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. 2022 Sep 5:13:954039.
doi: 10.3389/fimmu.2022.954039. eCollection 2022.

Intratumoral CD73: An immune checkpoint shaping an inhibitory tumor microenvironment and implicating poor prognosis in Chinese melanoma cohorts

Zixu Gao  1 , Lu Wang  1 , Zhengqing Song  2 , Ming Ren  1 , Yang Yang  1 , Jianrui Li  1 , Kangjie Shen  1 , Yinlam Li  1 , Yiteng Ding  1 , Yanwen Yang  1 , Yuhong Zhou  2 , Chuanyuan Wei  1 , Jianying Gu  1
Affiliations

Intratumoral CD73: An immune checkpoint shaping an inhibitory tumor microenvironment and implicating poor prognosis in Chinese melanoma cohorts

Zixu Gao et al. Front Immunol. .

Abstract

Background: As a novel immune checkpoint, CD73 has been reported to play prominent roles in several malignancies. However, the significance of CD73 in melanoma remains ambiguous. This study sought to reveal the impact of CD73 on the tumor microenvironment (TME) and patients' prognosis, and to investigate whether CD73 could be a therapeutic target in Chinese melanomas, which were dominated by acral and mucosal subtypes.

Methods: Two independent Chinese cohorts of 194 patients with melanoma were enrolled. CD73 and PD-L1 expression as well as CD8+ and CD56+ cell infiltrations were evaluated by immunohistochemistry in 194 resected melanoma samples. Clinical outcomes of patients were assessed utilizing the Kaplan-Meier plotter and Cox proportional hazard analysis. RNA-seq data was obtained from TCGA database. Gene set functional annotations were performed based on GO, KEGG and GSEA analysis. CIBERSORT, ssGSEA and TIMER were used to explore the association between CD73 and immune infiltration. These findings were validated by establishing tumor xenograft model, and functions of tumor-infiltrating immune cells were examined by flow cytometry and immunofluorescence.

Results: High CD73 expression showed poorer clinical outcomes and was identified as an independent prognostic indicator for survival in two cohorts. Expression of CD73 was more prevalent than PD-L1 in Chinese melanoma cohorts (54.6% vs 23.2%). Co-expression of both immune checkpoints was infrequent (12.9%) in melanoma, and 54.4% of PD-L1 negative cases showed elevated expression of CD73. CD73high tumors showed a microenvironment with fewer CD8+ T cells and CD56+ NK cells infiltration, which displayed a dysfunctional phenotype. With the treatment of CD73 inhibitor APCP, the amount of CD8+ T cells and CD56+ NK cells infiltrated in tumors was elevated and the immunosuppressive effect of CD73 was eliminated.

Conclusions: High CD73 expression was associated with an inhibitory TME and adverse clinical outcomes of melanoma. In comparison to PD-L1, CD73 was more prevalent and possessed more definite prognostic significance. Therefore, it may serve as a prognostic indicator and immunotherapeutic target next to PD-L1 in melanoma for Chinese population.

Keywords: CD73; CD8+ T cells; PD-L1; immunosuppressive; immunotherapy; melanoma; prognosis; tumor microenvironment.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
CD73 and PD-L1 expression in melanoma specimens. Representative micrographs of CD73 (A) and PD-L1 (B) expression within melanoma. The positive rate of CD73 in discovery and validation sets were 55.6 and 53.8% respectively (C). PD-L1 was elevated in 26.7 and 20.2% of cases in discovery and validation sets, respectively (D). No significant correlation was found between CD73 and PD-L1 expression (E).
Figure 2
Figure 2
Kaplan-Meier survival curves and forest plots for OS and DFS of patients with melanoma. High CD73 expression was significantly associated with poor overall survival (OS) and disease-free survival (DFS) in the discovery set and the relevance was confirmed in an independent validation set (A). PD-L1 expression in both of the discovery and validation sets failed to stratify OS and DFS (B). According to the Multivariate Cox regression analysis, CD73 could serve as an independent predictor of adverse clinical outcomes (C). The p-values were determined via log-rank test.
Figure 3
Figure 3
CD73 expression was markedly associated with tumorigenesis and tumor immunity. Significantly altered genes in CD73high tumor shown on volcano plot (A); Bar plots displayed the top 10 BP, MF and CC terms substantially correlated with CD73, and most of them were tumor-related and immune-related (B); The bubble plot showed the top 15 KEGG terms substantially correlated with CD73, and they were also tumor-related and immune-related (C); The results of GSEA revealed that most of the CD73-related pathways were related to tumorigenesis and activation of immune response (D).
Figure 4
Figure 4
Correlations between CD73 expression and immune infiltration levels in the TCGA cohort. The immune infiltration patterns in 471 tumor tissues arranged by CD73 expression (A). Differential analysis of immune cells between CD73high and CD73low groups in TCGA (B). Detection of the correlation between the CD73 expression level and a series of immune cells using ssGSEA analysis (C, D). CD73 expression was significantly inversely correlated with infiltration of CD8+ T cells, activated NK cells and follicular helper T cells, while considerably positively correlated with infiltration of resting memory CD4+ T cells (E). Heatmap showed the correlation between representative immune cells infiltration (F).
Figure 5
Figure 5
CD73 promoted melanoma growth and was associated with CD8+ T cell infiltration and exhaustion. Tumor xenografts of C57BL/6 mice were established by using CD73-NC and CD73-OE B16-F10 cells (A, B). CD8+T cell infiltration level in CD73high and CD73low subgroup. Expression of immune exhausted markers, activated markers and proliferative markers for total CD8+ T cells in CD73high and CD73low subgroup (C). CD8+ T cells and NK1.1+ cells infiltrated in CD73-NC and CD73-OE tumors (D, E). *p<0.05; **p<0.01; ***p<0.001; ns, no significance.
Figure 6
Figure 6
The function of CD73 inhibitor APCP on tumor growth and CD8+ T cell infiltration. Tumor xenografts of C57BL/6 mice by using B16-F10 cells were treated with PBS or CD73 inhibitor APCP (A–C). CD8+ T cell infiltration level in PBS/APCP subgroup. Expression of immune exhausted markers, activated markers and proliferative markers for total CD8+T cells in PBS/APCP subgroup (D). CD8+ T cells and NK1.1+ cells infiltrated in tumors treated with PBS and APCP (E, F). *p<0.05; **p<0.01; ***p<0.001.
Figure 7
Figure 7
Tumor infiltrating CD8+ T cells and CD56+ NK cells and their correlations between CD73 and PD-L1 expression, and prognostic value of intratumoral immune cells in CD73high and CD73low subgroups. Typical micrographs of positive CD8 (A), CD56 (B) staining and the corresponding intra-tumor negative controls. Original magnification × 40 for full views and × 400 for detailed views. Scatter plots depicted the correlation between CD8+ T cells and CD56+ NK cells and CD73 expression. High CD73 expression. was significantly correlated with lower intra-tumor counts of CD8+ T cells and CD56+ NK cells (C). PD-L1 expression had no significant correlation with counts of CD8+ T cells and CD56+ NK cells (D). Kaplan-Meier curves of OS and DFS in CD73high and CD73low subgroups according to CD8+ and CD56+ cells infiltration (E, F). *p<0.05; **p<0.01; ns, no significance.
Figure 8
Figure 8
Graphical abstract. In this study, we enrolled two cohorts of 194 melanoma patients from Zhongshan Hospital to explore the expression of CD73, PD-L1, CD8 and CD56 in their tumor tissues, and confirmed the poor OS and DFS in CD73high patient subgroup. We also investigated the functional enrichment and patterns of infiltrating immune cells related to CD73 by analyzing RNA-seq of 471 melanoma patients from TCGA (A). Furthermore, we established tumor xenograft of melanoma to explore the role of CD73 in tumor progression and its impact on TME (B). CD73high tumors showed an inhibitory TME with fewer CD8+ T cells infiltration displaying a dysfunctional phenotype, which could be reactivated when treated with CD73 inhibitor (C). OS, overall survival; DFS, disease-free survival; TCGA, the Cancer Genome Atlas; TME, tumor microenvironment.
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