Evasion of immunosurveillance by the upregulation of Siglec15 in bladder cancer

Abstract

Immunotherapy resistance in bladder cancer (BLCA) is associated with elevated levels of sialic acid–binding immunoglobulin-like lectin (Siglec15). This protein plays a crucial role in fostering a noninflammatory tumor microenvironment (TME), which is conducive to cancer progression. Our study confirmed that the overexpression of Siglec15 led to a reduction in CD8⁺ T cell infiltration. This effect was mediated by the downregulation of pro-inflammatory cytokines and chemokines, which in turn exacerbated BLCA malignancy. Furthermore, Siglec15 inhibited the cytotoxicity of effector T cell, contributing to immune evasion. An in vivo study demonstrated that Siglec15 overexpression induced a non-inflammatory TME and promoted resistance to immunotherapy. These findings highlight Siglec15 as a potential therapeutic target for BLCA. By modulating inflammation in the TME and CD8⁺ T cell function, targeting Siglec15 may offer a novel strategy for overcoming immunotherapy resistance and improving patient outcomes.

To editor

Immunotherapy has shown promise in bladder cancer (BLCA) [1, 2], due to its high immunogenicity and tumor mutation burden, however, most patients develop resistance [3,4,5]. Siglec15, a sialic acid-binding protein [6], promotes tumor growth by inhibiting CD8⁺ T cells and facilitating immune evasion [7,8,9]. We previously reported that Siglec15 levels inversely correlate with tumor immune status and predict response to immune checkpoint inhibitors in BLCA [10]. Further validation is required, prompting our investigation into the role of Siglec15 in the BLCA immune microenvironment.

Results and discussion

Siglec15 is overexpressed in BLCA cells, but not in immune or stromal cells within the TME

We evaluated Siglec15 expression in two single-cell cohorts, showing enrichment in BLCA epithelial cells and negligible expression in other TME components (Supplementary Fig. 1A, B). Multicolor immunofluorescence confirmed Siglec15 colocalization with CK19 in tumors, but not adjacent tissues (Supplementary Fig. 1C). Cancer tissues showed increased CK19⁺, Siglec15⁺, and double-positive cells compared to normal tissues, with no significant differences in other immune subsets (Supplementary Fig. 1D). Further analysis confirmed Siglec15 expression in CK19⁺ tumor cells (Supplementary Fig. 1E). In a tissue microarray of 45 BLCA samples, Siglec15 was positive in 50.37% of cases, with 31.44% of cells both Siglec15⁺ and CK19⁺ (Supplementary Fig. 1F), indicating a specific Siglec15 expression pattern in tumor cells.

Siglec15 regulates chemokine expression and lymphocyte migration in BLCA

We overexpressed Siglec15 in human T24 cells and mouse MB49 and MBT2 BLCA cells and knocked down in T24 cells (Supplementary Fig. 2A–D). GO and KEGG analyses revealed disrupted leukocyte migration and immune pathways following Siglec15 modulation (Supplementary Fig. 2E–F). High-throughput liquid-phase protein chip detection showed Siglec15 suppressed cytokine/chemokine secretion by T24 cells, whereas Siglec15 knockdown increased secretion (Fig. 1A). Cytokines levels varied in the supernatants of Siglec15-modulated T24 cells (Supplementary Fig. 2G), with decreasing in Siglec15-overexpressing cells and increasing in knockdown cells at both mRNA and protein levels (Fig. 1B, Supplementary Fig. 2H). These findings were validated in MB49 and MBT2 cells (Supplementary Fig. 3A–F), confirming the role of Siglec15 in modulating cytokine/chemokine secretion in BLCA.

Fig. 1

Evasion of immunosurveillance by the upregulation of Siglec15 in BLCA. A High-throughput liquid-phase protein chip detection shows the effects of different Siglec15 expression levels on the concentrations of 34 cytokines. B The effects of different Siglec15 expression levels on CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10 were determined by reverse transcription‒quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. C Transwell chemotaxis assay process diagram. D Natural killer cell, CD4⁺ T cell, and CD8⁺ T cell recruitment decreased with Siglec15 overexpression. E Panoramic scanning was used to analyze the co-expression and spatial distance between each cell. F Spatial distribution of CD8⁺ T cells and Siglec15⁺/CK19⁺ cells. G Schematic diagram of the CD8⁺ T cell and tumor cell coculture killing assay. H Residual tumor cells were stained with crystal violet. I Flow cytometry analysis of the expression levels of TNF-α and IFN-γ in CD8⁺ T cells. J Schematic diagram of the in vivo experimental process. K Tumor tissue photos at the endpoint of treatment. L Tumor growth curves of the mice in the different treatment groups. M Tumor volume statistics at the endpoint of treatment. N Survival curves of the mice in the different treatment groups. O Flow cytometry analysis of the expression levels of TNF-α, GZMB and Ki67 in different groups of tumor tissues. P Expression of cytokines in CD8⁺ T cells within the tumor tissues of different treatment groups. Q Immunofluorescence staining revealed different levels of CD8⁺ T-cell infiltration in the tumor tissues of different treatment groups. R Quantitative statistical analysis of the number of CD8⁺ T cells in Q

Siglec15 promotes a noninflamed immune microenvironment in BLCA

Transwell chemotaxis assays (Fig. 1C) revealed that Siglec15 overexpression reduced immune cell recruitment (Fig. 1D), while knockdown increased it (Supplementary Fig. 4A). Panoramic scanning (Fig. 1E) correlated Siglec15⁺ tumors and CD8⁺ T cell infiltration negatively (Fig. 1F, Supplementary Fig. 4B–C). Siglec15 overexpression hindered CD8⁺ T cell infiltration, while other immune cells showed different patterns. CD8⁺ T cell infiltration increased with distance from Siglec15⁺/CK19⁺ cells (Supplementary Fig. 4D), highlighting the role of Siglec15 in modulating CD8⁺ T cell infiltration. These results were validated in three independent cohorts: E-MTAB-4321, GSE48075, and GSE69795 (Supplementary Fig. 5–7).

Siglec15 promotes tumor growth, immune escape and immunotherapy resistance in vitro and in vivo

Siglec15 enhanced proliferation, invasion, and migration of BLCA cells whereas knockdown suppressed these processes (Supplementary Fig. 8A–E). Coculture assays (Fig. 1G) showed that Siglec15-knockdown cells were more susceptible to T cell killing (Fig. 1H), whereas Siglec15 overexpression inhibited cytotoxicity of T cell by suppressing TNF-α and IFN-γ (Fig. 1I).

In the allografted BLCA mouse model, Siglec15 overexpression led to larger tumors and immunotherapy resistance (Fig. 1J–N and Supplementary Fig. 8F). Flow cytometry and immunofluorescent staining showed that Siglec15 overexpression suppressed CD8⁺ T cell infiltration and function, which was partially restored by a PD-1 inhibitor (Fig. 1O–R, Supplementary Fig. 8G).

Siglec15 overexpression correlated with shorter survival time in patients with nasopharyngeal adenocarcinoma and glioblastoma, receiving immunotherapy, and showed a trend in urothelial cancer (Supplementary Fig. 8H–J). These findings highlight the role of Siglec15 in immune evasion and CD8⁺ T cell suppression in immunotherapy resistance.

Siglec15 levels correlate with a non-inflammatory TME and immunotherapy resistance in BLCA [10]. Siglec15 overexpression inhibits cytokines, hinders CD8⁺ T cell recruitment and inhibits CD8⁺ T cells cytotoxicity [10,11,12]. It promotes tumor progression and drives resistance to PD-1 inhibitors. Anti-Siglec15 therapy may enhance CD8⁺ T cell infiltration and improve immune checkpoint inhibitor efficacy.

Conclusions

This study confirms Siglec15 as a novel immunotherapeutic target for BLCA, inhibiting CD8⁺ T cell immunity in the TME and promoting immune escape and immunotherapy resistance.