Fig. 2
Accumulation of circTMEM181 forms an anti-PD1-resistant immune status in the HCC tumor microenvironment. a Levels of circTMEM181 were measured by qPCR in six different HCC cell lines. b Representative images of Transwell assays show metastatic ability after circTMEM181 manipulation in the two HCC cell lines (Left). Statistical results of Transwell assays are shown (Right, t test, ns: not significant). c Representative images of colony formation assay with crystal violet staining reveal the effects of circTMEM181 on the two HCC cell lines (Left). Statistical results of the colony formation assay are shown (Right, t test, ns: no statistical significance). d Representative fluorescence photography of the two groups at 22 days after anti-PD1 therapy in the orthotopic xenograft model of liver cancer (α-PD1: anti-PD1 therapy) (t test, **: p < 0.01). e Survival curves of six mice per group are shown (logrank test; death or tumor volume > 2.5 cm3 were defined as event happened). f H22 cell line overexpressing circTMEM181 (H22OE) shows more metastatic lung nodules than the control group (H22ctrl) in the C57 mouse model (n = 6 mice for each group, t test). g tSNE plot and statistical results depict different clusters of CD45+ tumor-infiltrating leukocytes from the H22OE and H22ctrl group after anti-PD1 therapy. h Representative mIHC images show macrophages and CD8+ T cells in tumors of the H22OE and H22ctrl group (Left). Statistical results show less CD8+ T cells and more CD163+ F4/80+ macrophages in the tumor microenvironment in the H22OE group than in the H22ctrl group after anti-PD1 therapy (t test). i IHC for CD4 T cells (CD4+), CD8 T cells (CD8+), NK cells (CD56+), B cells (CD19+), macrophages (CD68+), and M2 macrophages (CD163+) from two representative patients with different circTMEM181 expression from our 204 patient cohort. j Statistical results show a positive correlation between CD163+ macrophages and circTMEM181, but a negative correlation between CD8+ T cells and circTMEM181 in our 204 patient cohort (Pearson correlation)