SCUBE2 Antibody

What is SCUBE2 and what domains make it significant for antibody targeting?

SCUBE2 (Signal Peptide, CUB Domain, EGF-Like 2) is a multidomain protein of approximately 999 amino acids organized in a modular fashion. Its structure includes an N-terminal signal sequence, 9 copies of EGF-like repeats, a spacer region, 3 cysteine-rich motifs, and 1 CUB domain at the C-terminus . This protein can tether to cell surfaces as a peripheral membrane protein through two independent mechanisms: electrostatic interactions and lectin-glycan interactions .

For antibody development, the C-terminal region has proven to be a particularly important targeting region. Several validated antibodies are directed against this region, with immunogens consisting of specific peptide sequences such as KTPEAWNMSECGGLCQPGEYSADGFAPCQLCALGTFQPEAGRTSCFPCGG . The modular nature of SCUBE2 allows researchers to target different functional domains depending on the research question being investigated.

What experimental applications are validated for SCUBE2 antibodies?

SCUBE2 antibodies have been validated for multiple experimental applications, with varying reactivity across species:

When designing experiments, researchers should select antibodies with validated reactivity for their species of interest. Cross-reactivity predictions indicate high conservation across mammalian species, with predicted reactivity of 100% in human, rat, dog, guinea pig, horse, and rabbit; 93% in cow; 86% in mouse; and 83% in zebrafish .

How should SCUBE2 antibodies be stored and handled for optimal performance?

For optimal antibody performance, storage and handling protocols should follow these research-validated guidelines:

Storage recommendations:

• Long-term storage: -20°C in small aliquots to prevent freeze-thaw cycles

• Short-term storage (up to 1 week): 2-8°C

• Most SCUBE2 antibodies are supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose as stabilizers

Handling considerations:

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and reduced activity

• Work with aliquots rather than the stock vial when possible

• Sodium azide in the buffer is hazardous and should be handled by trained staff only

• Optimal working dilutions should be determined experimentally for each specific application and antibody lot

These protocols ensure maximum antibody stability and reproducibility in experimental results.

How can researchers utilize SCUBE2 antibodies to investigate tumor angiogenesis mechanisms?

SCUBE2 plays a critical role in tumor angiogenesis through its interaction with VEGFR2. Research methodologies for investigating this mechanism include:

Immunohistochemical analysis of tumor microenvironments:
Immunohistochemistry has revealed that SCUBE2 is highly expressed in endothelial cells (ECs) of numerous human carcinomas compared to adjacent normal tissue. This upregulation has been documented in xenograft tumors including prostate, sarcoma, bladder, breast, lung tumors, and melanoma . Researchers should use appropriate isotype-matched irrelevant antibodies as negative controls to ensure specificity of staining.

Functional studies using blocking antibodies:
The development of function-blocking monoclonal antibodies (mAbs) such as SP.B1 has enabled mechanistic studies of SCUBE2's role in angiogenesis. SP.B1 binds to SCUBE2 and induces its internalization for lysosomal degradation, thereby reducing cell surface levels and inhibiting VEGF-induced angiogenesis . This approach has demonstrated that:

• SCUBE2 acts as a coreceptor for VEGFR2 to facilitate VEGF binding

• SCUBE2 promotes VEGFR2 phosphorylation and downstream AKT/MAPK activation

• Blocking SCUBE2 impairs EC sprouting, proliferation, and tube formation

Experimental design considerations:
When studying SCUBE2's role in tumor angiogenesis, researchers should consider combining:

• In vitro tube formation assays with SCUBE2 antibody treatments

• Co-immunoprecipitation studies to investigate SCUBE2-VEGFR2 interactions

• Analysis of downstream signaling pathways using phospho-specific antibodies

• In vivo tumor growth models with endothelial-specific SCUBE2 knockout or antibody treatment

What methodologies are effective for investigating SCUBE2's role in cancer metastasis?

Recent research has established SCUBE2 as a mediator of bone metastasis in luminal breast cancer. Several methodological approaches have proven effective:

Genetic manipulation approaches:

• Stable knockdown of SCUBE2 in ER+ luminal cell lines (MCF7, T47D) significantly reduced bone metastasis burden in experimental models

• Overexpression of Scube2 in murine cancer cell line Py8119 increased bone metastasis in immunocompetent mice

• The effect of SCUBE2 on metastasis extends beyond the luminal subtype, as demonstrated by overexpression studies in triple-negative MDA-MB-231 cells

In vivo metastasis model systems:

Quantification techniques:

• Bioluminescent imaging (BLI) for longitudinal tracking of metastatic burden

• Micro-computed tomography (micro-CT) for bone lesion assessment

• Histological analysis with SCUBE2 and other marker antibodies

Studies show that SCUBE2 knockdown suppresses metastasis signal as early as one week after cancer cell inoculation, indicating its role in the early stage of metastatic colonization rather than in primary tumor growth .

How does antibody-induced SCUBE2 internalization occur, and what techniques can be used to study this process?

The SP.B1 monoclonal antibody against SCUBE2 has been shown to induce SCUBE2 internalization in endothelial cells. This process can be studied using several advanced techniques:

Flow cytometry for quantitative surface expression analysis:
SP.B1 dose-dependently reduces surface levels of SCUBE2 while minimally altering cell surface levels of other proteins like neuropilins and VEGFR2 . Flow cytometry using fluorescently-labeled SCUBE2 antibodies allows quantitative assessment of surface expression changes.

Confocal microscopy for internalization tracking:
Without treatment or after incubation with control IgG, SCUBE2 predominantly localizes on the plasma membrane at the EC peripheral rim. After SP.B1 treatment, SCUBE2 becomes detectable inside the cytoplasm, including the perinuclear region . This trafficking can be visualized using fluorescently-labeled antibodies.

Co-localization with endosomal-lysosomal markers:
Confocal microscopy with dual staining for SCUBE2 and markers of the endosomal-lysosomal pathway reveals:

• Co-localization with EEA1 (early endosomal marker)

• Co-localization with LAMP2 (lysosomal marker)

This indicates that SP.B1 promotes SCUBE2 internalization and trafficking to the endosomal-lysosomal compartment for degradation .

Surface biotinylation assay:
This technique provides a quantitative measure of cell surface protein levels. Biotinylated EC surface SCUBE2 levels decrease dose-dependently after incubation with SP.B1, confirming the reduction in surface expression observed by flow cytometry .

These methodologies together provide a comprehensive approach to studying antibody-induced receptor internalization mechanisms.

What factors should researchers consider when selecting a SCUBE2 antibody for their specific application?

When selecting a SCUBE2 antibody for research applications, multiple factors must be considered to ensure experimental success:

Epitope targeting and antibody specificity:

• C-terminal antibodies: Target regions such as amino acids KTPEAWNMSECGGLCQPGEYSADGFAPCQLCALGTFQPEAGRTSCFPCGG

• Internal region antibodies: Target other functional domains

• Consider epitope accessibility in your experimental system (native vs. denatured conditions)

Species reactivity and cross-reactivity:

• Human-specific antibodies are appropriate for clinical samples or human cell lines

• Antibodies with cross-reactivity to mouse/rat are essential for animal model studies

• Verify predicted reactivity: Human (100%), Rat (100%), Mouse (86%), and other species

Validated applications:
Select antibodies validated for your specific application (WB, IF, IHC, flow cytometry, or ELISA) .

Functional properties:

• For descriptive studies: Standard detection antibodies

• For functional blockade: Consider function-blocking antibodies like SP.B1

• For mechanistic studies: Antibodies targeting specific functional domains

Clonality considerations:

• Polyclonal antibodies: Broader epitope recognition, potentially higher sensitivity

• Monoclonal antibodies: Higher specificity, better reproducibility between experiments

Conjugation status:
Select appropriately conjugated antibodies (HRP, FITC, biotin) or unconjugated antibodies depending on your detection system requirements .

How can researchers optimize protocols for detecting SCUBE2 in tumor tissue samples?

Detecting SCUBE2 in tumor tissues requires careful optimization of immunohistochemical protocols:

Tissue preparation and processing:

• Freshly fixed tissues (formalin-fixed, paraffin-embedded) yield optimal results

• Antigen retrieval methods should be tested as SCUBE2 epitopes may be masked during fixation

• Consider tumor heterogeneity in sampling strategy

Staining optimization:

• Antibody dilution: Determine optimal working dilutions experimentally for each tissue type

• Incubation conditions: Typically overnight at 4°C for primary antibodies

• Detection systems: Use high-sensitivity detection systems for low-abundance expression

• Counterstaining: Select appropriate counterstains to visualize tissue context

Control samples:

• Positive controls: Include tissues known to express SCUBE2 (e.g., endothelial cells in carcinomas)

• Negative controls: Include isotype-matched irrelevant antibodies to confirm specificity

• Blocking peptide controls: Consider using immunizing peptide for competition studies

Analysis considerations:

• SCUBE2 is highly expressed in endothelial cells of numerous types of human carcinomas compared to adjacent normal tissue

• Expression has been documented in prostate, sarcoma, bladder, breast, lung tumors, and melanoma

• Consider dual staining with endothelial markers (CD31, CD34) to confirm endothelial localization

Quantification approaches:

• Scoring systems for expression intensity

• Digital image analysis for quantitative assessment

• Microvascular density correlations with SCUBE2 expression

What are the key considerations when designing experiments to investigate SCUBE2-VEGFR2 interactions?

SCUBE2 functions as a coreceptor for VEGFR2, facilitating VEGF binding and promoting its signaling activity. When investigating this interaction:

Co-immunoprecipitation studies:

• Use anti-SCUBE2 antibodies to pull down protein complexes and probe for VEGFR2

• Reciprocal approach: Use anti-VEGFR2 antibodies and probe for SCUBE2

• Include appropriate controls to confirm specificity

• Consider crosslinking approaches for transient interactions

Functional signaling assays:

• Analyze VEGFR2 phosphorylation in the presence/absence of SCUBE2

• Monitor downstream AKT or MAPK activation

• Compare VEGF-induced angiogenic responses with SCUBE2 modulation (knockdown, blockade, overexpression)

Binding studies:

• Determine if SCUBE2 enhances VEGF binding to VEGFR2

• Investigate direct binding between SCUBE2 and VEGFR2

• Identify domains involved in the interaction

Cell-based assays:

• Sprouting assays with ECs with/without SCUBE2 manipulation

• Proliferation and tube formation assays in response to VEGF

• Migration assays to assess chemotactic responses

Research findings indicate that SCUBE2 interacts with VEGFR2 in endothelial cells and functions as a coreceptor for VEGFR2 to facilitate VEGF binding and promote its signal activity. ECs isolated from EC-specific Scube2-knockout mice showed impaired ability to sprout, proliferate, and form tubes in response to VEGF treatment. Consistently, VEGF-induced VEGFR2 phosphorylation and AKT or MAPK activation were significantly inhibited in these knockout models .

What are common technical challenges when using SCUBE2 antibodies and how can they be addressed?

Researchers often encounter several technical challenges when working with SCUBE2 antibodies:

Nonspecific binding:

• Problem: High background staining in immunohistochemistry or Western blotting

• Solution: Optimize blocking conditions (5% BSA, normal serum matching secondary antibody host)

• Validation: Use isotype controls and knockout/knockdown samples as negative controls

Epitope masking in fixed tissues:

• Problem: Reduced or absent signal in fixed tissue samples

• Solution: Test different antigen retrieval methods (heat-induced epitope retrieval, enzymatic retrieval)

• Optimization: Compare different fixatives and fixation times for fresh samples

Variability between antibody lots:

• Problem: Inconsistent results between experiments using different antibody lots

• Solution: Validate each new lot against previously working conditions

• Strategy: Maintain internal standards and positive controls across experiments

Detection of membrane-associated SCUBE2:

• Problem: Difficulty detecting membrane-associated SCUBE2 due to its peripheral membrane association

• Solution: Use non-ionic detergents for gentle lysis; avoid harsh extraction conditions

• Alternative: Consider surface biotinylation approaches for specific detection of surface-associated SCUBE2

Cross-reactivity with other SCUBE family members:

• Problem: Potential cross-reactivity with SCUBE1 or SCUBE3

• Solution: Select antibodies specifically validated against other family members

• Verification: Include recombinant SCUBE1 and SCUBE3 proteins as specificity controls

Each of these challenges requires systematic optimization and appropriate controls to ensure reliable, reproducible results.

How can researchers distinguish between different forms and localizations of SCUBE2 protein?

SCUBE2 can exist in different forms and localizations within cells and tissues, requiring sophisticated approaches to distinguish between them:

Membrane-associated vs. internalized SCUBE2:

• Surface biotinylation assays quantitatively measure cell surface levels

• Flow cytometry with non-permeabilizing conditions detects only surface protein

• Confocal microscopy with membrane markers (e.g., WGA) visualizes colocalization

Subcellular fractionation approaches:

• Differential centrifugation to separate membrane fractions from cytosolic components

• Sucrose gradient fractionation for more refined separation

• Western blotting of fractions with compartment-specific markers as controls

Distinguishing SCUBE2 trafficking pathways:
Advanced microscopy techniques with markers for different compartments:

• Early endosomes: Co-staining with EEA1

• Lysosomes: Co-staining with LAMP2

• Recycling endosomes: Co-staining with Rab11

Research has shown that after SP.B1 antibody treatment, SCUBE2 is internalized and traffics through the endosomal-lysosomal pathway, colocalizing with both EEA1 and LAMP2 . This provides a model system for studying antibody-induced receptor internalization.

What experimental strategies address the hypoxia-dependent regulation of SCUBE2 in tumors?

SCUBE2 is hypoxia-inducible, and its expression is elevated in tumor endothelial cells. This presents unique experimental considerations:

In vitro hypoxia models:

• Hypoxic chambers with controlled O₂ levels (1-5%)

• Chemical hypoxia mimetics (e.g., CoCl₂, DMOG)

• HIF-1α stabilization assays to confirm hypoxic conditions

Analysis of HIF-dependent regulation:

• ChIP assays to identify HIF binding to SCUBE2 promoter regions

• HIF-1α knockdown/knockout studies to confirm regulation

• HIF reporter assays to assess activity under different conditions

In vivo hypoxia assessment:

• Pimonidazole staining of tumor sections to identify hypoxic regions

• Correlation of SCUBE2 expression with hypoxia markers

• Ex vivo analysis of tumor hypoxic zones for SCUBE2 expression

Functional consequences:

• Analyze how hypoxia-induced SCUBE2 affects VEGF signaling

• Determine if hypoxic regulation of SCUBE2 contributes to therapy resistance

• Investigate if blocking SCUBE2 is more effective in hypoxic tumor regions

Research indicates that many cancers contain areas of intratumoral hypoxia, and immunohistochemistry has revealed that SCUBE2 is highly expressed in ECs of numerous types of human carcinomas compared to adjacent normal tissue . This suggests hypoxia may be a key driver of SCUBE2 expression in the tumor microenvironment.

How might SCUBE2 antibodies be utilized in developing novel therapeutic strategies for cancer?

The functional role of SCUBE2 in tumor angiogenesis and metastasis suggests several potential therapeutic applications:

Anti-angiogenic approaches:

• Function-blocking antibodies like SP.B1 reduce surface SCUBE2 levels through internalization and lysosomal degradation

• This inhibits VEGF-induced angiogenesis, potentially restricting tumor growth

• Combining SCUBE2 blockade with existing anti-VEGF therapies may enhance efficacy

Targeting bone metastasis:

• SCUBE2 contributes to bone tropism of luminal breast cancer

• Antibodies blocking this function could potentially reduce bone metastasis burden

• Early intervention is critical as SCUBE2 appears important in the early stages of metastatic colonization

Antibody-drug conjugates (ADC) approach:

• SCUBE2's high expression in tumor endothelial cells makes it a potential ADC target

• The internalization mechanism observed with SP.B1 could be leveraged for drug delivery

• Tumor-specific vascular targeting while sparing normal vasculature

Combinatorial approaches:

• SCUBE2 blockade with immune checkpoint inhibitors

• Combination with radiation therapy, which can increase tumor hypoxia and potentially SCUBE2 expression

• Dual targeting of SCUBE2 and VEGFR2 pathways

Research indicates that endothelial deletion of SCUBE2 in mice inhibits tumor growth, with B16F10 tumors 70% smaller and LLC tumors 40% smaller in EC-KO versus control mice . These findings suggest significant therapeutic potential for SCUBE2-targeting strategies.

What methodological approaches can be used to investigate the relationship between SCUBE2 and hormone receptor status in breast cancer?

SCUBE2 expression has been linked to estrogen receptor (ER) status in breast cancer, suggesting hormone-dependent regulation:

Hormone-responsive studies:

• Treat ER+ breast cancer cells with estrogen or anti-estrogens and measure SCUBE2 expression

• Compare SCUBE2 levels in ER+ versus ER- cell lines and patient samples

• Use ChIP assays to investigate ER binding at SCUBE2 regulatory regions

Clinical correlation studies:

• Analyze SCUBE2 expression across breast cancer molecular subtypes

• Correlate SCUBE2 levels with hormone receptor status in patient samples

• Investigate if SCUBE2 expression predicts response to endocrine therapy

Functional implications:

• Determine if hormone-mediated SCUBE2 regulation affects:

  • Angiogenic potential of tumor cells

  • Metastatic propensity, particularly to bone

  • Response to anti-angiogenic therapies

Experimental models:

• Use hormone manipulation in xenograft models to assess SCUBE2 expression changes

• Develop models with inducible SCUBE2 expression to mimic hormone responsiveness

• Study SCUBE2 in the context of endocrine therapy resistance

Research has shown that SCUBE2 is ER-regulated in luminal breast cancer cells. In the murine Py8119 cell line (ER positive), Scube2 expression is responsive to estrogen treatment, highlighting the hormone-dependent regulation of this protein .