Cancer cell-derived exosomal circUSP7 induces CD8+ T cell dysfunction and anti-PD1 resistance by regulating the miR-934/SHP2 axis in NSCLC

Abstract

Background: CD8⁺ T cells play a critical role in the innate antitumour immune response. Recently, CD8⁺ T cell dysfunction has been verified in various malignant cancers, including non-small cell lung cancer (NSCLC). However, the molecular biological mechanisms of CD8⁺ T cell dysfunction in human NSCLC are still unclear.

Methods: The expression of circular ubiquitin-specific protease-7 (circUSP7) in NSCLC tissues, exosomes, and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Exosomes were isolated from the culture medium of NSCLC cells and the plasma of NSCLC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit. The exosomes were then characterized by transmission electronic microscopy (TEM), NanoSight and western blotting. The role of circUSP7 in CD8⁺ T cell dysfunction was assessed by enzyme-linked immunosorbent assay (ELISA). In vivo circular RNA (circRNA) precipitation (circRIP), RNA immunoprecipitation (RIP), and luciferase reporter assays were performed to explore the molecular mechanisms of circUSP7 in CD8⁺ T cells. In a retrospective study, the clinical characteristics and prognostic significance of circUSP7 in NSCLC tissues were determined.

Results: The expression levels of circUSP7 were higher in human NSCLC tissues than in matched adjacent nontumour tissues. Increased levels of circUSP7 indicate poor clinical prognosis and CD8⁺ T cell dysfunction in patients with NSCLC. The circUSP7 found in NSCLC patient plasma is predominantly secreted by NSCLC cells in an exosomal manner, and circUSP7 inhibits IFN-γ, TNF-α, Granzyme-B and Perforin secretion by CD8⁺ T cells. Furthermore, circUSP7 inhibits CD8⁺ T cell function by upregulating the expression of Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) via sponging miR-934. Finally, we show that circUSP7 may promote resistance to anti-PD1 immunotherapy in NSCLC patients.

Conclusions: Exosomal circUSP7 is predominantly secreted by NSCLC cells and contributes to immunosuppression by promoting CD8⁺ T cell dysfunction in NSCLC. CircUSP7 induces resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for NSCLC patients.

Keywords: Anti-PD1; Exosome; NSCLC; SHP2; circUSP7; miR-934.

Fig. 1

CircUSP7 is upregulated in NSCLC tissues. a Heatmap showing USP7 gene-derived circRNAs in NSCLC tissues compared with those in matched adjacent nontumour tissues, as analysed by qRT-PCR. b Diagram of the structure of circUSP7. c Differential expression of circUSP7 in the NSCLC tissues and adjacent nontumour tissues of 126 patients. d A total of 126 patients were divided into the ≤3 cm and >  3 cm size groups. The circUSP7 expression in each group is shown in the diagram. e A total of 126 NSCLC patients were divided into groups negative for lymph node metastasis or positive for lymph node metastasis. The diagram shows circUSP7 expression in each group. f and g Kaplan-Meier analysis of the OS and recurrence of 126 patients with NSCLC according to circUSP7 expression (log-rank test). The data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001

Fig. 2

The expression level of plasma exosomal circUSP7 is upregulated in NSCLC patients. a The expression level of circUSP7 in the NSCLC tissues and preoperative plasma exosomes of NSCLC patients (R² = 0.7023; P < 0.0001). b Exosomal circUSP7 expression in the plasma of healthy donors, patients before surgery, patients after surgery, and patients with recurrence. c The expression of plasma exosomal circUSP7 in the PDX or control group. d A negative correlation between the circUSP7 expression in plasma exosomes and the proportion of CD8⁺ T cells was observed in NSCLC patient tissues (R² = 0.1080; P = 0.0002). The data are presented as the mean ± SD; n = 3, *P < 0.05, **P < 0.01

Fig. 3

Characterization of exosomes and inhibition of CD8+ T cell function by circUSP7 expression. a High-resolution TEM image of NSCLC cell-derived exosomes (scale bar = 100 nm). b The expression of exosomal biomarkers in NSCLC cell-derived exosomes was detected by western blot. c CircUSP7 expression in NSCLC cell-derived exosomes was measured by qRT-PCR. d CircUSP7 expression in plasma exosomes derived from A549 cells was modified by transfection with shRNA to knock down its expression, and circUSP7 expression in plasma exosomes derived from NCI-H460 cells was modified by transfection with cDNA to increase its expression. e The secretion of IFN-γ, TNF-α, Perforin, and Granzyme-B by CD8⁺ T cells cocultured with exosomes derived from A549 cells transfected with shRNA or exosomes derived from NCI-H460 cells transfected with cDNA was analysed by ELISA. The data are presented as the mean ± SD. **P < 0.01. NS: not significant

Fig. 4

CircUSP7 inhibits the biological function of miR-934 in CD8+ T cells. a RIP was performed for circRNA in CD8⁺ T cells using a circUSP7 probe and a NC probe. b RIP experiments were carried out in CD8⁺ T cell extracts using an anti-AGO₂ antibody. c The level of circUSP7 in the streptavidin-captured fractions from CD8⁺ T cell lysates after transfection with biotinylated miR-934 or the NC. CircANRIL was used as the negative control. d Putative binding sites of miR-934 in the circUSP7 sequence were predicted by StarBase v3.0. e The luciferase activity of pGL3-circUSP7 in CD8⁺ T cells after cotransfection with miR-934. f CircUSP7 expression in CD8⁺ T cells cocultured with exosomes derived from A549 cells and NCI-H460 cells. g The relative level of miR-934 in CD8⁺ T cells transfected with NC, shcircUSP7, mock, or circUSP7 was measured by qRT-PCR. The data are presented as the mean ± SD; **P < 0.01, ***P < 0.001

Fig. 5

CircUSP7 regulates the miR-934/SHP2 pathway in CD8⁺ T cells. a The putative binding site of miR-934 in the SHP2 sequence was predicted by StarBase v3.0. b Luciferase activity of pGL3-SHP2 in CD8⁺ T cells cotransfected with miR-934. c and d The mRNA and protein levels of SHP2 were measured by qRT-PCR and western blotting, respectively, in CD8⁺ T cells transfected with miR-934 or miR-934 combined with circUSP7. e SHP2 in representative NSCLC patients was analysed by IHC staining. f A positive correlation between circUSP7 and SHP2 mRNA expression was observed in the CD8⁺ T cells from NSCLC patients (R2 = 0.6580; P < 0.0001). g A negative correlation between circUSP7 and miR-934 expression was observed in the CD8+ T cells from NSCLC patients (R2 = 0.2470; P = 0.0052). h A negative correlation between miR-934 and SHP2 mRNA expression was observed in the CD8⁺ T cells from NSCLC patients (R² = 0.2330; P = 0.0069). The data are presented as the mean ± SD; *P < 0.05, **P < 0.01, NS: not significant

Fig. 6

CircUSP7 promotes NSCLC progression in a CD8⁺ T cell-dependent manner and leads to resistance to anti-PD1 therapy. a The relative expression levels of circUSP7 in the serum exosomes from HuNSG mice. b and c The data are expressed as the mean tumour volume (the data are presented as the mean ± SD; n = 6). d The data are expressed as the percent of tumours with inhibited growth (the data are presented as the mean ± SD; n = 6). e Comparison of the OS curves for mice with xenograft NSCLC tumours with high and low circUSP7 expression that were treated with Opdivo. f The expression of circUSP7 in the CD8⁺ T cells from NSCLC patient tissues (n = 30). g CD8-positive cells in NCI-H460-circUSP7 or NCI-H460-mock cell-derived tissues were analysed by IHC. The data are presented as the mean ± SD; *P < 0.05, **P < 0.01

Fig. 7

Upregulation of CircUSP7 induces cytotoxic T lymphocyte exhaustion by interacting with the miR-934/SHP2 axis in NSCLC patients. a CD8 in tissues from representative NSCLC patients was analysed by IHC staining. b CD8⁺ T cells in 126 pairs of NSCLC tissues and matched nontumour tissues, shown as log2 (tumour/nontumour). c A positive correlation between circUSP7 expression and CD8-positive cell number was observed in the NSCLC tissues (R² = 0.1088; P = 0.0002). d A negative correlation between SHP2 expression and CD8-positive cell number was observed in the NSCLC tissues (R² = 0.1594; P < 0.0001). e A positive correlation between circUSP7 and SHP2 mRNA was observed in NSCLC tissues (R² = 0.2183; P < 0.0001). f The expression level of circUSP7 in the plasma exosomes of these patients after anti-PD1 treatment cycles. The data are presented as the mean ± SD; *P < 0.05, **P < 0.01