ccdc167 Antibody

What is CCDC167 and why is it significant in cancer research?

CCDC167 (Coiled-coil domain-containing protein 167) is a protein that has been identified as significantly upregulated in multiple cancer types, particularly breast cancer. The significance of CCDC167 in cancer research stems from several key findings:

Most significantly, bioinformatics analyses and experimental validation have revealed that CCDC167 appears to play a crucial role in regulating cell proliferation and growth in breast cancer, suggesting its potential as a therapeutic target .

What are the common applications for CCDC167 antibodies in research?

CCDC167 antibodies are versatile research tools that can be employed in numerous experimental applications:

Western Blotting (WB): For detecting and quantifying CCDC167 protein expression levels in cell or tissue lysates, allowing researchers to compare expression between normal and cancerous tissues or evaluate knockdown efficiency .

Immunofluorescence (IF): Both for cultured cells (IF-cc) and paraffin-embedded tissue sections (IF-p), enabling visualization of CCDC167 localization and expression patterns at the cellular and tissue levels .

Immunohistochemistry (IHC): For both frozen sections (IHC-fro) and paraffin-embedded sections (IHC-p), particularly valuable for examining CCDC167 expression patterns in clinical samples and tissue microarrays .

ELISA: For quantitative measurement of CCDC167 in solution, useful for serum-based biomarker studies .

Immunocytochemistry (ICC): For detailed cellular localization studies of CCDC167 protein in cultured cells .

These applications collectively enable comprehensive investigation of CCDC167's expression patterns, cellular localization, and potential role in cancer pathogenesis.

How can researchers validate CCDC167 antibody specificity?

Validating antibody specificity is critical for ensuring experimental reliability. For CCDC167 antibodies, researchers should implement the following validation approaches:

Positive and negative control samples: Use cell lines or tissues known to express high levels of CCDC167 (such as breast cancer cell lines, particularly MCF-7 based on research findings) as positive controls . For negative controls, consider using CCDC167 knockdown cells created using shRNA technology, which has been shown to effectively reduce CCDC167 expression .

Western blot validation: Confirm that the antibody detects a protein of the expected molecular weight for CCDC167. Compare results between control and CCDC167-knockdown samples to verify specificity .

Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (for the ABIN1714979 antibody, this would be a synthetic peptide derived from human C6orf129/CCDC167) . If the antibody is specific, this should abolish or significantly reduce signal in subsequent applications.

Cross-reactivity testing: While the antibody is predicted to react with CCDC167 from multiple species (human, mouse, rat, cow, sheep, pig) , it's advisable to validate cross-reactivity empirically if working with non-human samples.

Multiple antibody concordance: Where possible, compare results using multiple antibodies targeting different epitopes of CCDC167 to confirm consistent patterns of expression and localization.

What signaling pathways are associated with CCDC167 in cancer progression?

Research on CCDC167-associated signaling pathways reveals intricate connections to cancer progression through multiple mechanisms:

Cell cycle regulation pathways: Bioinformatics analysis of CCDC167 co-expressed genes in breast cancer identified cell cycle-related signaling as essential . Pathway mapping and GO enrichment analyses from both METABRIC and TCGA databases consistently showed that CCDC167 and its co-expressed genes are primarily involved in cell division progression, spindle assembly, and chromosome separation .

Immune response pathways: CCDC167 co-expressed genes affect immune-related pathways, including "immune response antigen presentation by MHC class I" and "immune response interferon (IFN)-alpha/beta signaling via phosphatidylinositol 3-kinase (PI3K) and nuclear factor (NF)-κB pathways" . This suggests potential immunomodulatory roles for CCDC167 in the tumor microenvironment.

Ubiquitination pathways: Co-expression analysis also revealed significant involvement in ubiquinone metabolism and ubiquitination-related pathways . Ubiquitination is a critical post-translational modification that regulates protein degradation and is frequently dysregulated in cancer.

Knockdown of CCDC167 in MCF-7 breast cancer cells significantly altered the expression of cell cycle-related and apoptosis-related genes, providing experimental validation of CCDC167's involvement in these pathways . Additionally, CCDC167 knockdown resulted in increased early and late apoptosis, suggesting its role in apoptotic resistance common in cancer progression .

How does CCDC167 expression correlate with breast cancer prognosis and treatment response?

CCDC167 expression demonstrates significant correlations with breast cancer prognosis and potential treatment response:

Correlation with cancer progression: CCDC167 expression levels increase progressively from nuclear grade I to III breast cancers, indicating a relationship between CCDC167 expression and tumor aggressiveness .

Chemotherapy response: Treatment of MCF-7 breast cancer cells with standard chemotherapeutic agents (fluorouracil, carboplatin, paclitaxel, and doxorubicin) resulted in decreased CCDC167 expression levels . This suggests that CCDC167 downregulation may be part of the mechanism by which these drugs exert their anticancer effects.

Treatment implications: The relationship between CCDC167 and response to established chemotherapeutics suggests that CCDC167 signaling might be a critical target for these cytotoxic drugs. Compounds specifically designed to target CCDC167 or its downstream pathways could potentially represent new therapeutic approaches for breast cancer patients .

These findings collectively indicate that CCDC167 could serve as both a prognostic biomarker and a potential therapeutic target in breast cancer.

What techniques can be used to study CCDC167 knockdown effects in cancer cell lines?

For investigating CCDC167 knockdown effects in cancer cell lines, researchers can employ several complementary techniques:

RNA interference (RNAi): Short hairpin RNA (shRNA) targeting CCDC167 has been successfully used to create stable knockdown in MCF-7 breast cancer cells . This approach allows for long-term suppression of CCDC167 expression, enabling studies of sustained effects on cancer cell phenotypes.

Cell proliferation assays:

• Short-term proliferation: The MTT assay has been effectively used to detect differences in short-term cell proliferation between CCDC167-knockdown and control cells .

• Long-term proliferation: Colony-formation assays provide assessment of long-term growth potential. This approach revealed that long-term cell growth was significantly suppressed in CCDC167-shRNA breast cancer cells .

Cell cycle analysis: Flow cytometry-based cell cycle analysis can reveal how CCDC167 knockdown affects cell cycle progression, which is particularly relevant given CCDC167's association with cell cycle-related pathways .

Apoptosis assessment: Flow cytometry with Annexin V and propidium iodide staining has demonstrated that CCDC167 knockdown increases both early and late apoptosis in breast cancer cells .

Gene expression analysis: Quantitative PCR (qPCR) analysis of CCDC167-knockdown cells has revealed significant alterations in cell cycle-related and apoptosis-related genes, providing insights into the molecular mechanisms underlying CCDC167's effects .

Xenograft models: Although not directly mentioned in the search results, in vivo validation of CCDC167 knockdown effects could be performed using xenograft models, where CCDC167-knockdown cancer cells are implanted into immunodeficient mice to assess tumor growth and progression.

What is the optimal protocol for using CCDC167 antibodies in Western blotting?

For optimal Western blotting with CCDC167 antibodies, researchers should consider the following protocol adaptations:

Sample preparation:

• Extract total protein from cells using a complete lysis buffer containing protease inhibitors.

• For breast cancer cell lines like MCF-7, a standard RIPA buffer with protease inhibitor cocktail is suitable.

• Quantify protein concentration using BCA or Bradford assay to ensure equal loading.

Gel electrophoresis and transfer:

• Load 20-30 μg of protein per lane on a 10-12% SDS-PAGE gel.

• After separation, transfer proteins to a PVDF or nitrocellulose membrane.

• Verify transfer efficiency with Ponceau S staining.

Antibody incubation:

• Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Incubate with anti-CCDC167 primary antibody (such as ABIN1714979) at the manufacturer's recommended dilution (typically 1:500 to 1:2000) overnight at 4°C .

• Wash thoroughly with TBST (3 times, 5 minutes each).

• Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit IgG for ABIN1714979) at 1:5000 dilution for 1 hour at room temperature .

• Wash extensively with TBST (4 times, 5 minutes each).

Detection and controls:

• Develop using ECL substrate and image using a digital imaging system.

• Include positive controls (e.g., MCF-7 cells with known CCDC167 expression) .

• Include negative controls (e.g., CCDC167 knockdown cells or non-expressing cell lines) .

• Use β-actin or GAPDH as loading controls to normalize expression levels.

Quantification:

• Perform densitometric analysis of bands using software like ImageJ.

• Normalize CCDC167 signal to the loading control for accurate quantification.

• Compare expression levels between experimental conditions using appropriate statistical tests.

How can researchers optimize immunofluorescence protocols for CCDC167 detection?

Optimizing immunofluorescence for CCDC167 detection requires attention to several critical parameters:

Sample preparation:

• For cultured cells (IF-cc): Grow cells on glass coverslips, fix with 4% paraformaldehyde for 15 minutes, and permeabilize with 0.1% Triton X-100 for 10 minutes .

• For paraffin-embedded sections (IF-p): Perform antigen retrieval using citrate buffer (pH 6.0) under pressure or heat treatment to unmask epitopes potentially obscured during fixation .

Blocking and antibody incubation:

• Block with 5% normal serum (matching the species of the secondary antibody) plus 1% BSA in PBS for 1 hour at room temperature.

• Incubate with anti-CCDC167 primary antibody (ABIN1714979 or equivalent) at dilutions typically ranging from 1:100 to 1:500 overnight at 4°C .

• Wash thoroughly with PBS (3 times, 5 minutes each).

• Incubate with fluorophore-conjugated secondary antibody at 1:500 dilution for 1 hour at room temperature in the dark .

• For simpler detection, directly conjugated antibodies (such as AbBy Fluor® 488, 555, or 647 conjugated anti-CCDC167) can be used to eliminate the secondary antibody step .

Counterstaining and mounting:

• Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes.

• Mount slides with anti-fade mounting medium to preserve fluorescence.

Imaging and controls:

• Capture images using a confocal or fluorescence microscope with appropriate filter sets.

• Include a secondary-only control to assess background autofluorescence and non-specific binding.

• Use CCDC167 knockdown cells as a negative control to confirm antibody specificity .

• Consider co-staining with markers of subcellular compartments to determine precise localization.

Troubleshooting:

• If signal is weak, increase antibody concentration or incubation time.

• If background is high, increase blocking time, adjust antibody dilutions, or add 0.1% Tween-20 to washing buffers.

• For tissue sections showing high autofluorescence, consider using Sudan Black B treatment or commercial autofluorescence quenching reagents.

How should experiments be designed to study the relationship between CCDC167 and chemotherapy response?

To effectively investigate CCDC167's role in chemotherapy response, researchers should design comprehensive experimental approaches:

Cell line selection:

• Primary model: MCF-7 breast cancer cells have demonstrated measurable CCDC167 expression and responsiveness to chemotherapy agents in previous studies .

• Validation models: Include additional breast cancer cell lines representing different molecular subtypes (e.g., MDA-MB-231 for triple-negative, SKBR3 for HER2+) to assess subtype-specific effects.

• Non-cancer control: Include normal breast epithelial cells (e.g., MCF-10A) for comparison.

Experimental conditions:

• Treatment groups: Test clinically relevant chemotherapeutic agents shown to affect CCDC167 expression, including fluorouracil, carboplatin, paclitaxel, and doxorubicin .

• Dose-response: Establish dose-response curves for each agent to identify IC50 values.

• Time-course: Examine both short-term (24-48h) and long-term (5-7 days) effects.

Key readouts:

• CCDC167 expression changes:

  • mRNA level: Quantitative PCR to measure CCDC167 transcript levels following drug treatment .

  • Protein level: Western blotting with anti-CCDC167 antibody to assess protein expression changes .

• Cell viability and proliferation:

  • MTT or resazurin assays for metabolic activity assessment .

  • Colony formation assays for long-term growth potential evaluation .

  • BrdU incorporation for cell proliferation analysis.

• Apoptosis assessment:

  • Annexin V/PI staining and flow cytometry to quantify early and late apoptotic populations .

  • Caspase activity assays to measure apoptotic pathway activation.

Mechanistic investigations:

• CCDC167 modulation: Compare chemotherapy response in parental versus CCDC167-knockdown and CCDC167-overexpressing cells .

• Pathway analysis: Examine changes in cell cycle-related and apoptosis-related genes following treatment to identify potential mechanism of action .

• Combination approaches: Test whether CCDC167 knockdown sensitizes cells to lower doses of chemotherapeutic agents.

Translational relevance:

• Patient-derived models: Where possible, include patient-derived xenografts or organoids to validate findings in more clinically relevant models.

• Correlation with clinical data: Analyze public datasets to correlate CCDC167 expression with documented chemotherapy response in breast cancer patients.

This experimental design provides a comprehensive framework for understanding how CCDC167 interacts with chemotherapy response mechanisms, potentially identifying strategies to improve treatment efficacy.

What are the best experimental controls when studying CCDC167 expression across various cancer types?

Rigorous experimental controls are essential for reliable CCDC167 expression studies across cancer types:

Tissue and cell line controls:

• Normal tissue controls: Include matched normal tissue from the same patient/donor whenever possible. For breast cancer studies, normal adjacent breast tissue serves as an appropriate control to account for patient-specific variables .

• Cell line panel: Include a diverse panel of cancer cell lines spanning multiple cancer types alongside their normal counterparts (e.g., MCF-7 vs. MCF-10A for breast studies) .

• Positive expression control: Include samples known to express high levels of CCDC167, such as breast cancer tissues with nuclear grade III as established by previous research .

Technical controls:

Analytical controls:

• Reference genes: For qPCR, use multiple validated reference genes appropriate for each tissue type being studied.

• Loading controls: For Western blotting, use appropriate loading controls (β-actin, GAPDH, or total protein staining) with verification of equal loading .

• Batch effect controls: Include standardized samples across experimental batches to detect and correct for technical variation.

Bioinformatic controls:

• Database integration: Cross-validate findings across multiple cancer databases (e.g., TCGA, METABRIC, Oncomine) to ensure robustness of expression patterns .

• Statistical thresholds: Establish appropriate statistical thresholds and corrections for multiple testing when analyzing expression across numerous cancer types.

• Subtypes consideration: Account for molecular subtypes within each cancer type, as CCDC167 expression may vary between subtypes (as demonstrated in breast cancer) .

Implementation of these comprehensive controls ensures reliable and reproducible assessment of CCDC167 expression patterns, strengthening the validity of comparative oncology studies.

What methodologies can be used to evaluate CCDC167 as a potential therapeutic target?

Evaluating CCDC167 as a therapeutic target requires a systematic, multi-faceted approach:

Target validation studies:

• Expression correlation: Comprehensive analysis of CCDC167 expression across cancer types and stages using tissue microarrays and public databases (TCGA, METABRIC, Oncomine) .

• Survival impact: Kaplan-Meier analysis to correlate CCDC167 expression with patient outcomes, as previously demonstrated for breast cancer .

• Functional necessity: CCDC167 knockdown experiments in multiple cancer cell lines to establish its necessity for cancer cell survival and proliferation .

Mechanistic investigations:

• Pathway analysis: Detailed characterization of CCDC167-dependent signaling pathways using phospho-proteomic approaches and pathway inhibitors .

• Interaction partners: Co-immunoprecipitation with anti-CCDC167 antibodies followed by mass spectrometry to identify protein interaction partners .

• Structure-function studies: Domain mapping to identify critical functional regions that could be targeted by small molecules or biologics.

Therapeutic intervention approaches:

• Small molecule screening:

  • High-throughput screening of compound libraries against CCDC167 function

  • Structure-based drug design if structural information becomes available

• RNA interference therapeutics:

  • Development of siRNA or shRNA delivery systems targeting CCDC167 mRNA

  • Testing in cell culture and animal models of cancer

• Antibody-based therapeutics:

  • Development of function-blocking antibodies against CCDC167

  • Antibody-drug conjugates targeting CCDC167-expressing cells

Translational research:

• Animal models: Testing promising therapeutic candidates in patient-derived xenograft models

• Combination approaches: Evaluation of CCDC167-targeting agents in combination with standard chemotherapeutics (fluorouracil, carboplatin, paclitaxel, and doxorubicin) that have shown interactions with CCDC167 expression

• Biomarker development: Development of companion diagnostics using validated anti-CCDC167 antibodies for patient stratification

Safety assessment:

• Expression profiling: Thorough analysis of CCDC167 expression in normal tissues to anticipate potential off-target effects

• Toxicity screening: In vitro and in vivo toxicity screening of CCDC167-targeting approaches

This comprehensive evaluation framework provides a roadmap for assessing CCDC167's potential as a therapeutic target, from basic validation through preclinical development of targeted interventions.

How can CCDC167 antibodies be used in patient stratification for clinical trials?

CCDC167 antibodies offer powerful tools for patient stratification in clinical trials through several methodological approaches:

Tissue-based expression analysis:

• Immunohistochemistry (IHC): Using validated anti-CCDC167 antibodies to analyze expression in patient tumor samples . This allows for semi-quantitative scoring of expression levels (0, 1+, 2+, 3+) similar to HER2 testing in breast cancer.

• Multiplexed immunofluorescence: Combining CCDC167 detection with other relevant markers (e.g., estrogen receptor, HER2) to identify expression patterns across tumor subtypes .

Scoring and classification systems:

• Expression threshold determination: Establishing clinically relevant CCDC167 expression thresholds based on correlation with survival data . This could classify patients as "CCDC167-high" or "CCDC167-low" for stratification purposes.

• Digital pathology: Employing automated image analysis with machine learning algorithms to standardize and quantify CCDC167 immunostaining, reducing inter-observer variability.

Clinical application strategies:

• Prognostic stratification:

  • Given the established correlation between high CCDC167 expression and poor prognosis in breast cancer , patients could be stratified into risk categories for clinical trial enrollment.

  • This approach would be particularly valuable for trials evaluating adjuvant therapies in early-stage disease.

• Predictive biomarker development:

  • For trials evaluating CCDC167-targeting agents, expression levels could serve as a predictive biomarker.

  • For chemotherapy trials, CCDC167 expression might predict response to standard agents like fluorouracil, carboplatin, paclitaxel, and doxorubicin, which have been shown to modulate CCDC167 expression .

• Patient enrichment strategies:

  • First-line trials: Enriching for patients with high CCDC167 expression who may benefit most from targeted therapies.

  • Resistance-focused trials: Selecting patients who progressed after standard therapies and show persistent high CCDC167 expression.

Quality control considerations:

• Reference standards: Inclusion of cell line controls with known CCDC167 expression levels on each testing batch .

• Interlaboratory validation: Ensuring consistent CCDC167 detection across multiple testing sites through standardized protocols and proficiency testing.

• Complementary technologies: Validating IHC results with orthogonal methods like qPCR or RNA-seq in a subset of samples to confirm expression patterns.

By implementing these methodologies, clinical researchers can effectively leverage CCDC167 antibodies for patient stratification, potentially improving trial outcomes and accelerating the development of precision medicine approaches.

What is known about the relationship between CCDC167 and other members of the coiled-coil domain-containing protein family in cancer?

The relationship between CCDC167 and other CCDC family members in cancer represents an emerging research area with several important insights:

Expression patterns across CCDC family members:
Research has revealed that several CCDC family members, not just CCDC167, show significant overexpression in various breast cancer subtypes compared to normal breast tissues . This suggests potential functional redundancy or cooperative mechanisms among CCDC family proteins in cancer development.

Subtype-specific expression:
Comparative analysis of multiple CCDC family members across breast cancer subtypes (including claudin-low, basal, HER2, luminal A, and luminal B) has shown distinct expression patterns . This differential expression might indicate specialized roles for different CCDC family members in specific cancer subtypes.

Functional implications:
The significant overexpression of multiple CCDC members in breast cancer subtypes suggests this protein family may have a substantial collective impact on tumor development . This observation points to potential functional redundancy that could affect therapeutic approaches targeting individual CCDC proteins.

Research opportunities:
Further investigation is needed to determine whether:

• Multiple CCDC proteins function in the same signaling networks or represent distinct oncogenic pathways

• Combinations of CCDC family members have synergistic effects on cancer progression

• Targeting multiple CCDC proteins simultaneously might provide therapeutic advantages over single-target approaches

• Specific CCDC combinations correlate with particular cancer phenotypes or treatment responses

Methodological approaches for comparative studies:

• Multiplexed analysis: Simultaneous detection of multiple CCDC family members using antibody panels in tissue microarrays

• Co-expression network analysis: Bioinformatic investigation of potential functional relationships between CCDC family members

• Combinatorial knockdown experiments: Assessing the effects of silencing multiple CCDC family members simultaneously

This emerging research area highlights the importance of considering CCDC167 within the broader context of the CCDC protein family, potentially revealing more comprehensive understanding of coiled-coil domain proteins in cancer biology.

How might new antibody discovery technologies advance CCDC167-targeted therapeutics?

Recent advances in antibody discovery technologies offer promising approaches for developing CCDC167-targeted therapeutics:

Microfluidics-enabled antibody screening:
Recent technological developments utilize droplet microfluidics to encapsulate single antibody-secreting cells (ASCs) at extremely high throughput (10^7 cells per hour) . This approach enables concentration of secreted antibodies and simple addition and removal of detection reagents . When combined with fluorescence-activated cell sorting (FACS), this technology allows isolation of antigen-specific ASCs for single-cell sequencing and recombinant antibody expression .

Benefits for CCDC167-targeted therapeutic development:

• Accelerated discovery timeline: The microfluidics-FACS approach could generate CCDC167-specific antibodies within 2 weeks, dramatically accelerating the early development process compared to traditional methods .

• High hit rate: Recent applications of this technology have achieved >85% target binding among characterized antibodies, suggesting efficient identification of CCDC167-binding candidates would be possible .

• Superior affinity: The approach has yielded antibodies with subpicomolar affinity (<1 pM) in other applications, indicating potential for developing high-affinity anti-CCDC167 antibodies .

• Enhanced functional properties: Previous applications have generated antibodies with excellent neutralizing capacity (<100 ng/ml), suggesting this approach could yield functionally active anti-CCDC167 antibodies with therapeutic potential .

Implementation strategy for CCDC167:

• Immunization of mice with CCDC167 recombinant protein or peptides

• Isolation of CD138+ plasma cells from immunized animals

• Single-cell encapsulation in BG-agarose using microfluidics

• Functionalization with anti-mouse κ VHH–SNAP for antibody capture

• FACS-based selection of CCDC167-binding antibodies

• Single-cell sequencing and recombinant expression of selected antibodies

Therapeutic development pathways:

• Direct antagonists: Development of antibodies that directly block CCDC167 function

• Antibody-drug conjugates: Conjugation of cytotoxic payloads to CCDC167-targeting antibodies for selective delivery to cancer cells

• Bispecific antibodies: Engineering bispecific antibodies linking CCDC167 recognition with immune cell recruitment

This application of cutting-edge antibody discovery technology to CCDC167 could significantly advance therapeutic development by reducing discovery timelines while improving antibody quality characteristics.