Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: Galectin 9 , REA435 , 50 , 130-124-237 , PE (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 expression is substantially increased in patients with colorectal cancer (CRC) resistant to neoadjuvant therapy. ST6GAL1 expression was measured in 14 surgically removed fresh CRC tumor tissues and matched adjacent normal tissues using RT‒qPCR, WB and IHC after. The correlation between ST6GAL1 expression and sensitivity to neoadjuvant therapy was analyzed in combination with the clinical information. ( A ) ST6GAL1 mRNA expression was quantified by RT‒qPCR in tumor tissues compared with adjacent normal tissues from 14 clinical CRC patients. ( B ) Statistical analysis of the S T6GAL1 mRNA level in the CRC patients in A. ( C ) ST6GAL1 protein levels were measured in 14 clinical samples from CRC patients by WB. ( D ) Statistical analysis of the ST6GAL1 protein levels in CRC patients in C. ( E ) Representative images of HE staining and IHC staining of ST6GAL1 in colorectal tumor tissue and adjacent normal colorectal tissue from CRC patients (scale bars, 625 μm; scale bars, 50 μm). ( F ) Statistical results for the relative intensity of ST6GAL1 staining shown in E. ( G ) Statistical analysis of the correlation between ST6GAL1 mRNA expression and sensitivity to neoadjuvant therapy. ( H ) Representative images of IHC staining for ST6AGL1 in colorectal tumor tissue from neoadjuvant therapy-sensitive and nonsensitive CRC patients (scale bars, 625 μm; scale bars, 50 μm). ( I ) Statistical results for the relative intensity of ST6GAL1 staining shown in H. Statistical significance was determined using Student’s t test ( B , D , F and G )

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 expression is negatively correlated with the immune response in CRC. scRNA-seq data from public CRC databases were used to analyze ST6GAL1 expression in different cell subsets and its correlation with immune response pathways. ( A ) Uniform manifold approximation and projection (UMAP) plots showing the clusters of cells in the tumor tissue. ( B ) ST6GAL1 expression in different cell clusters. ( C ) GSEA of immune response-associated pathways between ST6GAL1 + cells and ST6GAL1 − cells in tumor tissues. ST6GAL1 expression and IFNG sensitivity in different CRC cell lines were further verified by RT‒qPCR and WB. ( D ) ST6GAL1 mRNA expression in the different CRC cell lines was quantified by RT‒qPCR. ( E ) ST6GAL1 protein levels were measured in 4 CRC cell lines using WB. ( F ) The mRNA levels of molecules associated with the IFNG response were measured by RT‒qPCR in different CRC cell lines treated with 100 ng/µl IFNG for 24 h

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Expressing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 expression decreases IFNG sensitivity in CRC cell lines. After ST6GAL1 overexpression (OE) in the SW48 cell line (SW48-OE) and the HT29 cell line (HT29-OE) and ST6GAL1 knockdown (KD) in the Caco2 cell line (Caco2-KD) and the HT29 cell line (HT29-KD), as well as the construction of the related empty virus (EV) control cell lines SW48-EV, Caco2-KD and HT29-EV, the levels of molecules associated with the IFNG response were measured. ( A , B , C and D ) The mRNA levels of IFNG response-associated molecules were measured by RT‒qPCR in the SW48-EV and SW48-OE cell lines ( A ), the HT29-EV and HT29-OE cell lines ( B ), the Caco2-EV and Caco2-KD cell lines ( C ), and the HT29-EV and HT29-KD cell lines ( D ) after treatment with 100 ng/µl IFNG for 24 h. ( E , F , and G ) GBP1 and IRF1 protein levels in the SW48-EV and SW48-OE cell lines ( E ), the Caco2-EV and Caco2-KD cell lines ( F ), and the HT29-EV, HT29-OE and HT29-KD cell lines ( G ) were measured by WB after treatment with 100 ng/µl IFNG for 12 and 24 h

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Expressing, Over Expression, Knockdown, Virus, Control

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 overexpression promotes the immune escape of CRC tumor cells in vivo. An ST6GAL1 -OE MC38 cell line (MC38-OE) and the related empty virus (EV)-expressing control cell line MC38-EV were constructed. We used MC38-EV ( n = 4) and MC38-OE ( n = 4) cells to construct an animal model of subcutaneous tumorigenesis in wild-type C57BL/6 mice. When the tumor grew to an appropriate size, the tumor was removed, and the change in cell count was detected by flow cytometry. ( A ) Statistical analysis of the differences in the numbers of cells in tumors, draining lymph nodes and PBMCs between the ST6GAL1-EV and ST6GAL1-OE groups. ( B ) Flow cytometry analyses of CD3 + T, CD4 + T, CD8 + T and Treg cells in CD45-gated cells from tumor tissues from the ST6GAL1-EV and ST6GAL1-OE groups. ( C , D and E ) Statistical results for CD3 + T-cell numbers, CD4 + and CD8 + T-cell numbers ( D ) and Treg numbers ( E ) between the ST6GAL1-EV and ST6GAL1-OE groups. ( F , H and J ) Flow cytometry analyses of GZMB from CD8 + T cells ( F ), IFNG from CD4 + T cells ( H ) and IFNG from CD8 + T cells ( J ) in tumor tissues from the ST6GAL1-EV and ST6GAL1-OE groups. ( G , I and K ) Statistical results for GZMB from CD8 + T cells ( G ), IFNG from CD4 + T cells ( I ) and IFNG from CD8 + T cells ( K ) in tumor tissues from the ST6GAL1-EV and ST6GAL1-OE groups. Statistical significance was determined using Student’s t test ( A , C , D , E , G , I and K )

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Over Expression, In Vivo, Virus, Expressing, Control, Construct, Animal Model, Cell Counting, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: Multiomics analysis revealed that ST6GAL1 inhibits the activity of the IFNG signaling pathway. SW48-EV ( n = 3) and SW48-OE ( n = 3) cell lines were subjected to RNA-sequencing (RNA-seq) after treatment with 100 ng/µl IFNG for 24 h ( A , B and C ). ( A ) Volcano plot showing all genes expressed in the SW48-EV and SW48-OE cell lines. The x-axis shows the log 2 fold change (SW48-OE vs. SW48-EV cells), and the y-axis shows the -log 10 P value, which represents the threshold values in log transformation. Each dot represents a differentially expressed gene (DEG). The red dots indicate significantly upregulated DEGs, the blue dots indicate significantly downregulated DEGs, and the gray dots represent DEGs whose differential expression was not statistically significant. ( B ) Heatmap showing the relative expression of selected genes in three different functional modules (immune-related genes, inflammatory factor-related genes and IFNG response-related genes) in the S6TGAL1-OE cell line. ( C ) Gene set enrichment analysis (GSEA) of the SW48-OE cell line revealed an upregulated signaling pathway compared with that of the SW48-EV cell line. Kinase phosphorylation array detection of the SW48-EV and SW48-OE cell lines was subsequently performed ( D and E ). ( D ) Results of the IFNGR1-pTyr457 measurement via kinase phosphorylation array in the SW48-EV and SW48-OE cell lines. ( E ) KEGG pathway analysis of the differentially phosphorylated proteins in SW48-EV and SW48-OE cell lines identified via kinase phosphorylation array detection

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Activity Assay, RNA Sequencing, Transformation Assay, Quantitative Proteomics, Expressing, Functional Assay, Phospho-proteomics

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 regulates the IFNGR1/JAK1/STAT1 signaling pathway by affecting IFNGR1 and BICD2 binding. ( A , C and E ) WBs showing the levels of the pIFNGR1, IFNGR1, and pSTAT1 proteins in the SW48-EV and SW48-OE cell lines ( A ), the Caco2-EV and Caco2-KD cell lines ( C ), and the HT29-EV, HT29-OE and HT29-KD cell lines ( E ) after treatment with 100 ng/µl IFNG for 15 and 30 min ( B , D , and F ) Statistical results for the gray values shown in A ( B ), C ( D ) and E ( F ). ( G ) IP results for the HT29-EV and HT29-OE cell lines. ( H ) Immunofluorescence staining for SNA-FITC (green) and BICD2 (red) in the Caco2-EV and Caco2-KD cell lines. Nuclei were counterstained with DAPI (blue). Scale bars, 20 μm

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Binding Assay, Immunofluorescence, Staining

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: The mechanism by which ST6GAL1 promotes tumor immune evasion. ST6GAL1, an enzyme that adds sialic acid to proteins, is abundantly localized in the tumor microenvironment, where it inhibits IFNGR1 phosphorylation through the sialylation of BICD2, thereby suppressing the activation of the IFNGR1/JAK1/STAT1 signaling pathway. This interaction leads to resistance to the antitumor immune response and CRC development. *BICD2, BICD cargo adaptor 2; GBP1, guanylate binding protein 1; IDO1, indoleamine 2,3-dioxygenase 1; ISGs, IFN-stimulating genes; IRF, interferon regulatory factor; IFNG, interferon gamma; IFNGR, interferon gamma receptor; JAK, Janus kinase; NK, natural killer; STAT1, signal transducer and activator of transcription 1; ST6GAL1, ST6 β-galactoside α2,6-sialyltransferase; TH1, helper T

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Phospho-proteomics, Activation Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: Expression of PNA-reactive glycans by germinal center B cells is correlated with downregulation of the α2,3-sialyltransferase ST3Gal1. (A) Gating strategy for analyzing tonsillar naïve (N), germinal center (GC), memory (M), and plasmablast (PB) B cells by flow cytometry. Representative fluorescence minus one (FMO) controls used for gating of CD27 are shown. (B) Analysis of peanut lectin binding to human tonsil B cells. Representative histograms of results are shown ( left ) as well as quantification of geometric mean fluorescence intensities (MFI) ( right ). (C) Schematic of synthesis of potential PNA-reactive O-linked glycans on B cells. O-glycan synthesis is initiated in the Golgi apparatus by polypeptide N-acetylgalactosamine transferases ( GALNTs ), which transfer a single GalNAc to select serine/threonine residues of a polypeptide backbone. The initiating GalNAc can be terminally sialylated, or further extended by C1GalT1 ( C1GALT1 ) to form the simplest PNA-reactive epitope, a Core 1 O-glycan termed “T-antigen.” This core T-antigen moiety can be branched and elongated by other glycosyltransferases to form extended Core 2 O-glycans, which retain binding to PNA, or modified with sialic acid by the α2,3-sialyltransferase ST3Gal1 ( ST3GAL1 ), which destroys PNA reactivity. Endogenous (or exogenous) sialidases may remove sialic acids and restore PNA binding. (D) Analysis of ST3GAL1 expression in tonsillar B cells by quantitative real-time reverse transcription PCR (qRT-PCR), sorted as in (A) . Data are normalized to the housekeeping gene VCP and presented relative to naïve B cells. Data are representative of eight (B) or three (D) distinct tonsil specimens pooled from two (B) or three (D) independent experiments. Statistics were calculated using a Kruskal–Wallis test with Dunn's multiple comparisons test (B) or One-way analysis of variance (ANOVA) and Tukey's multiple comparisons test. Throughout, bars and error bars depict the mean and SEM, respectively. ns = not significant, *** p ≤ 0.001. ΔMFI, background subtracted geometric mean fluorescence intensity; GalNAc, N-acetylgalactosamine; Gal, galactose; Sia, sialic acid.

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Expressing, Flow Cytometry, Fluorescence, Binding Assay, Glycoproteomics, Modification, Reverse Transcription, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: ST3Gal1 regulates PNA binding in B cells by sialylating Core 1 O-glycans. (A) Validation of ST3GAL1 overexpression in Ramos B cells by qRT-PCR. Data were normalized to housekeeping control VCP and presented relative to vector control. (B) Representative histogram ( left ) and quantification ( right ) of flow cytometric analysis of PNA binding to vector control and ST3Gal1OE Ramos B cells. The Core 1 O-glycan/ T-antigen specificity of PNA is depicted at top right. (C) Representative histogram ( left ) and quantification ( right ) of flow cytometric analysis of MAL-II plant lectin binding to vector control and ST3Gal1OE Ramos B cells. The α2,3-sialylated Core 1 O-glycan/sialylated T-antigen glycan favored by MAL-II lectin is depicted at top right. (D) Representative histogram ( left ) and quantification ( right ) of PNA binding to vector control ( top ) or ST3Gal1OE Ramos B cells ( bottom) before and after removal of sialic acids by intact cell treatment with Arthrobacter ureafaciens sialidase. (E) Representative histogram ( left ) and quantification ( right ) of MAL-II binding to vector control ( top ) or ST3Gal1OE ( bottom) Ramos B cells before and after sialidase treatment, as in (D) . Data in (B–E) are from three independent experiments with three biological replicates in total. Statistics were calculated using Welch's unpaired, two-tailed t -test (B–E) . Throughout, bars and error bars depict the mean and SEM, respectively. ns = not significant, * p ≤ 0.05, ** p < 0.01, ΔMFI, background subtracted geometric mean fluorescence intensity; Ctrl, vector control. ST3OE; ST3Gal1 overexpression.

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Binding Assay, Biomarker Discovery, Over Expression, Quantitative RT-PCR, Control, Plasmid Preparation, Glycoproteomics, Two Tailed Test, Fluorescence

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: Overexpression of ST3Gal1 in B cells modulates binding of glycosylation sensitive CD45 antibodies. (A) Representative histogram ( left ) and quantification ( right ) of binding of two glycosylation-sensitive CD45 antibodies (MEM55 and B220) and one glycosylation-insensitive antibody (total CD45, clone HI30) to vector control and ST3Gal1OE Ramos B cells by flow cytometry. (B) Western blot analysis of binding of CD45 mAb MEM55 (800 nm fluorescence channel) and CD45 mAb HI30 (700 nm fluorescence channel) to Ramos vector control and ST3Gal1OE lysates. (C) Representative histograms depicting flow cytometric analysis of CD45 antibody binding to vector control and ST3Gal1OE Ramos B cells, before and after treatment with Arthrobacter ureafaciens sialidase. Data in (A,C) are from three independent experiments with three biological replicates. Data in (B,C) are representative of similar results from three (B) or two (C) independent experiments. Statistics in (A) were calculated using Welch's unpaired, two-tailed t -test. Throughout, bars and error bars depict the mean and SEM, respectively. ns, not significant, ** p ≤ 0.01, *** p ≤ 0.001, ΔMFI, background subtracted geometric mean fluorescence intensity; Ctrl, vector control; ST3OE, ST3Gal1OE.

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Over Expression, Binding Assay, Glycoproteomics, Plasmid Preparation, Control, Flow Cytometry, Western Blot, Fluorescence, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: Overexpression of ST3Gal1 truncates O-glycans in B cells. (A) Representative histograms ( left ) and quantification ( right ) of binding of the Core 2 poly-LacNAc binding lectin Solanum tuberosum agglutinin (STA) to vector control and ST3Gal1OE Ramos B cells by flow cytometry. (B) Representative histograms ( left ) and quantification ( right ) of binding of the CD43 mAb 1D4 (Core 2 O-glycan-specific glycoform) to vector control and ST3Gal1OE Ramos B cells by flow cytometry. (C) Cellular O-glycome Reporter/Analysis (CORA) of untransduced Ramos, Ramos vector control, and Ramos ST3Gal1OE B cells. Depicted are MALDI-TOF MS spectra of peracetylated Benzyl-α-GalNAc-linked O-glycans. Structures above a bracket have not been unequivocally defined. Indicated areas in the spectra have a 20-fold magnification. “M” and “m” designations indicate major and minor abundances, respectively. Cartoon structures were drawn according to http://www.functionalglycomics.org guidelines and are representative from repeat experiments on two different biological replicates. Structure assignments are based on composition, tandem mass spectrometry, and biosynthetic knowledge. Full methods for MS analysis can be found in Materials and Methods. Data depict results from three (A,B) or two (C) biological replicates. Statistics in (A) and (B) were calculated using Welch's unpaired, two-tailed t -test. Throughout, bars and error bars depict the mean and SEM, respectively. ** p ≤ 0.01, ΔMFI, background subtracted geometric mean fluorescence intensity. Ctrl, vector control; ST3OE, ST3Gal1OE; Fuc, fucose; Man, mannose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid (sialic acid).

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Over Expression, Binding Assay, Plasmid Preparation, Control, Flow Cytometry, Glycoproteomics, Mass Spectrometry, Two Tailed Test, Fluorescence