Siglec-15 recognition of sialoglycans on tumor cell lines can occur independently of sialyl Tn antigen expression

Abstract Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors, which plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-β. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-β secretion following co-culture of Siglec-15-expressing monocytic cell lines with tumor cells expressing sTn or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.


Figure S2
Supplementary Figure S2. Three different clones of anti-sTn antibodies were incubated with K562 cells at increasing concentrations (0, 3.3, 10 and 30 µg/ml) and tested for binding using PEconjugated anti-mouse IgG secondary antibody. Sialidase-treated K562 cells were used as the negative control. MFI values were plotted against concentration of the anti-sTn antibody. Representative of two independent experiments.

Figure S3
Supplementary Figure S3. STn expression on lung (A) and breast (B) carcinoma cell lines was analysed by incubating the cells with anti-sTn antibody clone 3F1 followed by APC-conjugated anti-mouse IgG secondary antibody. K562 cells were used as a positive control and isotype-matched mouse IgG was used as a negative control. Histograms are representative of three independent experiments.

Figure S4
Supplementary Figure S4. (A) H460 wildtype and ST6GalNAc-I transferase-expressing cells, pretreated without or with sialidase, were tested for sTn expression using anti-sTn antibody (clone 3F1) followed by PE-conjugated anti-mouse IgG secondary antibody. (B) H460 cells were tested for expression of α2-3-linked sialic acids using biotinylated MAL-II (2 µg/ml) followed by streptavidin-FITC. (C) H460 cells with or without pre-treatment with sialidase were incubated with Siglec-15-Fc wildtype or R143A mutant precomplexed with anti-human IgG Fc-FITC. (D) sTn expressed on the surface of K562 cells was blocked with anti-sTn antibody (30 µg/ml) followed by Siglec-15-Fc wildtype or R143A mutant precomplexed with anti-human IgG Fc-FITC. Human isotype-matched IgG precomplexed with anti-IgG Fc-FITC was used as negative control. Histograms are representative of three independent experiments.

Figure S5
Supplementary Figure S5. Human monocytic cell lines THP-1 and U937 stably expressing human Siglec-15 were generated using the retroviral system. Expression of Siglec-15 on these cell lines was analysed by using two mouse monoclonal antibodies (clones 25E9 and A9E8) against Siglec-15 followed by anti-mouse IgG conjugated to PE.

Description of Sample
Sample names: Human Siglec-15 wildtype (hSiglec-15 WT) and Arg134Ala mutant (hSiglec-15 ArgR143Ala) are recombinant and were expressed as Fc-fusion proteins as described in materials methods section in the main text.
Sample modifications Not relevant.
In brief, the arrayed slides were blocked with 2% w/v BSA in 100 mM Phosphate Buffer Saline pH 7.4 (2%BSA/PBS). The Siglec-15 chimeras were analysed as multimers at 2 µg/ml, prepared by pre-complexing with the biotinylated goat anti-human IgG (VECTOR, BA300) at 1:1 ratio (by weight). The Siglec-15-antibody complex was prepared by pre-incubating the protein and antibody, diluted in the final required volume of blocking buffer, for 1h at 4ºC. The anti-sialyl Tn antibody 3F1 was analysed at 50µg/ml prepared in binding buffer (1% BSA/PBS), followed by incubation (1h) with 10 µg/ml biotinylated anti-mouse IgG (SIGMA, B-7264) .

Glycan description for defined glycans
Sixty amino-terminating glycans (aminoalkyl-, phenyl-or glycine-terminating, or natural serine or threonine terminating amino acids) were used to prepare neoglycolipids (NGLs). These are listed and referenced below, together with 22 reducing oligosaccharides or glycosylceramides used as reference glycans.

Amino-terminating glycans
W Chai and colleagues (submitted)
Glycan description for undefined glycans Not applicable.

Description of surface
Nitrocellulose-coated glass microarray slides.
Custom preparation of surface Not relevant.
Dispensing mechanism Non-contact liquid delivery with four dispensing tips.
Glycan deposition Approximately 0.33 nl was printed per spot.
Each glycan probe was printed at 2 levels (2 and 5 fmol per spot), in duplicate.

Printing conditions
The printing solutions were aqueous-based. Printing was performed at ambient temperature and relative humidity of 58%.
The NGL printing solutions contained 100 pmol/l of DHPC and cholesterol (both from SIGMA) as lipid carriers in addition to the lipid-linked glycan probes. The concentrations of the NGL probes were 5 and 15 pmol/l for the 2 and 5 fmol per spot levels, respectively.
The printing solutions also contained Cy3 NHS ester (GE Healthcare) at 20 ng/ml (26 fmol/l) as a marker to monitor the printing process.

Array layout
Each array slide contained 16-pad subarrays. Each pad was set up for printing 64 probes maximum, each at 2 levels in duplicate (four spots for one probe in a row); up to 256 spots (16x16) in total for each subarray. The probes were printed on multiple subarrays for parallel binding analyses.

6. Detector and Data Processing
Scanning hardware GenePix 4300A (Molecular Devices)

Scanner settings
Scanning resolution: 10 m / pixel; Laser channel: red (scan wavelength 635nm); PMT Voltages: 350 Scan power: Adjusted for each sample to achieve maximum signal without saturation of any single spot.
Image analysis software GenePix® Pro 7 (Molecular Devices)

Data processing
The gpr file was entered into an in-house microarray database using software (designed by Mark Stoll, http://www.beilstein-institut.de/en/publications/proceedings/glyco-2009) for data processing. No particular normalization method or statistical analysis was used.

Data presentation
In Figure 4, the binding results are presented as 2D bar graphs with bars representing averaged mean signal of duplicate spots at 5 fmol/spot and error bars representing standard deviation. The fluorescence intensity numerical scores are depicted in Supplementary Table S2.

8. Interpretation and Conclusion from Microarray Data
Data interpretation No software or algorithms were used to interpret processed data.

Conclusions
Human Siglec-15 is shown to bind to sialylated glycans apart from sTn. Mutation of the conserved Arg143 that is critical for interaction with sialic acid to Ala markedly reduced but not abolished the binding. The binding of hSiglec-15 contrasts with the restricted binding observed with the 3F1-IgG antibody to the sTn antigen.