FUT8 Antibody

Basic Research Questions

• What experimental methods validate FUT8 antibody specificity in glycan modification studies?

  • Methodological answer: Validate FUT8 antibodies using:

    • Western blotting with FUT8-knockout (KO) cell lysates (e.g., CHO or cancer cell lines) to confirm band disappearance .

    • Immunohistochemistry (IHC) paired with enzymatic deglycosylation to distinguish core fucosylation signals from nonspecific binding .

    • Flow cytometry with FUT8-overexpressing vs. wild-type cells to quantify membrane-associated FUT8 .

    • Table 1: Common validation controls for FUT8 antibodies:

      Control Type	Application	Expected Outcome (FUT8-KO vs. WT)
      CRISPR-KO cell lysate	Western blot	Band absence in KO lysate
      Lectin-binding assay	Glycan profiling	Reduced LCA reactivity in KO
      Co-staining with FUT8	IHC/IF	Colocalization with ER/Golgi markers

    • Key citations: .

• How does FUT8 knockout influence global glycoproteomic profiles in CHO cells?

  • Methodological answer:

    • Perform large-scale glycoproteomics using hydrophilic interaction chromatography (HILIC) and LC-MS/MS to compare FUT8-KO vs. wild-type CHO cells .

    • Key findings from :

      • Core fucosylation: Reduced by >99% in FUT8-KO cells.

      • Sialylation: Decreased sialyltransferase activity (e.g., ST6GAL1 down 2.1-fold).

      • Glycosite occupancy: 26.7% of glycosites showed altered occupancy (e.g., increased high-mannose glycans).

• What cancer models demonstrate FUT8’s role in immune evasion?

  • Methodological answer:

    • Use syngeneic mouse models (e.g., lung adenocarcinoma) with FUT8-KO tumor cells. Assess:

      • Immune checkpoint expression: PD-L1/PD-1 downregulation via flow cytometry .

      • Tumor-infiltrating lymphocytes (TILs): Increased CD8+ T cells in FUT8-KO tumors .

    • Table 2: FUT8-associated immune markers in cancer:

      Cancer Type	Observed Change (FUT8 Inhibition)	Mechanism
      Lung	↓ PD-L1, ↑ T cell infiltration	Altered TGF-β signaling
      Prostate	↓ EGFR stability	Impaired growth factor binding
      Lymphoma	↓ B7-H3 expression	Reduced T cell exhaustion

    • Key citations: .

Advanced Research Questions

• How to resolve contradictions in FUT8’s pro-metastatic vs. anti-metastatic roles across cancers?

  • Methodological answer:

    • Context-dependent analysis:

      • Organ-specific glycosylation: Profile FUT8 substrates (e.g., integrins in melanoma vs. cadherins in prostate cancer) .

      • Microenvironment modulation: Co-culture FUT8-KO tumor cells with fibroblasts/immune cells to assess ECM remodeling .

    • Statistical approach: Apply multivariate regression to isolate FUT8’s effect from confounding factors (e.g., hypoxia, inflammation) .

    • Key data: In prostate cancer, FUT8 overexpression increased metastasis by stabilizing EGFR, while in melanoma, KO enhanced PD-1 suppression .

• What strategies improve antibody-dependent cellular cytotoxicity (ADCC) via FUT8 modulation?

  • Methodological answer:

    • Co-expression systems: Use CHO cells with anti-FUT8 intrabodies to reduce core fucosylation to <5% while maintaining titer .

    • Glycoengineering: Combine FUT8-KO with β-galactosidase overexpression to optimize FcγRIIIa binding .

    • Table 3: ADCC enhancement strategies:

      Strategy	Fucosylation Level	ADCC Increase (vs. WT)	Limitations
      FUT8-KO CHO cells	0%	10–50x	Altered cell growth
      Anti-FUT8 intrabodies	<5%	5–20x	Transient transfection
      Lectin selection	1–3%	3–10x	Low yield

    • Key citations: .

• How to integrate FUT8 glycomics data with transcriptomic/proteomic datasets?

  • Methodological answer:

    • Multi-omics pipeline:

      1. Glycoproteomics: Identify FUT8-regulated glycosites (e.g., 928 sites in CHO cells) .

      2. RNA-seq: Correlate FUT8 expression with EMT markers (e.g., SNAI1, VIM) .

      3. Kinase profiling: Link core fucosylation to MAPK/ERK activation using phosphoproteomics .

    • Tool recommendation: Use GlyConnect or UniCarb-DB for glycan structure annotation.

Data Contradiction Analysis

• Why do some studies report FUT8 as oncogenic while others show tumor-suppressive effects?

  • Critical factors:

    • Cell type specificity: FUT8 promotes metastasis in prostate cancer but suppresses it in colorectal cancer via divergent Wnt/β-catenin modulation .

    • Experimental endpoint: Short-term vs. long-term FUT8 inhibition may differentially affect immune checkpoint dynamics .

  • Resolution strategy:

    • Perform time-course experiments to track FUT8’s dual roles in early tumorigenesis vs. metastatic colonization.

    • Use conditional KO models to isolate tissue-specific effects.