Fig. 5
Spatially isolated activation of the ATP–adenosine pathway by macrophages and HCC cell cooperation impairs antitumor immunity. a WB analysis shows CD39, CD73, and GAPDH expression across the six HCC cell lines (Huh-7, PLC/PRF/5, Li-7, HepG2, 97H, and HCCLM3) and THP-1circOE. b Flow cytometry analysis shows CD39 expression on THP-1 or TAM increased after co-culturing with Li-7OE, but was rescued after incubating with GW4869. CD73 was detected on Li-7OE co-cultured with/without THP-1, but not detected on TAM or THP-1 with/without co-culturing conditions. c Heatmap shows the level of eATP, AMP, and ADO in medium with different conditions. d Detection of the function of sorted TAM after co-culturing with Li-7OE or with CD39 inhibitor POM-1. TAM showed an M2 polarization, expressing higher CD163, secreting more IL-10 and less TNFα after co-culturing with Li-7OE (one-way ANOVA: ***: p < 0.001, **: p < 0.01, *: p < 0.05). e, f Flow cytometry analysis and statistics show proliferation of CFSE-labeled CD8+ T cells in medium with different conditions. Top left to right: CD8+ T cells cultured with the supernatant from THP-1 (Ctrl), CD8+ T cells cultured with ADO (ADO), CD8+ T cells cultured with the supernatant from THP-1 and Li-7OE co-cultured medium (SPNT(Co)), CD8+ T cells cultured with the supernatant from THP-1 and Li-7OE co-cultured medium pre-adding POM1 (SPNT(Co + POM1)). Bottom left to right: CD8+ T cells cultured with Li-7OE (Ctrl(co-Li-7OE)), CD8+ T cells cultured with Li-7OE and AMP (AMP(co-Li-7OE)), CD8+ T cells cultured with Li-7OE and ADP (ADP(co-Li-7OE)), CD8+ T cells cultured with Li-7OE and ATP (ATP(co-Li-7OE)) (one-way ANOVA: ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: not significant). g Flow cytometry analysis of PD1, TIM3 expression on CD8+ T cells with/without ADO. (MFI, mean fluorescent intensity; t test: **: p < 0.01, *: p < 0.05). h Multi-label immunofluorescence showing CD39 expression (red) on CD68+ macrophages (purple), and CD73 expression (yellow) on CK8+ HCC tumor cells (Green) in the HCC tumor microenvironment (white arrow indicates spatial isolated CD73+ CK8+ HCC cells and CD39+ CD68+ macrophages; dotted line circle indicates the niches where CD39+ CD68+ macrophages are surrounded by CD73+ CK8+ HCC tumor cells). i Graphical abstract showing the activation of the spatial isolated ATP–adenosine pathway by tumor–macrophage communication in the HCC tumor microenvironment