突破结直肠癌免疫治疗瓶颈！铁死亡纳米疗法靶向 CAFs，Nat Commun.助力机制验证

Fig. 1

a Schematic illustration of the antitumor process of NPs through inducing the ferroptosis of CAFs and remodeling the CAFs-related immune TME. b Individuals in the GSE17538 cohort are stratified into low and high CAF groups based on MCP-counter derived CAF abundance by the optimal cutoff value. Box plots depict the median (centre line), interquartile range (25th-75th percentiles; box), and 1.5 × interquartile range (whiskers). For the low group: min = 7.682, Q1 = 8.675, median = 8.873, Q3 = 9.165, max = 9.414 (n = 134 patients); for the high group: min = 9.424, Q1 = 9.547, median = 9.673, Q3 = 9.826, max = 10.854 (n = 98 patients). Statistical significance is assessed using a two-sided Mann-Whitney U test. c Kaplan-Meier curves of 232 colon cancer patients from the GSE17538 cohort stratified by CAF abundance assessed using the MCP-counter method. Tick marks indicate censored events. P values are calculated using a two-sided log-rank test, and hazard ratios (HRs) with 95% confidence intervals are estimated using univariate Cox proportional hazards regression. Source data are provided as a Source Data file.

Fig. 2

a Images displaying the isolation of NFs from adjacent normal tissues and CAFs from CRC tumor tissues. b Western blot confirming successful isolation of NFs from three normal colonic tissues and CAFs from three CRC specimens, validated by antibodies against fibroblast activation protein-α (FAP), Vimentin, and α-smooth muscle actin (SMA); β-actin as a loading control. c Cytotoxicity assessment of NPs in NFs and CAFs for 24 h at varying concentrations. d Bio-TEM images of NFs and CAFs incubated with NPs for 24 h; Red dashed boxes marking NPs location. e Bright-field and fluorescent images of CAFs stained with Calcein-AM (live cells, green fluorescence) and PI (dead cells, red fluorescence) after treatment with phosphate buffer saline (PBS), Laser, NPs, and NPs+Laser. PBS as control (NC) (left); Statistical analysis of cell viability (right). f, g Fluorescent images of (f) intracellular Fe2+ levels and (g) LPO in CAFs treated without or with NPs for 24 h; Nuclei stained with Hoechst 33342 (blue). h Western blot of GPX4 expression in CAFs treated with increasing concentrations of NPs for 24 h; β-actin as a loading control. i Relative cell viability of CAFs treated with NPs alone, NPs with DFO, NPs with TM, and NPs with both DFO and TM for 24 h by CCK8 assay. j, k Fluorescent images of (j) intracellular Fe2+ levels and (k) LPO in CAFs treated as in (i); Nuclei stained with Hoechst 33342 (blue). l Western blot of GPX4 expression in CAFs treated as in (i); β-actin as a loading control. n = 3 independent experiments per group in (c–g) and (i–k). Western blot experiments in (b, h, l), FerroOrange and Liperfluo staining experiments in (f, g, j, k) are repeated independently three times with similar results. Data are presented as means ± standard deviation (SD). Statistical analyses are performed using one-way analysis of variance (ANOVA) with multiple comparisons (e). Source data are provided as a Source Data file.

Fig. 3

a Schematic illustration of cell viability and transwell migration assays using HCT116 cells treated with supernatants from NFs, CAFs, or NPs-treated CAFs. b Transwell migration images presenting the number of migrated HCT116 cells treated with CM from NFs, CAFs, or NPs-treated CAFs for 24 h. c Cell viability of HCT116 cells treated as in (b) by the CCK8 assay. d Western blot of E-cadherin, N-cadherin, and Vimentin expression in HCT116 cells treated with CM from NFs, CAFs, or NPs-treated CAFs for 24 h; β-actin as a loading control. e, f Bubble plots of (e) GO and (f) KEGG pathway enrichment analyses for differentially expressed genes (DEGs) in NPs-treated CAFs compared to controls. g, h MSigDB Hallmark GSEA analysis showing enrichment of (g) chemokine activity and (h) ferroptosis pathways in NPs-treated CAFs compared to controls. ES: enrichment score; NES: normalized enrichment score. i Heatmap and histogram displaying gene expression levels of DEGs (log₂|FC | ≥ 1) from three chemokine-related GO pathways (GO:0016493, GO:0048020, GO:0008009) in NPs-treated CAFs compared to controls, ranked by differential expression. j QRT-PCR quantification of CCL3, CXCL12, and CCR7 expression in NPs-treated CAFs compared to controls. k, l Anti-human ELISA quantification of (k) CCL3 and (l) CXCL12 concentrations in supernatants from CAFs treated without or with NPs for 24 h. m Western blot of MAPK, p-MAPK (Thr180/Tyr182), NF-κB, p-NF-κB (Ser536), AKT, and p-AKT (Ser473) expression in CAFs treated without or with NPs for 24 h; β-actin as a loading control. n = 3 independent experiments in (b, c) and (j–l). Western blot experiments in (d, m) are repeated independently three times with similar results. Data are presented as means ± SD. Statistical analyses are performed using one-way ANOVA with multiple comparisons for (b, c); and two-tailed unpaired Student's t-test for (j–l). Source data are provided as a Source Data file.

Fig. 4

a Schematic illustration of the treatment strategy and related immune assessment in vivo based on NPs for the CT26 and the CT26 + BALB/3T3 group. b Individual tumor growth curves and (c) average tumor growth curves of the CT26 and the CT26 + BALB/3T3 group without or with the treatment of NPs. d Representative images of mice bearing CT26 cells and CT26 + BALB/3T3 cells without or with the treatment of NPs. e Body weight curves of CT26 cells- and CT26 + BALB/3T3 cells-bearing mice without or with the treatment of NPs. f Quantified expression of CCL3 and CXCL12 in homogenized tumor tissues from the CT26 and the CT26 + BALB/3T3 groups without or with the treatment of NPs. g Flow cytometry and corresponding statistical analysis of CD11c+MHC II+CD80+ and CD11c+MHC II+CD86+ cells (in total CD45+ cells) around tumor tissues of mice in four groups. h Statistical analysis of flow cytometry of CD3+CD8+Eomes+ and CD3+CD8+T-bet+ cells around tumor tissues of mice in four groups. i Statistical analysis of flow cytometry of CD3+CD8+CD107a+, CD3+CD8+IFN-γ+, and CD3+CD8+TNF-α+ cells around tumor tissues of mice in four groups. n = 5 mice per group in (b–e) and n = 3 independent experiments per group in (f–i). Data are presented as means ± SD. Statistical analyses are performed using two-way ANOVA with multiple comparisons for (c); and one-way ANOVA with multiple comparisons for (f–i). Source data are provided as a Source Data file.

Fig. 7

a Real-time thermal infrared images of the PDX model after intravenous injection of PBS, NPs, and NPs-AS1411 for 24 h under 808 nm laser irradiation. b Representative images of tumors of the PDX model in six groups. c Average tumor growth curves of the PDX model in six groups. d Bright-field and fluorescent images of CEA, Ki67, α-SMA, and Vimentin expression in co-cultured tumor cells and CAFs from PDX model tumor tissues. e Bright-field and fluorescent images of co-cultured tumor cells and CAFs from PDX model tumor tissues stained with Calcein-AM (live cells, green fluorescence) and PI (dead cells, red fluorescence) after treatment with PBS, Laser, NPs, NPs-AS1411, NPs + Laser, and NPs-AS1411 + Laser. f Bright-field and H&E staining images of organoids from normal colonic epithelia and CRC. g H&E staining and immunohistochemical images of expression of Ki67, CDX2, CK20, CK7, and CEA of normal colonic organoids and CRC organoids. h, i Confocal bright-field and fluorescent images of Calcein-AM (live cells, green fluorescence) and PI (dead cells, red fluorescence) staining in (h) normal colonic organoids and (i) CRC organoids after treatment with PBS, Laser, NPs, NPs-AS1411, NPs + Laser, and NPs-AS1411 + Laser. n = 5 mice per group in (a–c). Immunofluorescent experiments (d, e), H&E and immunohistochemical experiments (f, g), and Calcein-AM/PI staining experiments (h, i) are repeated three times with similar results. Data are presented as means ± SD. Statistical analyses are performed using two-way ANOVA with multiple comparisons for (c). Source data are provided as a Source Data file.