Hypoxia-induced ZEB1 promotes cervical cancer immune evasion by strengthening the CD47-SIRPα axis

Abstract

Background: The dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear.

Methods: Exosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed.

Results: Our previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα⁺ TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα⁺ TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression.

Conclusions: Our results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity.

Keywords: CD47-SIRPα axis; Cervical cancer; Exosome; Hypoxia; Immune evasion.

Fig. 1

ZEB1 expression positively correlates with CD47 and SIRPα expression in the hypoxic TME of CSCC. A Representative images of CAIX (red), ZEB1 (purple), CD47 (green) and DAPI (blue) fluorescence staining in CSCC tissues. B Representative images of CAIX (red), CD163⁺ TAMs (purple), SIRPα⁺ TAMs (green) and DAPI (blue) fluorescence staining in CSCC tissues. C Representative images of CAIX (red), SIRPα⁺ TAMs (purple), CD47 (green) and DAPI (blue) fluorescence staining in CSCC tissues. Images at 400 × magnification are shown (scale bar, 50 μm). D Statistical analysis showing the differences in the expression levels of ZEB1, CD47 and SIRPα between normoxic and hypoxic tissues. *P < 0.05 by Student’s t test. E, G The clinical relevance of ZEB1, CD47 and SIRPα was evaluated by Pearson correlation analysis

Fig. 2

Hypoxia increases CD47 expression in a ZEB1-dependent manner. A Representative images of ZEB1 (green), CD47 (red) and DAPI (blue) immunofluorescence staining in normoxic and hypoxic SiHa and C33a cells (scale bar, 50 μm). B Western blot analysis results showing the protein levels of ZEB1 and CD47 in SiHa^ZEB1/Nx, SiHa^NC/Nx, SiHa^sh−NC/Hx, and SiHa^sh−ZEB1/Hx cells. C RT‒qPCR analysis results showing the RNA levels of ZEB1 and CD47 in SiHa^ZEB1/Nx, SiHa^NC/Nx, SiHa^sh−NC/Hx, and SiHa^sh−ZEB1/Hx cells. D A dual-luciferase reporter assay system was used to demonstrate the direct binding of ZEB1 to the CD47 promoter region. *P < 0.05. The ZEB1 and NC superscripts indicate the ZEB1 overexpression and control plasmid, respectively. The sh-ZEB1 and sh-NC superscripts indicate the ZEB1 knockdown and negative control shRNAs, respectively. Nx, normoxia; Hx, hypoxia

Fig. 3

ZEB1 can be enriched in hypoxic CSCC cell-derived exosomes and transferred to macrophages. A The morphology exosomes secreted from hypoxic/normoxic SiHa and C33a cells was confirmed by transmission electron microscopy (TEM). Scale bar, 100 nm. B Positive expression of exosomal markers (CD63, EpCAM and CD81), negative expression of the exosome exclusion marker Calnexin and ZEB1 expression in exosomes was detected by western blotting. C ZEB1 expression levels in the indicated cells and paired exosomes were measured by western blotting (relative grayscale analysis). *P < 0.05 by Student’s t test. ns, no significance. D THP-1 macrophages pretreated with PKH67-labelled exosomes (green) secreted by hypoxic/normoxic CSCC cells or with PBS for 48 h were stained with phalloidin (red) and DAPI (blue) for confocal microscopy. Scale bar, 20 µm. E ZEB1 levels in THP-1 macrophages pretreated with the indicated exosomes or PBS for 48 h were detected by western blotting. *, P < 0.05. Exo, exosome; Nx, normoxia; Hx, hypoxia

Fig. 4

Hypoxia-induced exosomal ZEB1 promotes SIRPα⁺ TAM polarization through STAT3 signalling in vitro. A Western blot analysis showing the expression of ZEB1, CD163 and SIRPα in THP-1 macrophages under different conditions. B Related downstream signals for exosomal ZEB1 in THP-1 macrophages were evaluated by western blotting. C The protein levels of STAT3, p-STAT3, CD163 and SIRPα were measured in THP-1 macrophages incubated with 5,15-DPP (a specific inhibitor of STAT3 signalling) by western blotting. *, P < 0.05. Exo, exosome; Nx, normoxia; Hx, hypoxia

Fig. 5

Hypoxia-induced exosomal ZEB1 promotes SIRPα⁺ TAM polarization through STAT3 signalling in vivo. An in vivo CSCC xenograft model was established in nude mice by inoculating SiHa cells (5 × 10⁶ cells/mouse) into the flank. When the tumour volume reached 50 mm³, exosomes (10 µg) secreted by SiHa^NC/Nx, SiHa^ZEB1/Nx, SiHa^sh−NC/Hx, and SiHa^sh−ZEB1/Hx cells were injected into the tumour centres (n = 3 mice/group) every other day for three weeks. The tumour volume was recorded every 4 days. After three weeks, the nude mice were euthanized using a barbiturate overdose, and the tumours were collected for analysis. A Representative images of STAT3 (purple), CD206 (red), SIRPα (green) and DAPI (blue) fluorescence staining in primary tumours from different treatment groups. Scale bar, 100 µm. B MFI analysis showing the relative expression of CD206 and SIRPα in the tumours described in (A). *, P < 0.05. MFI, mean fluorescence intensity. C M2-like TAMs were isolated from tumours and characterized ex vivo by western blot analysis of the levels of STAT3 and p-STAT3. Exo, exosome; Nx, normoxia; Hx, hypoxia

Fig. 6

ZEB1 activity affects the phagocytosis of CSCC cells by regulating CD47 and SIRPα expression. DiI-labelled THP-1 macrophages were incubated with CFSE-labelled CSCC cells. After a 4-h incubation, phagocytic events were imaged under an inverted microscope. A THP-1 macrophages were incubated with exosomes secreted by normoxic SiHa cells transfected with the ZEB1 overexpression (ZEB1/Nx-exo) or control plasmid (NC/Nx-exo) before a phagocytosis assay was performed. B Statistical analysis showing the phagocytic index of the cells described in (A). C THP-1 macrophages were incubated with exosomes secreted by hypoxic SiHa cells infected with the ZEB1 knockdown virus (sh-ZEB1/Hx-exo) or the negative control virus (sh-NC/Hx-exo) before a phagocytosis assay was performed. D Statistical analysis showing the phagocytic index of the cells described in (C). E Phagocytosis assays were performed in the presence of an anti-CD47 blocking antibody or control IgG. F Statistical analysis showing the phagocytic index of the cells described in (E). Scale bar, 50 μm. *, P < 0.05. Exo, exosome; Nx, normoxia; Hx, hypoxia