Enhanced Photodynamic Therapy Synergizing with Inhibition of Tumor Neutrophil Ferroptosis Boosts Anti‐PD‐1 Therapy of Gastric Cancer

Abstract For tumor treatment, the ultimate goal in tumor therapy is to eliminate the primary tumor, manage potential metastases, and trigger an antitumor immune response, resulting in the complete clearance of all malignant cells. Tumor microenvironment (TME) refers to the local biological environment of solid tumors and has increasingly become an attractive target for cancer therapy. Neutrophils within TME of gastric cancer (GC) spontaneously undergo ferroptosis, and this process releases oxidized lipids that limit T cell activity. Enhanced photodynamic therapy (PDT) mediated by di‐iodinated IR780 (Icy7) significantly increases the production of reactive oxygen species (ROS). Meanwhile, neutrophil ferroptosis can be triggered by increased ROS generation in the TME. In this study, a liposome encapsulating both ferroptosis inhibitor Liproxstatin‐1 and modified photosensitizer Icy7, denoted LLI, significantly inhibits tumor growth of GC. LLI internalizes into MFC cells to generate ROS causing immunogenic cell death (ICD). Simultaneously, liposome‐deliver Liproxstatin‐1 effectively inhibits the ferroptosis of tumor neutrophils. LLI‐based immunogenic PDT and neutrophil‐targeting immunotherapy synergistically boost the anti‐PD‐1 treatment to elicit potent TME and systemic antitumor immune response with abscopal effects. In conclusion, LLI holds great potential for GC immunotherapy.


Figure S1 .
Figure S1.Gating strategies were used to investigate proportion of various immune cells in human gastric cancer samples.(A) CD71 expression was gated form CD11b + CD15 + cells in tumors.(B) CD8 + T cells and CD4 + T cells were gated from CD3 + T cells in tumors.(C) M1 TAMs

Figure S2 . 3 Figure S3 .
Figure S2.Ferroptosis of cell types from tumor microenvironment of gastric cancer.(A and B) Different cell populations in the tumor microenvironment gated by relevant markers.(C to G) Statistical analysis of CD71 expression on different cell populations in tumor microenvironment of gastric cancer.(Data were presented as the mean  SD, ***p<0.001and ****p<0.0001).

Figure S4 .
Figure S4.Stability and absorption spectra of nanodrugs.(A) Stability of LLI in phosphate buffer saline (PBS) containing 10% FBS at 37 C, n=3.(B) Absorption spectra of LL, LI, LLI, and free Icy7 in MeOH.(C) The red fluorescence of DiI begins to fade after 12 hours, indicating the release of nanodrugs after cocultured with cells (DiO; blue), and (DiI; red), n=3.Scale bar, 50 m.(Data were presented as the mean  SD).

Figure S6 .
Figure S6.The cell uptake, apoptosis, and expression of CD71 in MFC cells were evaluated by 3D tumor spheroid models.(A) Apoptosis rate of MFC cells treated with PBS, LL, LI, and LLI without light irradiation, n=3.(B) Statistical analysis of apoptosis rate of MFC cells in different groups, n=3.(C) The formation of 3D tumor spheroid model.Scale bar, 500 m.(D and E) The cell apoptosis of MFC cells was evaluated by 3D tumor spheroid models.(F and G) The uptake of nanodrugs in MFC cells was evaluated by 3D tumor spheroid models.(H and I) Expression of CD71 in MFC cells was evaluated by 3D tumor spheroid models.(Data were presented as the mean  SD, **p<0.01,***p<0.001,****p<0.0001,ns, not significant).

Figure S7 .
Figure S7.Mitochondria membrane potential of MFC cells determined by flow cytometry in different groups.(A) JC-1 aggregates of MFC cells determined by flow cytometry in different groups, n=3.(B) Statistical analysis of JC-1 aggregates of MFC cells in different groups, n=3.(C) JC-1 monomers of MFC cells determined by flow cytometry in different groups, n=3.(D) Statistical analysis of JC-1 monomers of MFC cells in different groups, n=3.(Data were presented as the mean  SD, **p<0.01,***p<0.001,and ns, not significant).

Figure S10 .
Figure S10.ROS production in MFC cells detected by confocal microscope or flow cytometry.(A) Fluorescence imaging of ROS production in MFC cells incubated with PBS, LL, LI, and LLI without light irradiation, n=3.Scale bar, 100 m.(B and C) ROS production in MFC cells detected by flow cytometry after various treatments, n=3.(Data were presented as the mean  SD, ns, not significant).

Figure S13 .
Figure S13.ATP and HMGB1 from cell supernatants were measured by using enzyme-linked immunosorbent assay (ELISA).(A and B) ATP from cell supernatants was measured by ELISA after various treatments, n=3.(C and D) HMGB1 from cell supernatants was measured by ELISA after various treatments, n=3.(Data were presented as the mean  SD, **p<0.01,***p<0.001,and ns, not significant).

Figure S14 .
Figure S14.Ferroptosis of neutrophils in TME and spleen of MFC tumor-bearing mice.(A) Macroscopic image of tumor tissue and spleen from MFC tumor-bearing mice, n=6.(B and C) Ferroptosis of neutrophils detected by flow cytometry in TME and spleen of MFC tumorbearing mice, n=6.(Data were presented as the mean  SD, ****p<0.0001).

Figure S16 .
Figure S16.Viability and purity of CD8 + cells from spleen of mice.(A) Viability of CD8 + cells sorted by flow cytometry from spleen of mice.(B) Purity of CD8 + cells sorted by flow cytometry from spleen of mice.(Data were presented as the mean  SD).

Figure S19 .
Figure S19.Biosafety evaluation of nanodrugs in vivo.(A) H&E staining of major organs of the treated mice, including the heart, liver, spleen, lung, and kidney, n=6.Scale bar, 50 m.(B to E) Serum levels of liver function (ALT and AST) and renal function (Cr and BUN) were detected at the end of the treatments, n=6.(Data were presented as the mean  SD).

Figure S20 .
Figure S20.Gating strategies were used to investigate proportion of various immune cells in tumors of mice.(A) CD71 expression was gated form CD11b + Ly6G + cells in tumors.(B) CD8 + T cells and CD4 + T cells were gated from CD3 + T cells in tumors.(C) M1 TAMs and M2 TAMs

Figure S21 .
Figure S21.Gating strategies were used to investigate proportion of various immune cells in tumor or spleen of mice.(A) PD-L1 expression was gated from CD11b + cells or CD11c + cells.

Figure S22 .
Figure S22.Nanodrugs treatments with abscopal effect.(A) Macroscopic image of primary tumor tissues from mice receiving various treatment, n=6.(B) Tumor weight of primary tumor tissues from mice receiving various treatment, n=6.(C) Tumor growth curve of primary tumor tissues from mice receiving various treatment, n=6.(D) Macroscopic image of distant tumor tissues from mice receiving various treatment, n=6.(E) Tumor weight of distant tumor tissues from mice receiving various treatment, n=6.(F) Tumor growth curve of distant tumor tissues from mice receiving various treatment, n=6.(G) Representative images of distant tumor tissue sections showed tumor morphology (H&E staining), cell proliferation (Ki67), and apoptosis (TUNEL), n=6.Scale bar, 50 m.(H) Statistical analysis of TUNEL in distant tumor section after different treatments.(I) Statistical analysis of Ki67 in distant tumor section after different treatments.(Data were presented as the mean  SD, *p<0.05,**p<0.01,***p<0.001,and ****p<0.0001).

Figure S23 .
Figure S23.Nanodrugs enhanced anti-tumor immune response in distant tumors of mice.(A and B) CD71 expression of neutrophils in distant tumor tissues determined by flow cytometry after different treatments (gated on CD11b + Ly6G + cells), n=6.(C to E) The percentage of CD4 +