Siglec-15 Promotes Progression of Clear Renal Cell Carcinoma
Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma, accounting for approximately 80% of cases. While early-stage ccRCC can often be cured through surgical intervention, a significant proportion of patients are diagnosed at advanced stages, with over 30% presenting with distant metastases at the time of diagnosis. For these patients, the prognosis is grim, with a 5-year survival rate of only 12%. Immunotherapy has emerged as a promising treatment strategy, particularly for advanced ccRCC, by selectively correcting tumor microenvironment (TME) immunity. However, only a small subset of patients responds effectively to current immunotherapy regimens, underscoring the need for novel therapeutic targets.
Siglec-15, a sialic acid-binding immunoglobulin-like lectin, has recently garnered attention as a potential immunotherapy target. Drugs targeting Siglec-15 are currently under development, and previous studies have shown that Siglec-15 operates independently of the B7-H1/PD-1 pathway. Despite this, the expression and role of Siglec-15 in ccRCC remain poorly understood, particularly at the translational level. This study aimed to elucidate the expression of Siglec-15 in ccRCC at both transcriptional and translational levels and to explore its association with clinicopathological prognostic parameters.
The study was conducted with approval from the Ethics Committee of Peking University People’s Hospital. Informed consent was obtained from all participants. A tissue microarray was constructed using 150 ccRCC samples and 30 paired adjacent normal tissues. These samples were supplemented with immunohistochemistry (IHC) staining scores for programmed death ligand-1 (PD-L1) and B-cell lymphoma-2 (BCL-2), obtained from Shanghai Outdo Biotech. Additionally, 32 ccRCC samples and 25 adjacent normal renal tissues were collected from the study center, and fibrosis levels were quantified using second harmonic generation/two-photon excitation fluorescence (SHG/TPEF). The SHG signal intensity positively correlates with collagen deposition, indicating abnormal tissue fibrosis.
Siglec-15 expression was evaluated using a polyclonal antibody in IHC staining. The total staining score was calculated as the product of the positive staining rate score and the staining intensity score. The positive staining rate was scored based on the percentage of positive cells, while the staining intensity was graded by two independent pathologists. RNA sequencing data and clinical information from 537 ccRCC patients and 72 adjacent normal tissues were obtained from The Cancer Genome Atlas (TCGA). The median expression level of Siglec-15 was used as the cutoff to divide patients into high-expression and low-expression groups.
To investigate the relationship between Siglec-15 and immune cell infiltration in ccRCC, the xCell method was employed to analyze the transcriptome profiles of 534 tumors. This method deconvolutes immune, stromal, and microenvironmental cell gene characteristics. Higher immune scores and lower stromal scores were associated with increased CD8+ T cell infiltration and elevated expression of immune therapy markers such as PD1, PD-L1, PD-L2, and CTLA4.
Statistical analyses were performed using SPSS, R software, and GraphPad Prism. Continuous data were presented as mean ± standard error. Differences in Siglec-15 expression between tumor and adjacent normal tissues were analyzed using paired t-tests. The association between pathological grade and Siglec-15 expression was evaluated using chi-square tests. Co-expressed genes with Siglec-15 in TCGA data were analyzed using the Limma package, and functional annotation analysis was conducted using the Metascape website. Bivariate correlation and linear regression were used to correlate Siglec-15 IHC scores with PD-L1 and BCL-2 expression. Kaplan-Meier survival analysis was performed to assess survival differences, with overall survival time calculated from the date of surgery until death.
The study found that Siglec-15 was frequently expressed in ccRCC, with localization in both tumor and stromal cell membranes. Siglec-15 was translationally overexpressed in tumor tissues compared to adjacent normal tissues in both the tissue microarray and the study center’s cohort. Notably, Siglec-15 expression was not significantly correlated with PD-L1 or BCL-2 expression. High expression of Siglec-15 was associated with a higher Fuhrman grade at both the protein and transcript levels, but no significant correlations were found with sex, age, or tumor stage. Multivariate analysis indicated that Siglec-15 overexpression was an independent risk factor for higher Fuhrman grades.
Kaplan-Meier survival analysis revealed that patients with high Siglec-15 expression had shorter overall survival periods, although this was not statistically significant at the transcript level. However, a statistically significant decrease in overall survival was observed at the protein level. Overexpression of Siglec-15 at the transcript level was positively associated with higher immune and microenvironment scores but negatively associated with stromal scores. This suggests that the TME in patients with high Siglec-15 expression is significantly immunosuppressed, potentially making them more responsive to immunotherapy.
Further analysis using the xCell method showed that Siglec-15 expression was positively correlated with the infiltration of tumor-associated macrophages and tumor-associated fibroblasts in ccRCC. Additionally, Siglec-15 was inversely associated with tumor fibrosis levels, as quantified by SHG/TPEF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Pathway analyses indicated that Siglec-15 may be involved in myeloid leukocyte activation, immune response, cell adhesion regulation, extracellular matrix organization, and supramolecular fiber organization.
Siglec-15 shares a protein sequence similarity with PD-L1 and has been implicated in osteoclast differentiation, making it a potential therapeutic target for osteoporosis. Recent studies have also highlighted its role in inhibiting tumor immune responses by binding to T cell receptors and suppressing T cell activity. Genetic ablation or antibody blockade of Siglec-15 has been shown to enhance anti-tumor immunity and inhibit tumor growth in non-small-cell lung cancer. Importantly, Siglec-15 may serve as a primary immunosuppressive factor in PD-L1-negative tumors, offering a potential immunotherapy option when PD-1/PD-L1 treatments are ineffective.
This study demonstrated that Siglec-15 is frequently expressed in ccRCC and is significantly overexpressed in tumor tissues compared to adjacent normal tissues. Siglec-15 expression was positively associated with higher pathology grades and poorer prognosis, a finding not previously reported. The lack of correlation between Siglec-15 and PD-L1 expression suggests that Siglec-15 could be a valuable immunotherapy target, particularly for patients who do not respond to PD-1/PD-L1 inhibitors.
Previous proteomic studies have identified ccRCC with higher immune scores and lower stromal scores as the CD8+ inflamed subtype, characterized by elevated expression of immunosuppressive molecules. This study found that high Siglec-15 expression was associated with higher immune scores and lower stromal scores, further supporting its role in immunosuppression. The positive correlation between Siglec-15 expression and the infiltration of tumor-associated macrophages and fibroblasts underscores its involvement in shaping the TME. Additionally, the inverse relationship between Siglec-15 and tumor fibrosis highlights its potential role in the fibrosis process, which is associated with poor prognosis in ccRCC.
The study has several limitations. First, it did not include biological experiments to validate the role of Siglec-15 in ccRCC in vivo or in vitro. Second, the study was initiated without access to a commercial monoclonal antibody against Siglec-15. Finally, the sample sizes for IHC and SHG/TPEF analyses were relatively small, although the use of samples from different centers enhances the reliability of the findings.
In conclusion, this study provides compelling evidence that Siglec-15 is a potential immunotherapy target in ccRCC. Its frequent expression in tumor tissues, association with higher pathology grades, and role in immunosuppression make it a promising candidate for further research and therapeutic development. The findings suggest that targeting Siglec-15 could expand the immunotherapy benefit cohort for ccRCC patients, particularly those who do not respond to existing treatments.
doi.org/10.1097/CM9.0000000000001752
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