Research ArticleOncology Open Access | 10.1172/JCI180282

Ru M. Wen,1 Jessica C. Stark,2,3 G. Edward W. Marti,4 Zenghua Fan,5,6 Aram Lyu,5,6 Fernando Jose Garcia Marques,7,8 Xiangyue Zhang,9 Nicholas M. Riley,3 Sarah M. Totten,7,8 Abel Bermudez,7,8 Rosalie Nolley,1 Hongjuan Zhao,1 Lawrence Fong,5,6 Edgar G. Engleman,9 Sharon J. Pitteri,7,8 Carolyn R. Bertozzi,2,10 and James D. Brooks1,8

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

Find articles by Zhang, X. in: JCI | PubMed | Google Scholar

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

Find articles by Nolley, R. in: JCI | PubMed | Google Scholar

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

Find articles by Zhao, H. in: JCI | PubMed | Google Scholar

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

Find articles by Fong, L. in: JCI | PubMed | Google Scholar

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

Find articles by Engleman, E. in: JCI | PubMed | Google Scholar |

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

Find articles by Pitteri, S. in: JCI | PubMed | Google Scholar

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

Find articles by Bertozzi, C. in: JCI | PubMed | Google Scholar

1Department of Urology,

2Department of Chemistry, and Sarafan ChEM-H, Stanford University, Stanford, California, USA.

3Department of Biological Engineering, Department of Chemical Engineering, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

4Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

5Department of Medicine, UCSF, San Francisco, California, USA.

6Parker Institute for Cancer Immunotherapy, San Francisco, California, USA.

7Department of Radiology,

8Canary Center at Stanford for Cancer Early Detection, and

9Department of Pathology, Stanford University, Stanford, California, USA.

10Howard Hughes Medical Institute, Stanford, California, USA.

Address correspondence to: Ru M Wen, Department of Urology, Stanford University School of Medicine, 3172 Porter Dr, Palo Alto, California 94304 Email: r.wen@stanford.edu. Or to: James D Brooks, Center of Academic Medicine, 453 Quarry Rd, Urology-5656, Palo Alto, California 94304. Phone:650.725.5746. Email: jdbrooks@stanford.edu.

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Published October 22, 2024 - More info

Published in Volume 134, Issue 24 on December 16, 2024
J Clin Invest. 2024;134(24):e180282. https://doi.org/10.1172/JCI180282.
© 2024 Wen et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 22, 2024 - Version history
Received: February 13, 2024; Accepted: October 15, 2024 View PDF Abstract

Prostate cancer is the second leading cause of male cancer death in the U.S. Current immune checkpoint inhibitor–based immunotherapies have improved survival for many malignancies; however, they have failed to prolong survival for prostate cancer. Siglecs (sialic acid–binding immunoglobulin-like lectins) are expressed on immune cells and regulate their function. Siglec-7 and Siglec-9 contribute to immune evasion in cancer by interacting with sialic acid–containing glycoprotein ligands on cancer cells. However, the role of Siglec-7/9 receptors and their ligands in prostate cancer remains poorly understood. Here, we find that Siglec-7 and Siglec-9 are associated with poor prognosis in patients with prostate cancer and are highly expressed in myeloid cells, including macrophages, in prostate tumor tissues. Siglec-7 and -9 ligands were expressed in prostate cancer cells and human prostate tumor tissues. Blocking the interactions between Siglec-7/9 and sialic acids inhibited prostate cancer xenograft growth and increased immune cell infiltration in humanized mice in vivo. Using a CRISPRi screen and mass spectrometry, we identified CD59 as a candidate Siglec-9 ligand in prostate cancer. The identification of Siglec-7 and -9 as potential therapeutic targets, including the CD59/Siglec-9 axis, opens up opportunities for immune-based interventions in prostate cancer.

Graphical Abstract Introduction

Prostate cancer (PCa) is one of the most prevalent cancers in men worldwide, with a 5-year survival rate of under 30% for men with metastatic disease, despite treatment with androgen deprivation therapies (1–3). While immune checkpoint inhibitor–based (ICI-based) immunotherapies have quickly become the standard of care for several malignancies (4, 5), they have been ineffective in treating PCa, except for a small subset (approximately 1%) with mismatch repair gene mutations (6–9). However, dendritic cell therapies, such as sipuleucel-T, can prolong survival in many advanced PCas and suggest that alternative immune checkpoint pathways could be utilized during disease development and progression (10, 11).

Siglecs, sialic acid–binding immunoglobulin-like lectins, are a family of glycan-binding immunoreceptors that recognize glycans or glycoconjugates bearing the sialic acid monosaccharide (12). The Siglec family is composed of both activating and inhibitory receptors, which each bind discrete sialoglycan ligands. The majority of CD33-related Siglecs function as inhibitory receptors, including Siglec-5, Siglec-8, Siglec-7, Siglec-9, and Siglec-10 (13–16). Siglec-7 and Siglec-9 are expressed by NK cells, monocytes, dendritic cells (DCs), macrophages, and subsets of T cells in the tumor microenvironment (17–22), and a growing body of evidence implicates these Siglecs as potential immune checkpoints in cancer (23–28).

Aberrant expression of sialic acid–containing glycans (sialoglycans) by cancer cells was initially observed in 1960s (29). More recently, these sialoglycans have been identified as Siglec ligands that can bind directly to Siglec receptors on immune cells in the tumor microenvironment and cause immune suppression (27, 30–32). For example, sialylated glycans on the surface of melanoma, lung cancer, and pancreatic cancer cells have been shown to bind to Siglec-7/9 on immune cells to inhibit the immune response (20, 33, 34). Previous work has demonstrated that cancer cell lines PC3 and DU145 express ligands for Siglec-7 and -9 (35, 36); however, the role of the Siglec-7 and -9–sialic acid pathway in PCa remains poorly understood.

To better understand whether Siglec-7 and -9 could have a role in immune-suppressive pathways in PCa, we analyzed the expression profiles of Siglec isoforms in patient samples and examined their correlation with clinicopathological parameters. We also examined the expression levels of Siglec-7 and Siglec-9 ligands in PCa cells and tumors and interrogated their functional effects in vivo. Finally, we used discovery-based approaches to identify candidate Siglec ligands expressed by PCa cells and evaluated their potential as therapeutic targets.

Results

Elevated Siglec-7/9 expression is correlated with higher recurrence rates in patients with PCa. To determine the association of Siglec-7 and Siglec-9 expression with recurrence-free survival (RFS) in PCa, we assessed the association of Siglec expression with clinical outcomes in publicly available datasets. In the Cancer Genome Atlas–Prostate Adenocarcinoma (TCGA-PRAD) dataset, transcript levels of Siglec-1, -6, -7, -9, -15, and -16 were significantly higher in tumor samples compared with normal tissues (Figure 1, A and B and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI180282DS1). Increased expression levels of Siglec-1, Siglec-7, and Siglec-9 were associated with higher cancer grade (Gleason scores) in PCa (Figure 1, C and D and Supplemental Figure 2). Moreover, the expression levels of Siglec-7 and Siglec-9 showed a significant association with RFS in 488 PCa patients (Figure 1E). Patients with high expression levels of Siglec-7 and Siglec-9 also exhibited worse RFS outcomes in a cohort of 140 patients with PCa in the Memorial Sloan Kettering Cancer Center (MSKCC)dataset (37) (Figure 1F), suggesting Siglec-7 and Siglec-9 may play a promoting role in PCa progression.

Figure 1

High Siglec-7/9 expression is correlated with worse clinical outcome in patients with PCa. Violin plots showing that (A) Siglec-7 and (B) Siglec-9 mRNA expression are significantly higher in tumor tissues (n = 497) than normal tissues (n = 53). Data were analyzed by unpaired student’s t test. (C) Siglec-7, and (D) Siglec-9 are correlated with Gleason Score. Data were analyzed by 1-way ANOVA with post hoc Tukey’s test and presented as mean ± SEM. (E) High Siglec-7 and Siglec-9 expression is correlated with worse survival in patients with PCa in the TCGA-PRAD database (n = 488). (F) High Siglec-7 and Siglec-9 expression are correlated with worse survival in patients with PCa in MSKCC database (n = 140). Survival analysis was conducted using the Kaplan–Meier (log-rank test). Median expression was used as cutoff between high and low Siglec-7 and -9 expressing groups. (G) Siglec-7, and (H) Siglec-9 are highly expressed in monocyte, dendritic cells, and CD8+ T cells identified by CIBERSORTx from the TCGA-PRAD database. (I and J) Scatter plot of RNA expression of (I) Siglec-7 and (J) Siglec-9 compared with CD68 in prostate tumor tissues (n = 494), according to TCGA data. Siglec-7, and Siglec-9 are correlated with macrophage marker CD68. Data were analyzed by Pearson’s correlation. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.0001.

To gain insights into the immune cell populations associated with Siglec expression and clinical outcomes, CIBERSORTx, an analytical tool employing machine learning techniques, was utilized to deconvolute the composition of cell populations based on gene expression data (38). CIBERSORTx analysis of TCGA-PRAD dataset showed that tumor infiltrating immune cells expressed different Siglec profiles in PCa (Supplemental Figure 3). Specifically, DCs, monocytes, and CD8+ T cells were predominantly associated with the expression of Siglec-7 and Siglec-9, while tumor infiltrating neutrophils expressed high levels of Siglec-9 (Figure 1, G and H). Due to the limitations of immune cell type definitions by the CIBERSORTx algorithm, Siglec-7 and Siglec-9 expression in certain immune cells, including macrophages, could not be identified. Therefore, we performed a Pearson correlation analysis between Siglec-7 and Siglec-9 expression and a macrophage marker and found that both are correlated with the macrophage marker CD68 (Figure 1, I and J). These results suggest that Siglec-7 and Siglec-9 may play an important role in immune cell functions in the PCa tumor environment.

High Siglec-7/9 expression is found in myeloid cells in human PCa tumors. To elucidate the Siglec profile of PCas, we analyzed 3 different stages of PCa, including 5 primary tumors (localized), 14 metastatic hormone-sensitive tumors and 6 metastatic castration-resistant tumors by single-cell RNA-Seq (scRNA-seq). The cells were annotated (Figure 2A) using published gene signatures (39, 40). Uniform manifold approximation and projection (UMAP) visualization showed that Siglec-7 and -9 are exclusively expressed on immune cells (Figure 2, B and C), mainly on myeloid cells, including macrophages,and myeloid-derived suppressor cells (MDSCs), and sparsely expressed on CD4+ T and CD8+ T cells (Figure 2, B–E and Supplemental Figure 4) in castration-resistant PCa (CRPC) tumor tissues. Siglec-7 was also highly expressed on DCs and NK cells (Figure 2D). Furthermore, Siglec-7 and Siglec-9 were observed in all stages of PCa, including localized, hormone-sensitive PCa (HSPC) and CRPC (Figure 2, F–H and Supplemental Figures 5 and 6). Additionally, Siglec-10 was highly expressed in macrophages, MDSCs, and B cells across all stages of PCa (Figure 2H and Supplemental Figure 4–6). To validate the expression of Siglec-7 and Siglec-9 on immune cells in PCa, we performed immunofluorescence and confocal microscopy on PCa metastases in bone tissues and found Siglec-7 and Siglec-9 expression on macrophages, with a colocalization coefficient of approximately 65% with CD68 (Figure 2, I and J).

Figure 2

Siglec-7 and Siglec-9 are coexpressed on myeloid cells in human prostate tumors by single-cell RNA-seq. (A) UMAP plot showing the distribution of cell types. (B) UMAP profiles highlighting Siglec-7 expression, and (C) Siglec-9 expression in immune cells of CRPC tumor tissues (n = 6). (D) Siglec-7 is predominantly expressed in dendritic cells (DCs), macrophages, myeloid-derived suppressor cells (MDSCs), and natural killer (NK) cells in CRPC. (E) Siglec-9 is primarily expressed in macrophages and MDSCs in CRPC specimens. (F) Siglec-7 and (G) Siglec-9 are expressed in macrophages in human tumor tissues from patients with localized PCa (n = 5), metastatic HSPC (n = 14), and metastatic CRPC (n = 6). (H) Dot plot illustrates fractional profiles of Siglec expression in immune cells across localized PCa (n = 5), metastatic HSPC (n = 14), and metastatic CRPC tumor tissues (n = 6). (I) Confocal microscopy images of a PCa bone metastasis showing the coexpression of Siglec-7 and Siglec-9 on macrophages, observed at 40 × magnification. (J) Colocalization coefficient of Siglec-7 or Siglec-9 with the macrophage marker CD68 (n = 7).

Sialylated glycans are detected in PCa cells. To explore the presence of sialic acids on the surface of PCa cells, we conducted a sialic acid fluorometric assay using an enzyme-coupled reaction in which the oxidation of free sialic acid generates an intermediate that reacts with a probe, resulting in the production of a detectable fluorescent product. Cell surface sialic acids were present in all tested PCa cell lines (Figure 3A). Furthermore, treatment with sialidase, an enzyme that cleaves sialic acids, led to a significant reduction in surface sialic acid levels in all PCa cells (Figure 3, B–D and Supplemental Figure 7, A and B).

Figure 3

Sialic acid is expressed on the surface of PCa cells. (A) surface sialic acid is detected in tested PCa cell lines. Sialidase treatment reduces the surface sialic acid levels in (B) PC3, (C) DU145, (D) LNCaP. (E) The expression levels of α2,6-linked and α2,3-linked sialic acids in PC3 cells were analyzed by flow cytometry using Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin II (MALII) lectins, respectively. (F) Quantification was assessed using MFI for SNA and MALII staining on PCa cells. (G) Sialylated spectral counts versus total spectral counts of PCa tumor tissues and adjacent normal tissues by mass spectrometry. (H) Summary of glycosylation changes by glycosite in PCa compared with normal prostate samples. (I) Schematics of glycan structures. The schematic diagram was created with Biorender.com. (J) GO term pathway analysis of sialylated proteins in PCa tumor tissues and adjacent normal tissues by mass spectrometry show enrichment in immune response/antigen presentation pathways. Protein names displayed on the right side with the glycosites in parentheses. (K) Analysis of transcript data from TCGA-PRAD database of sialyglycan gene expression demonstrates 7 significantly upregulated genes and 12 downregulated genes in cancer samples compared with noncancerous prostate tissues. Data were analyzed by unpaired Student’s t test and presented as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ***P ≤ 0.0001.

To determine whether the sialic acids present on the cell surface of the PCa cells were α2,3-linked and α2,6-linked, we utilized Sambucus nigra agglutinin (SNA) to detect α2,6-linked sialic acids and Maackia amurensis agglutinin II (MALII) to detect α2,3-linked sialic acids by flow cytometry. All PCa cells stained with both SNA and MALII lectins had higher expression levels of SNA compared with MALII (Figure 3, E and F and Supplemental Figure 7, C–F). This suggests that PCa cells are decorated with both α2,3-linked and α2,6-linked sialic acids, with the surface sialic acids primarily consisting of α2,6-linked sialic acids.

We investigated the sialylation status of proteins in PCa tumor tissues and their adjacent normal tissues. Intact glycoproteomics analysis demonstrated that cancer tissues displayed significantly more sialylated spectral counts (approximately 30%) compared with adjacent normal prostatic tissues (approximately 20%) (Figure 3G). Remarkably, more than 85% of the differential glycosylation between PCa and normal prostate were due to increases in sialylation, including complex sialylated, complex fucosylated and sialylated, or both (Figure 3, H and I). Gene Ontology (GO) term pathway analysis of these glycosylated proteins, including sialylated proteins, demonstrated enrichment in pathways associated with immune response/antigen presentation, metabolism, cell adhesion and communication, and others (Figure 3J). Analysis of transcript expression in sialoglycan biosynthetic genes within tumor tissues from patients with PCa reveals upregulation in 7 genes and a corresponding downregulation in 12 genes, as indicated by the tumor-to-normal tissue ratio (Figure 3K). Across the samples, α-2,3- and α-2,6-sia