Membranolytic peptide programs immunogenic cell death for cancer therapy

a, Schematic illustration of aMP C12 -CA 50 and aMP C6 -CA 50 . b, Time-dependent helicity recovery of aMP Cn -CA 50 (100 µg ml−1) upon incubation in buffers of different pH. c, Dye leakage from liposomes upon incubation with aMP Cn -CA 50 (160 µg ml−1) following preincubation under different pH conditions overnight. d, Concentration- and time-dependent cytotoxicity of aMP Cn -CA 50 against MC38 cells at pH 7.4 and 6.8 (n = 3 biological replicates). e, Confocal imaging of MC38 LAMP1-mCherry cells incubated with aMP Cn -CA 50 -FITC (160 µg ml−1) at pH 6.8 for 1 h. Scale bars, 2 µm. f–k, Time-lapse confocal imaging of MC38 Gal3-GFP cells treated with aMP Cn -CA 50 (160 µg ml−1) and PI (red) at pH 6.8. f, Representative images. Yellow arrowheads indicate the same cell at different time points. Scale bar, 5 µm. g,h, Percentage of MC38 Gal3-GFP cells exhibiting Gal3-GFP puncta (g) and PI-positive staining (h). i,j, Time for Gal3-GFP puncta appearance (i) and PI-positive staining (j). k, Time interval between Gal3-GFP puncta appearance and PI-positive staining in each cell. ND refers to not detectable. l,m, Analysis of CD8+ T cells activation. OT-I T cells were co-cultured with BMDCs that had been pre-incubated with MC38 OVA cells treated with aMP Cn -CA 50 (160 µg ml−1) at pH 6.8 for 24 h. Flow cytometry analysis of CD8+ T-cell proliferation (l) and expression of IFN-γ, granzyme B, and perforin in CD8+ T cells (m). n, Confocal imaging of MC38 LAMP1-mCherry&Gal3-GFP cells treated with GPN (320 µg ml−1) at pH 6.8 for 1 h. Scale bars, 5 and 1 µm for regular and magnified images, respectively. o, Concentration-dependent cytotoxic effects of GPN on MC38 cells upon incubation at pH 7.4 or 6.8 for 24 h. p,q, RNA-seq analysis of MC38 cells treated with GPN (320 µg ml−1) at pH 6.8 for 24 h versus untreated controls. p, Volcano scatter plots comparing gene expression levels. Upregulated (fold change >2, P < 0.05) and downregulated (fold change <0.5, P < 0.05) genes are depicted as red and blue dots, respectively. q, GSEA of transcriptional profiles using the hallmark gene set from MSigDB. NES: normalized enrichment scores. FDR: false-discovery rate. r–x, Combination of GPN and MP C16 effectively enhances the ability of MC38 cells to stimulate antigen presentation and T cell activation. r, Schematic illustration of sample preparation strategy. MC38 OVA or MC38 cells were treated with (1) GPN, (2) the combination of GPN and MP C16 , or (3) aMP C16 -CA 50 at varying concentrations for 24 h; MP C16 (160 µg ml−1) was applied for 1 h. BMDCs were co-incubated with pretreated MC38 OVA or MC38 cells, and subsequent with OT-I T cells. Comparisons were made between the combination of MP C16 and GPN versus GPN treatment (s–u), and between the combination of MP C16 and GPN versus aMP C16 -CA 50 treatment (v–x). Flow cytometry analysis of H-2Kb–SIINFEKL expression on BMDCs (s,v), CD8+ T-cell proliferation (t,w), and expression of IFN-γ, granzyme B, and perforin in CD8+ T cells (u,x). In c,l,m,o,s–x, data are mean ± s.d., and n = 3 biological replicates. In e,f,n, representative images from at least two independent experiments. Statistical significance was determined using DESeq2 (two-sided Wald tests without adjustment) (p), permutation testing with FDR for GSEA (q), one-way ANOVA (l,m), and two-way ANOVA (s–x) with Tukey’s multiple-comparison test. Diagrams in r were created using BioRender; Xiong, M. https://biorender.com/p8xag3c.