ccbe1 Antibody

What is CCBE1 and why is it important in research?

CCBE1 is a 406 amino acid (44 kDa) secreted protein containing two collagen-like domains and one calcium-binding epidermal growth factor (EGF-like) domain . It plays essential roles in:

• Lymphatic vasculature development during embryogenesis

• Cardiac development and specification

• Cancer progression, particularly in lymphatic metastasis

The gene encoding CCBE1 is located on chromosome 18 in a region frequently deleted in breast and prostate cancers . Mutations in CCBE1 cause Hennekam syndrome, a rare autosomal recessive disorder characterized by lymphatic malformations and heart defects .

What are the validated applications for CCBE1 antibodies?

CCBE1 antibodies have been validated for multiple experimental techniques:

Researchers should validate each antibody for their specific application and sample type, as reactivity can vary between species (human, mouse, rat) and tissue types .

What controls should be used when validating a new CCBE1 antibody?

For proper CCBE1 antibody validation:

• Positive controls: Tissues with known CCBE1 expression, including fibroblasts, kidney tissue, and lymphatic endothelial cells

• Negative controls: Use tissues from CCBE1 knockout models or cells treated with CCBE1-targeting siRNA

• Blocking peptide: Consider using a blocking peptide corresponding to the immunogen to confirm specificity

• Antibody concentrations: Test multiple dilutions to determine optimal signal-to-noise ratio

When validating in cell lines, SW837 and SW480 colorectal cancer cells have been used successfully for CCBE1 expression studies .

How can CCBE1 antibodies be used to study its role in tumor lymphangiogenesis?

CCBE1 has been identified as a promoter of tumor lymphangiogenesis and lymphatic metastasis, particularly in colorectal cancer (CRC) . Research protocols to study this relationship include:

• Dual immunostaining: Use CCBE1 antibodies alongside lymphatic markers (PDPN, LYVE-1) to correlate CCBE1 expression with lymphatic vessel density in tumor samples

• Cell-specific expression analysis: Distinguish between CCBE1 expression in tumor cells versus cancer-associated fibroblasts (CAFs) in the tumor microenvironment

• Functional assays: Use CCBE1 antibodies to block protein function in tube formation and migration assays of human lymphatic endothelial cells (HLECs)

Research has shown that CCBE1 expression in both tumor cells and tumor stroma correlates with increased lymphatic vessel density, lymph node metastasis, and poor prognosis in CRC .

What experimental approaches can be used to study CCBE1's role in cardiac development?

CCBE1 plays a critical role in cardiac specification and development. Key experimental approaches include:

• Embryonic stem cell (ESC) differentiation models:

  • Use CCBE1 knockdown ESC lines to study cardiac lineage specification

  • Supplement culture medium with CCBE1-blocking antibody (100 ng/mL) to inhibit CCBE1 activity during differentiation

• Marker expression analysis:

  • Monitor expression of cardiac lineage markers following CCBE1 inhibition:

    • Early mesoderm marker: BraT

    • Cardiac mesoderm markers: Mesp1, Isl1

    • Cardiac progenitor marker: Nkx2.5

    • Cardiomyocyte markers: αMhc, cTnT

• Functional readouts:

  • Embryoid body size measurement

  • Counting of beating foci

  • Cell proliferation and apoptosis assays

Studies have shown that CCBE1 knockdown or antibody blockade results in reduced cardiac mesoderm formation, decreased cardiomyocyte differentiation, and impaired embryoid body growth .

How does CCBE1 interact with the VEGF-C/VEGFR-3 pathway and how can this be studied?

CCBE1 enhances VEGF-C proteolysis and signaling, which is crucial for lymphangiogenesis. Experimental approaches to study this interaction include:

• VEGF-C processing assays:

  • Western blot analysis to detect pro-VEGF-C and mature VEGF-C forms

  • Co-culture experiments with CCBE1-expressing cells and VEGF-C-producing cells

• ADAMTS3 interaction studies:

  • Co-immunoprecipitation to detect CCBE1-ADAMTS3 complexes

  • Functional assays with ADAMTS3 inhibition

• VEGFR-3 activation analysis:

  • Phosphorylation assays of VEGFR-3 in the presence/absence of CCBE1

  • Downstream signaling pathway activation studies

CCBE1 has been shown to enhance VEGFC proteolysis in vitro, facilitating tube formation and migration of lymphatic endothelial cells, and promoting tumor lymphangiogenesis and lymphatic metastasis in vivo .

What are the optimal conditions for detecting CCBE1 protein in different sample types?

For optimal detection in tissues, note that CCBE1 expression is particularly high in fibroblasts, renal tissues, and lymphatic structures .

How can researchers distinguish between CCBE1 expressed by different cell types within a tumor microenvironment?

Detecting cell-specific CCBE1 expression in complex tissues requires:

• Dual immunofluorescence staining:

  • Combine CCBE1 antibody with cell-type-specific markers:

    • Cancer-associated fibroblasts (CAFs): α-SMA, FAP

    • Cancer cells: Cytokeratin, EpCAM

    • Lymphatic vessels: PDPN, LYVE-1

• Laser capture microdissection:

  • Isolate specific cell populations from tumor sections

  • Perform protein or RNA analysis on isolated cells

• In situ approaches:

  • RNAscope for CCBE1 mRNA detection combined with immunostaining for cell-type markers

  • Analysis of spatial distribution within tumor regions (tumor core vs. margin)

Research has demonstrated that both cancer cells and CAFs express and secrete CCBE1 in colorectal cancer, contributing to VEGFC maturation and tumor lymphangiogenesis .

What experimental controls should be used when studying CCBE1 function through antibody blockade?

When using CCBE1 antibodies to block protein function:

• Essential controls:

  • Buffer-only control: Use equivalent amounts of antibody buffer alone

  • Isotype control: Use matched irrelevant antibody of the same isotype

  • Concentration series: Test multiple antibody concentrations (e.g., 50-200 ng/mL)

• Validation of blocking effect:

  • Confirm reduced VEGF-C processing by Western blot

  • Verify impaired lymphatic endothelial cell tube formation

  • Assess impact on known CCBE1-dependent processes

• Complementary approaches:

  • Compare antibody blockade with genetic knockdown

  • Rescue experiments with recombinant CCBE1 protein

Studies have successfully used CCBE1 antibody at 100 ng/mL to block protein function during embryonic stem cell differentiation, confirming specificity through parallel shRNA knockdown experiments .

How might CCBE1 antibodies be used to study its potential therapeutic applications in cancer and cardiac disease?

Current research suggests several emerging applications:

• Cancer metastasis intervention:

  • Evaluate CCBE1 blockade on lymphangiogenesis and metastasis in xenograft models

  • Investigate the relationship between CCBE1 and TGF-β pathway modulation in cancer progression

• Cardiac development and regeneration:

  • Use CCBE1 antibodies to study its role in cardiac progenitor mobilization

  • Investigate CCBE1's potential in enhancing cardiac repair after injury

• Biomarker development:

  • Correlate CCBE1 expression patterns with clinical outcomes

  • Develop tissue-specific CCBE1 detection methods for prognostic applications

Studies have demonstrated that CCBE1 expression correlates with lymph node metastasis and poor prognosis in colorectal cancer, suggesting potential as a prognostic biomarker .

What are the optimal experimental protocols for studying CCBE1's role in both promoting and inhibiting various cancer types?

CCBE1 has shown context-dependent roles in different cancer types. Recommended approaches:

• Expression analysis across cancer types:

  • Use tissue microarrays with CCBE1 antibodies to screen multiple cancers

  • Correlate with clinical parameters and outcomes

• Functional studies:

  • Gain-of-function: Recombinant CCBE1 supplementation

  • Loss-of-function: Antibody blocking, shRNA knockdown

  • Domain-specific analysis: Target specific CCBE1 domains with domain-specific antibodies

• Pathway interaction analysis:

  • Study CCBE1 in relation to TGF-β signaling (which downregulates CCBE1)

  • Investigate VEGF-C/VEGFR-3 axis modulation by CCBE1 in different tumor microenvironments

Research has shown that CCBE1 is downregulated in breast cancer but may be overexpressed in colorectal cancer, where it promotes lymphangiogenesis .

What methodological approaches can be used to study the developmental role of CCBE1 in hematopoiesis?

CCBE1 has been implicated in fetal erythropoiesis. Key experimental approaches include:

• Hematopoietic colony assays:

  • Methylcellulose colony assays with fetal liver cells

  • Analysis of CFU-E, BFU-E, and myeloid colony formation in the presence/absence of CCBE1

• Transplantation studies:

  • Transplantation of fetal liver cells from CCBE1-deficient donors

  • Assessment of hematopoietic reconstitution capacity

• Expression and localization studies:

  • Use CCBE1 reporter systems to track expression in hematopoietic compartments

  • Conditional deletion approaches to assess tissue-specific requirements

Research has demonstrated that CCBE1 is essential for fetal but not postnatal erythropoiesis, affecting erythroblastic island formation and function through a cell-nonautonomous mechanism .