ccdc25 Antibody

What is CCDC25 and why is it significant in cancer research?

CCDC25 is a 208-amino acid transmembrane protein (24-26 kDa) encoded by the CCDC25 gene located on chromosome 8p21.1 . It functions as a transmembrane receptor that can sense neutrophil extracellular traps (NETs) and trigger the ILK-PARVB pathway to enhance cell motility . Its significance in cancer research stems from its differential expression and diverse functional roles across multiple cancer types.

Recent studies have revealed contradictory roles for CCDC25 in different cancers:

• In cholangiocarcinoma (CCA), CCDC25 is significantly elevated in patient sera and has been proposed as a diagnostic biomarker

• In clear cell renal cell carcinoma (ccRCC), some research suggests CCDC25 acts as a tumor suppressor by inhibiting cell proliferation through the Hippo signaling pathway , while other studies indicate it promotes cancer progression via the ILK-NF-κB pathway

This multifaceted involvement makes CCDC25 an intriguing target for both diagnostic development and therapeutic intervention in cancer research.

What applications are CCDC25 antibodies commonly used for?

CCDC25 antibodies are utilized across multiple experimental applications in cancer research:

For optimal results, WB detection is validated in multiple cell lines including A549, HeLa, Jurkat, K-562, and MCF-7 cells, while IHC applications show positive results in human stomach tissue . When performing IHC, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may be used as an alternative .

How should researchers select between polyclonal and monoclonal CCDC25 antibodies?

The selection between polyclonal and monoclonal CCDC25 antibodies should be guided by the specific experimental requirements:

Polyclonal CCDC25 antibodies (e.g., 21209-1-AP):

• Recognize multiple epitopes on the CCDC25 protein

• Provide higher sensitivity due to binding to multiple epitopes

• Best suited for initial protein characterization and applications requiring robust signal detection

• Advantageous for detecting denatured proteins in applications like Western blot

• Show greater lot-to-lot variation

Monoclonal CCDC25 antibodies (e.g., GM-Tg-hg-MP2394-Ab):

• Target a single epitope with high specificity

• Offer consistent performance across experiments with minimal batch variation

• Preferred for applications requiring high reproducibility and specificity

• Superior for developing diagnostic assays, particularly serum-based biomarker tests

• Available with specific species reactivity for human, mouse, rat, and other organisms

For serum biomarker studies like those investigating CCDC25 in cholangiocarcinoma diagnosis, mouse monoclonal antibodies have been successfully employed . For mechanistic studies exploring CCDC25's role in signaling pathways, rabbit polyclonal antibodies provide excellent sensitivity for detecting protein interactions .

How can researchers optimize CCDC25 detection in serum for biomarker development?

Optimizing CCDC25 detection in serum samples requires careful consideration of multiple parameters based on successful biomarker studies:

Serum Sample Preparation:

• For cancer types with high CCDC25 expression (e.g., CCA), dilute samples appropriately (1:3 with normal saline solution)

• For healthy controls or cancers with lower CCDC25 expression, neat sera may be sufficient

Quantitative Dot Blot Methodology:

• Prepare standard curves using recombinant CCDC25 protein at serial dilutions (0.5-0.0156 ng/μl)

• Include pooled positive control sera for normalization across blots

• Spot 2 μl of each sample onto membranes

• Incubate with primary anti-CCDC25 antibody (1:1,000 dilution) overnight at 4°C

• Follow with HRP-conjugated secondary antibody (1:2,000 dilution) for 1 hour

• Detect and quantify chemiluminescent signals

Cutoff Value Determination:
ROC analysis and Youden's Index calculation from previous studies established optimal cutoff values for distinguishing:

• CCA from healthy controls: 0.017 ng/μl (100% sensitivity, 100% specificity)

• CCA from CRC: 0.017 ng/μl (99% sensitivity, 100% specificity)

• CCA from BC: 0.017 ng/μl (94% sensitivity, 98% specificity)

• CCA from HCC: 0.017 ng/μl (94% sensitivity, 95% specificity)

These parameters provide a methodological framework for developing CCDC25 as a serum biomarker, with particular promise for cholangiocarcinoma diagnosis where serum CCDC25 levels (0.193±0.039 ng/μl) significantly exceed those in other cancers and healthy controls .

What experimental approaches can resolve contradictory findings on CCDC25's role in cancer?

The contradictory findings regarding CCDC25's role in cancer progression, particularly in ccRCC where it has been reported as both tumor suppressor and promoter , necessitate systematic experimental approaches:

1. Comprehensive Expression Analysis:

• Perform multi-cohort analysis of CCDC25 expression across different cancer stages

• Correlate expression with patient outcomes using Kaplan-Meier survival analysis

• Stratify patients by molecular subtypes to identify context-dependent roles

2. Pathway-Specific Functional Studies:

• Evaluate CCDC25's effect on Hippo pathway components (reported in ccRCC suppression) :

  • Measure YAP/TAZ phosphorylation and nuclear localization

  • Assess expression of Hippo target genes (CTGF, CYR61)

• Analyze ILK-NF-κB pathway activation (reported in ccRCC promotion) :

  • Examine ILK-CCDC25 binding through co-immunoprecipitation

  • Measure NF-κB nuclear translocation and target gene expression

  • Perform rescue experiments with pathway inhibitors

3. Cell-Based Functional Assays:

• Conduct parallel studies using consistent methodologies:

  • Cell proliferation: CCK8 assay and EDU staining

  • Migration: Transwell assay and wound healing assay

  • Apoptosis: Flow cytometry with Annexin V/PI staining

  • Colony formation: Soft agar and plate-based assays

4. In Vivo Models:

• Develop both xenograft and genetic models with CCDC25 manipulation

• Analyze tumor initiation, growth rate, and metastatic potential

• Compare results across different cancer types and genetic backgrounds

5. Clinical Correlation:

• Correlate serum CCDC25 levels with tissue expression patterns

• Analyze associations with treatment response and clinical outcomes

• Integrate with other molecular markers for enhanced prognostic value

This multifaceted approach can help reconcile contradictory findings by identifying context-dependent mechanisms of CCDC25 action across different cancer types and stages.

How can CCDC25 antibodies be utilized in mechanistic studies of signaling pathways?

CCDC25 has been implicated in multiple signaling pathways, including the Hippo pathway and ILK-NF-κB pathway . CCDC25 antibodies can be strategically employed to elucidate these mechanisms:

1. Protein-Protein Interaction Studies:

• Co-immunoprecipitation (Co-IP):

  • Use CCDC25 antibodies for pull-down followed by immunoblotting for suspected interacting partners

  • Perform reverse Co-IP to confirm interactions with ILK or Hippo pathway components

  • Consider antibody epitope location to avoid interference with binding domains

• Proximity Ligation Assay (PLA):

  • Visualize endogenous protein interactions in situ

  • Combine CCDC25 antibodies with antibodies against pathway components

  • Quantify interaction signals across different cellular conditions

2. Subcellular Localization Studies:

• Immunofluorescence microscopy:

  • Track CCDC25 translocation in response to pathway activation

  • Co-stain with compartment markers to determine precise localization

  • Apply super-resolution microscopy for detailed analysis

3. CCDC25 Activation/Modification Detection:

• Phospho-specific antibodies (if available):

  • Monitor post-translational modifications affecting CCDC25 function

  • Correlate modifications with pathway activation status

4. Functional Validation:

• CCDC25 knockdown/knockout strategies:

  • CRISPR/Cas9 KO plasmids available for human and mouse

  • Analyze effects on downstream pathway components

  • Perform rescue experiments with wild-type vs. mutant CCDC25

• CCDC25 overexpression approaches:

  • CRISPR activation plasmids available for targeted upregulation

  • Assess pathway component activation in response to increased CCDC25

5. Chromatin Immunoprecipitation (ChIP):

• For transcription factors in CCDC25-regulated pathways:

  • Analyze binding to target gene promoters after CCDC25 manipulation

  • Combine with expression analysis to link pathway activation to transcriptional outcomes

These methodological approaches provide a comprehensive strategy for investigating CCDC25's mechanistic role in cancer-related signaling pathways.

What is the potential of CCDC25 as a cancer biomarker?

CCDC25 has emerged as a promising cancer biomarker, particularly for cholangiocarcinoma, with substantial evidence supporting its diagnostic utility:

Diagnostic Performance Across Cancer Types:
The following table summarizes CCDC25's diagnostic capabilities based on serum levels:

Advantages as a Biomarker:

• Independent biomarker showing no correlation with conventional markers (ALP, CEA, CA19-9)

• High sensitivity and specificity for differential diagnosis between CCA and other cancers

• Quantifiable in serum using standardized methodologies

• Demonstrates correlation between tissue expression and serum levels (r²=0.52, p=0.01)

Clinical Applications:

The exceptional performance of CCDC25 in distinguishing CCA from other cancers and healthy controls positions it as a particularly valuable biomarker for this difficult-to-diagnose cancer type, potentially enabling earlier detection and improved patient outcomes.

What methodological considerations are crucial for developing CCDC25-based diagnostic assays?

Developing robust CCDC25-based diagnostic assays requires careful attention to several methodological aspects:

1. Sample Collection and Processing:

• Standardize collection protocols (fasting vs. non-fasting, time of day)

• Establish consistent processing timelines to minimize degradation

• Define appropriate storage conditions (temperature, freeze-thaw cycles)

• Consider pre-analytical variables affecting serum protein stability

2. Assay Platform Selection:

• Quantitative dot blot:

  • Suitable for research but less practical for clinical implementation

  • Requires standardization with recombinant CCDC25 protein

• ELISA development:

  • More feasible for clinical adoption and high-throughput screening

  • Requires optimized antibody pairs (capture and detection)

  • Needs validation across diverse patient populations

3. Antibody Selection Criteria:

• Specificity: Minimal cross-reactivity with related proteins

• Sensitivity: Ability to detect CCDC25 at physiologically relevant concentrations

• Consistency: Minimal lot-to-lot variation

• Recognition domain: Accessible epitopes in native serum conditions

4. Reference Standards:

• Recombinant CCDC25 protein (e.g., orb424527) for standard curve generation

• Pooled positive control sera for inter-assay normalization

• Quality control samples spanning relevant concentration ranges

5. Clinical Validation Strategy:

• Multi-center studies with diverse patient populations

• Inclusion of appropriate disease controls and confounding conditions

• Longitudinal sampling to assess temporal stability

• Integration with conventional biomarkers for enhanced diagnostic accuracy

6. Data Analysis Framework:

• ROC analysis for determining optimal cut-off values

• Youden's Index calculation for maximizing sensitivity and specificity

• Development of multivariate models incorporating clinical factors

• Assessment of positive and negative predictive values in target populations

These methodological considerations provide a framework for translating the promising research findings on CCDC25 as a biomarker into clinically applicable diagnostic assays.

What optimization strategies improve CCDC25 detection in immunohistochemistry?

Optimizing CCDC25 detection in immunohistochemistry (IHC) requires careful attention to multiple technical parameters:

Antigen Retrieval Methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative approach: Citrate buffer pH 6.0

• Optimization based on tissue type and fixation conditions is essential

Antibody Selection and Dilution:

• Recommended dilution range: 1:50-1:500

• Start with manufacturer's recommendation and titrate for optimal signal-to-noise ratio

• Consider tissue-specific optimization (e.g., validated for human stomach tissue)

Detection Systems:

• HRP-conjugated secondary antibodies with appropriate species reactivity

• Amplification systems (e.g., tyramide signal amplification) for low-abundance detection

• Chromogens selected based on desired sensitivity and multiplexing needs

Controls and Validation:

• Positive control tissues: Use tissues with known CCDC25 expression

• Negative controls: Primary antibody omission and CCDC25-negative tissues

• CCDC25 knockdown/knockout tissues as definitive negative controls

• Peptide competition assays to confirm specificity

Counterstaining Considerations:

• Adjust hematoxylin intensity to maintain CCDC25 signal visibility

• Consider nuclear counterstains for co-localization studies

Image Analysis:

• Quantitative scoring methods (H-score, Allred score) for reproducible assessment

• Digital pathology approaches for automated quantification

• Machine learning algorithms for pattern recognition in complex tissues

These optimization strategies enable reliable CCDC25 detection in tissue samples, supporting both diagnostic applications and mechanistic studies in cancer research.

What challenges exist in detecting CCDC25 in different subcellular compartments?

As a transmembrane protein with potential dynamic localization, detecting CCDC25 across different subcellular compartments presents several technical challenges:

1. Membrane Protein Extraction Challenges:

• Require specialized buffers containing appropriate detergents

• Need to balance solubilization efficiency with preservation of protein structure

• Differential centrifugation protocols must be optimized for enrichment

• Verification of fraction purity using compartment-specific markers is essential

2. Epitope Accessibility Issues:

• Transmembrane domains may be masked in native conformations

• Different fixation methods affect epitope exposure differently:

  • Paraformaldehyde: Better preserves membrane structures but may reduce accessibility

  • Methanol/acetone: Enhances some epitopes but disrupts membrane integrity

• Antibodies targeting different CCDC25 domains may show varying efficacy across compartments

3. Immunofluorescence Optimization:

• Permeabilization conditions critically affect membrane protein detection:

  • Triton X-100: Effective for intracellular compartments

  • Saponin: Gentler for membrane proteins but may require continuous presence

  • Digitonin: Selective for plasma membrane with minimal internal membrane disruption

• Signal-to-noise ratio optimization through blocking conditions

• Multi-labeling approaches require careful antibody selection to avoid cross-reactivity

4. Dynamic Trafficking Analysis:

• Live-cell imaging requires fluorescently tagged CCDC25 constructs

• Tag position (N- vs. C-terminal) may affect localization and function

• Photobleaching techniques (FRAP/FLIP) require optimization for membrane proteins

• Inhibitors of specific trafficking pathways help elucidate regulatory mechanisms

5. Post-translational Modification Considerations:

• Modifications may affect antibody recognition in specific compartments

• Phosphorylation, glycosylation, or proteolytic processing may alter localization

• Modification-specific antibodies may be required for comprehensive analysis

Addressing these challenges requires systematic optimization and validation to ensure accurate detection of CCDC25 across different subcellular compartments, enabling more complete understanding of its functional roles.

How might CCDC25 research contribute to personalized cancer medicine?

CCDC25 research shows significant potential to advance personalized cancer medicine through multiple avenues:

Diagnostic Stratification:

• CCDC25 serum levels could identify patient subgroups requiring different diagnostic approaches

• Combined with conventional markers, CCDC25 may enhance early detection in high-risk populations

• Differential expression across cancer types could aid in identifying cancer of unknown primary

Prognostic Applications:

Therapeutic Targeting:

• Understanding CCDC25's role in the ILK-NF-κB pathway presents opportunities for targeted intervention

• The contradictory roles in different cancers suggest context-dependent therapeutic approaches:

  • Inhibition in cancers where CCDC25 promotes progression

  • Enhancement in cancers where CCDC25 acts as a tumor suppressor

• CCDC25's transmembrane localization makes it potentially accessible to antibody-based therapeutics

Treatment Response Prediction:

• CCDC25 expression patterns might predict response to specific therapeutic approaches

• Serial measurement of serum CCDC25 could monitor treatment efficacy

• Changes in CCDC25-regulated pathways may indicate developing resistance mechanisms

Immunotherapy Connections:

• CCDC25's reported role in sensing neutrophil extracellular traps (NETs) suggests potential immunological functions

• This connection to immune mechanisms could inform immunotherapy approaches

• Understanding CCDC25's immunological interactions may reveal new combinatorial strategies

These diverse applications highlight how continued CCDC25 research could significantly impact personalized medicine approaches across multiple cancer types, with particular promise in cholangiocarcinoma where diagnostic and therapeutic options remain limited.