Fig. 2

Exosomal circZNF451 reshapes the TME by inducing M2 polarization of macrophages and exhausting CD8⁺ T cells. A Infiltration of CD4⁺ T, CD8⁺ T, Foxp3⁺Treg, CD56⁺NK and CD11C⁺dendritic cells and CD86⁺ and CD163⁺ macrophages in the TMA containing the tumor tissues of 113 LUAD patients was analyzed by IHC; scale bar, 100 μm. A Spearman correlation analysis was applied. B LLC-circZNF451 and LLC-mock cells (5 × 10⁶) were subcutaneously implanted into C57BL/6 J mice (n = 5 per group). Tumor volumes were measured every 3 days for 2 weeks. Tumor volume equals 1/2 length × width². C LLC-circZNF451 and LLC-mock cells were subcutaneously implanted into C57BL/6 J mice (n = 5 per group), and survival was recorded. The Kaplan–Meier and log-rank tests were used for comparison. D After subcutaneous implantation of LLC-circZNF451 and LLC-mock cells, immune infiltration profiles, including CD8⁺ T cells, CD4⁺ T cells, Treg cells, NK cells, DCs, and TAMs, in the tumor tissue were detected by flow cytometry. E Immune checkpoints, including PD1, TIM3, and TIGIT, in CD8⁺ T cells were detected by flow cytometry in the circZNF451 and WT groups. F Cytotoxic activities of CD8⁺ T cells, including IFN-γ and GZMB, were detected by flow cytometry in the circZNF451 and WT groups. G Infiltration of M1 (F4/80⁺CD11b⁺CD86⁺) and M2 (F4/80⁺CD11b⁺CD163⁺) polarized macrophages in tumor tissues of circZNF451 and WT groups was detected by flow cytometry. H Diagram of the coculture model. H1395-circZNF451/control cells (5 × 10⁵) were cocultured with macrophages (1 × 10⁶; PMA stimulated THP-1 cells for 24 h), or CD8⁺ T cells (1 × 10⁵) from healthy donors for 48 h. I The M2 phenotype of macrophages (CD163⁺) after coculture with H1395-circZNF451 cells for 48 h was detected by flow cytometry. J Cytokine and chemokine profiles in the supernatant of LPS-stimulated macrophages were measured by the human cytokine/chemokine microarray after coculturing with H1395-control and H1395-circZNF451 cells for 48 h. B, D, E–G and I were analyzed using a two-tailed, Student’s t test. All experiments with statistical analysis have been repeated for at least three times