure S12.Detection of ferroptosis induced by CMArg@Lip with light treatment.(A) Calcein-AM/PI staining for detection of CMArg@Lip+L treatment preincubated with different PCD inhibitors in QBC-939.Scale bar: 100 μm.(B) Statistical analysis of Calcein-AM/PI staining.(C) Statistical analysis of GPX4, XCT and ACSL4 protein levels by WB detection after different treatments.(D) Flow cytometry analysis of lipid peroxidation in QBC-939 cells after different treatments by C11-BODIPY.(E) Detection of MDA levels in QBC-939 after different treatments.

Figure S14 .
Figure S14.WB and Elisa detection of ICD effects after different treatments.(A) WB analysis of CRT, HMGB1 and HSP70 in QBC-939 after different treatments.(B), (C) and (D) Statistical analysis of WB detection CRT, HMGB1 and HSP70.(E) and (F) Elisa assays for extracellular HMGB1 and ATP.

Figure S15 .
Figure S15.Detection of DC maturation in vitro.(A) Schematic diagram of coculture in vitro of immature DC and QBC-939 cells.(B) Flow cytometry detection of mature DC (CD11C + CD80 + CD86 + ) cells in co-culture system.

Figure S16 .
Figure S16.PDT up regulates PD-L1 expression through NRF2.(A) WB analysis of HIF1α, NRF2 and PD-L1 after PDT or combined with ML385 treatment in oxygen enriched

Figure S18 .
Figure S18.CMArg@Lip effectively reversed the upregulation of PD-L1.(A) WB analysis of PD-L1 in QBC-939 cells after receiving different treatments.(The right side shows the results of the statistical analysis of grayscale values).(B) Analysis of the relative expression of PD-L1 on the cell membrane in Figure 4A.(C) Flow cytometry analysis of PD-L1 on QBC-939 cell membranes after receiving different treatments.(The right side shows the statistical analysis of the mean fluorescence intensity).(D) 7-AAD assays for QBC-939 cell death after coincubation of QBC-939 after treatment with T cells.

Figure S19 .
Figure S19.Analysis of gray value after detecting the protein (cGAS, p-TBK1 and p-IRF3) of sting pathway in MDSC by WB.

Figure S20 .
Figure S20.Organ imagings at different time after intravenous injection of CMArg@Lip in nude mice bearing tumor.

Figure S21 .
Figure S21.Verification of the model of homograft of CCA.The figures showed the comparison of HE and CK7 staining of specimens from patients with CCA and mouse homologous transplanted tumors.Bar: 100 μm.

Figure S24 .
Figure S24.Flow cytometry analysis of the proportion of mature DC cells in tumors of mice receiving different treatments.

Figure S25 .
Figure S25.Statistical analysis of immunofluorescence staining images in Figure 6A and B.

Figure S27 .
Figure S27.Detection of hemolysis at different time points after in vitro treatment of mouse blood with different doses of CMArg@Lip.(Positive control: water, negative control: PBS)

Figure S29 .
Figure S29.CMArg@Lip had no significant toxic side effects in vivo.(A) HE staining images of the heart, liver, spleen, lungs and kidneys of tumor-bearing mice after receiving different treatments.Bar: 100 μm.(B) Blood parameters of tumor-bearing mice after different treatments.