ARI7 Antibody

What is AR-V7 and why is it important in prostate cancer research?

AR-V7 is a splice variant of the androgen receptor that lacks the C-terminal ligand-binding domain but retains the N-terminal transcriptionally active domain. This structural configuration enables AR-V7 to drive the expression of androgen-responsive genes through an androgen-independent pathway . The protein has 16 distinctive C-terminal amino acids encoded by an alternate cryptic exon 3, producing a unique AR-V7 C-terminal protein domain .

What types of AR-V7 antibodies are commercially available?

Currently, at least seven commercially available antibodies are designed to specifically detect the AR-V7 protein. These include:

• Clone EPR15656 (Abcam)

• Clone E308L (Cell Signalling)

• "Polyclonal antibody" (Cell Signalling)

• Clone SN8 (Creative Diagnostic)

• Clone DHH-1 (RQ4683, Assay Matrix)

• Clone RM7 (RevMab Biosciences)

• Clone AG10008 (Precision Antibody)

These antibodies have been raised against C-terminal peptides corresponding to the unique 16 amino acid sequence (EKFRVGNCKHLKMTRP) specific to AR-V7, although the exact antigens used for antibody generation vary between manufacturers .

How does AR-V7 expression relate to prostate cancer progression?

Multivariate analysis further confirmed AR-V7 as an independent risk factor for shorter PFS (HR, 3.76; 95% CI, 1.63 to 8.70; P = 0.002), shorter CSS (HR: 9.17; 95% CI, 1.48 to 55.56; P = 0.017), and shorter OS (HR: 4.81; 95% CI, 1.28 to 17.86; P = 0.020) . These findings highlight the prognostic significance of AR-V7 expression in guiding treatment decisions for prostate cancer patients.

How do different AR-V7 antibody clones compare in specificity and sensitivity?

Comparison studies of commercially available AR-V7 antibodies have revealed significant differences in specificity and sensitivity. A comprehensive evaluation of seven antibodies using western blotting and immunocytostaining on prostate cancer cell lines with known AR/AR-V7 status found that:

The antibody clone E308L emerged as the "cleanest" antibody with negligible cross-reactivity in AR-V7 negative cell lines and a specific nuclear signal, making it the most suitable choice for AR-V7 detection in circulating tumor cells (CTCs) .

What methodological challenges exist in AR-V7 detection in clinical samples?

Several methodological challenges complicate reliable AR-V7 detection in clinical samples:

These challenges underscore the need for standardized protocols and careful antibody validation before implementing AR-V7 detection in clinical decision-making.

How does AR-V7 subcellular localization impact clinical interpretation?

The subcellular localization of AR-V7 provides important information that may correlate with disease progression and therapy response. While AR-V7 mRNA detection is well-established, protein detection adds value by revealing subcellular distribution patterns that may indicate functional activity.

Nuclear localization of AR-V7 is generally associated with active transcriptional function, potentially driving resistance to androgen-directed therapies. Studies have shown that information regarding AR-V7 subcellular localization within CTCs may add important information correlating to disease progression and therapy response .

Researchers should note that different antibodies demonstrate varying abilities to detect nuclear versus cytoplasmic AR-V7. For example, the E308L antibody provides a specific nuclear signal with minimal background staining, making it particularly suitable for assessing AR-V7's transcriptional activity . The distinct visualization of nuclear versus cytoplasmic AR-V7 enables researchers to correlate localization patterns with clinical outcomes, potentially refining the biomarker's predictive value.

What is the optimal protocol for AR-V7 immunocytostaining in CTCs?

Based on the comparative studies in the search results, the following protocol represents an optimized approach for AR-V7 immunocytostaining in CTCs:

• CTC enrichment: Use of RosetteSep™ CTC enrichment cocktail containing anti-CD36 for negative depletion of leukocytes from peripheral blood (2 × 9 mL collected in EDTA vacutubes and processed within 24 hours) .

• Sample preparation: After enrichment, cells should be spun onto glass slides (200 × g, 10 min) and fixed in 4% paraformaldehyde for 10 minutes .

• Antibody selection: The anti-AR-V7 antibody clone E308L is recommended for optimal specificity and sensitivity, demonstrating the best signal-to-noise ratio with a specific nuclear signal .

• Staining procedure:

  • Permeabilize cells with 0.2% Triton X-100 in PBS for 10 minutes

  • Block with 5% BSA in PBS for 30 minutes

  • Incubate with primary anti-AR-V7 antibody (E308L) at manufacturer's recommended dilution overnight at 4°C

  • Wash and incubate with appropriate fluorophore-conjugated secondary antibody

  • Co-stain with anti-CD45 to exclude lymphocytes and Hoechst to identify nucleated cells

• Imaging and analysis: Use fluorescence microscopy and automated image analysis software (such as CellProfiler) to quantitatively assess AR-V7 staining intensity and subcellular localization .

• CTC identification criteria: Define CTCs as CD45-negative, AR-V7-positive, and Hoechst-positive events, with AR-V7 positivity determined relative to a positive control (e.g., 22RV1 cells processed in parallel) .

This protocol has been validated for detecting AR-V7 in CTCs from CRPC patients and enables subsequent correlation studies between AR-V7 subcellular localization and clinical outcomes.

How can contradictory AR-V7 staining results be reconciled?

Contradictory AR-V7 staining results between different antibodies represent a significant challenge in clinical research. Several approaches can help reconcile such discrepancies:

• Parallel validation with multiple detection methods: Combine immunohistochemistry/immunocytochemistry with molecular techniques such as RT-PCR or droplet digital PCR (ddPCR) to confirm AR-V7 status . This multi-modal approach provides complementary evidence that can clarify ambiguous staining patterns.

• Use of well-characterized positive and negative controls: Include cell lines with known AR-V7 status (e.g., 22RV1 as high AR-V7 expressing, VCaP as moderate AR-V7 expressing, and LNCaP as AR-V7 negative) in each staining run to establish threshold values for positivity .

• Quantitative image analysis: Implement automated image analysis software like CellProfiler to objectively quantify AR-V7 staining intensity and localization patterns, reducing subjective interpretation biases .

• Correlation with clinical outcomes: When faced with discordant antibody results, examine which antibody's staining pattern correlates better with established clinical endpoints. For instance, in one study comparing AG10008 and RM7 antibodies, only AG10008 staining conveyed prognostic information and was associated with shorter progression-free patient survival .

• Western blot validation: For research purposes, confirm the specificity of antibodies by western blotting using cell line controls to ensure detection of the expected ~80 kDa AR-V7 protein band with minimal cross-reactivity .

By implementing these strategies, researchers can better navigate the challenges posed by variable antibody performance and establish more reliable AR-V7 detection protocols.

What controls should be incorporated in AR-V7 detection experiments?

Proper controls are essential for reliable AR-V7 detection experiments. Based on the provided search results, the following controls should be incorporated:

• Positive cell line controls:

  • 22RV1 cells (high AR-V7 expression)

  • VCaP cells (moderate AR-V7 expression)

• Negative cell line controls:

  • LNCaP cells (AR-positive but AR-V7 negative)

  • PC3 cells (AR-negative and AR-V7 negative)

• Technical controls:

  • Secondary antibody-only control to assess non-specific binding

  • Isotype control to evaluate background staining

  • AR-FL staining in parallel to distinguish AR-V7 from full-length AR

• Process controls for CTC detection:

  • CD45 staining to exclude leukocytes

  • Nuclear staining (e.g., Hoechst) to confirm cellular identity

  • Spiking experiments with known quantities of AR-V7-positive cells to determine detection threshold and recovery rate

• Cross-validation controls:

  • Parallel assessment with multiple AR-V7 antibodies when feasible

  • Correlation with mRNA detection methods (e.g., ddPCR)

These comprehensive controls help establish assay validity, determine appropriate positivity thresholds, and ensure reliable interpretation of AR-V7 detection results in experimental and clinical samples.

How can AR-V7 status guide treatment decisions in prostate cancer patients?

AR-V7 status has emerged as a valuable biomarker for guiding treatment decisions in prostate cancer, particularly for patients with advanced disease. Evidence-based strategies for incorporating AR-V7 testing into clinical decision-making include:

It's important to note that while AR-V7 testing shows promise for clinical decision-making, standardization of detection methods and prospective validation in large clinical cohorts are still needed for optimal implementation in routine clinical practice.

Is AR-V7 expression in primary prostate cancer predictive of future castration resistance?

Emerging evidence suggests that AR-V7 expression in primary prostate cancer tissue may indeed predict future development of castration resistance, challenging earlier notions that AR-V7 becomes detectable only after progression to CRPC.

A study examining AR-V7 expression in primary prostate cancer tissue prior to long-term androgen deprivation found that AR-V7 can be detected in a subset of primary tumors . Specifically, using two different antibodies (AG10008 and RM7), researchers observed AR-V7 positivity in 24.9% and 21% of primary tumor cores, respectively .

More significantly, AR-V7 positivity in primary tumors (as detected by antibody AG10008) was associated with shorter progression-free survival, suggesting its potential value as a predictive biomarker for castration resistance . This finding indicates that AR-V7 expression may identify a subset of hormone-naïve prostate cancers with inherent resistance mechanisms that predispose them to earlier development of castration resistance.

What is the relationship between AR-V7 detection in CTCs and tissue biopsies?

The relationship between AR-V7 detection in circulating tumor cells (CTCs) and tissue biopsies represents an important area of investigation, particularly as liquid biopsies gain prominence as a less invasive alternative to tissue sampling. Based on the available research:

• Complementary but not identical information: AR-V7 detection in CTCs and tissue biopsies provides complementary but not always concordant information. CTCs may represent the current state of disease, potentially capturing heterogeneity from multiple metastatic sites, while tissue biopsies provide deeper information about a specific tumor location .

• CTC advantage in advanced disease: In advanced prostate cancer, particularly CRPC, tissue biopsies from metastatic sites are often difficult to obtain. CTCs offer a practical alternative for biomarker analysis, with studies demonstrating that AR-V7 detection in CTCs correlates with clinical outcomes and treatment responses . As noted in the research, "given the general lack of matching tumor tissue for biomarker analysis at the CRPC stage these methods have been used to successfully detect AR-V7 transcripts from liquid biopsies" .

• Methodological considerations: AR-V7 detection methods differ between CTCs and tissue samples. In tissue, immunohistochemistry is commonly employed, while CTCs can be analyzed through immunocytostaining or RNA-based methods. The choice of antibody significantly impacts results in both sample types, as demonstrated by comparative studies .

• Clinical utility: Both sample types have demonstrated clinical utility, but in different contexts. AR-V7 in primary tumor tissue appears valuable for long-term prognostication following radical prostatectomy , while CTC-based AR-V7 detection may be more useful for real-time treatment decisions in metastatic disease .

• Evolving landscape: The field is moving toward integrating information from both sample types when available, with emerging evidence suggesting that combined analysis may provide more comprehensive biomarker information to guide personalized treatment strategies .

As research in this area continues to evolve, standardized protocols for AR-V7 detection in both CTCs and tissue samples will be essential for reliable cross-platform comparisons and clinical implementation.

What emerging technologies might improve AR-V7 detection sensitivity and specificity?

Several emerging technologies hold promise for enhancing AR-V7 detection sensitivity and specificity:

• Novel antibody development technologies: Advanced recombinant antibody engineering approaches may yield next-generation AR-V7 antibodies with superior specificity for the unique C-terminal domain, potentially reducing the cross-reactivity issues observed with current antibodies .

• Multiplex imaging platforms: Technologies like multiplexed immunofluorescence or mass cytometry (CyTOF) could enable simultaneous detection of AR-V7 alongside other relevant biomarkers, providing more comprehensive characterization of tumor biology while internal controls improve specificity .

• Digital pathology and artificial intelligence: Machine learning algorithms trained on large datasets of AR-V7 staining patterns could improve standardization of interpretation and potentially identify subtle staining patterns that correlate with clinical outcomes better than traditional visual assessment .

• Single-cell technologies: Methods like single-cell RNA sequencing or single-cell proteomics may allow for more sensitive detection of AR-V7 at the individual cell level, potentially revealing clinically significant heterogeneity that might be missed by bulk analysis methods .

• Circulating tumor DNA (ctDNA) approaches: Beyond CTC analysis, emerging liquid biopsy techniques targeting AR-V7 in ctDNA might offer complementary or alternative approaches with potentially higher sensitivity for detecting AR-V7-expressing clones .

• Proximity ligation assays (PLA): This technology could enable more specific detection of AR-V7 protein by requiring simultaneous binding of two different antibodies targeting distinct epitopes, thereby reducing false positives from cross-reactivity .

These technological advances may address current limitations in AR-V7 detection and ultimately improve its utility as a biomarker for guiding prostate cancer treatment decisions.

How might AR-V7 detection be integrated into precision medicine approaches for prostate cancer?

The integration of AR-V7 detection into precision medicine frameworks for prostate cancer represents a promising frontier. Several potential approaches include:

• Biomarker-driven treatment algorithms: Developing clinical decision support tools that incorporate AR-V7 status alongside other molecular markers (e.g., PTEN loss, TMPRSS2-ERG fusion, DNA repair defects) to guide personalized treatment selection .

• Sequential liquid biopsy monitoring: Implementing regular AR-V7 testing in CTCs throughout treatment to dynamically track emerging resistance and adjust therapeutic strategies accordingly, potentially detecting resistance before clinical or radiographic progression .

• Combination therapy rationalization: Using AR-V7 status to identify patients who might benefit from novel combination approaches targeting both AR-dependent and AR-independent pathways simultaneously, rather than sequential monotherapies .

• Neoadjuvant therapy selection: Incorporating AR-V7 testing in primary tumors to inform decisions about neoadjuvant therapy before radical prostatectomy, potentially intensifying treatment for AR-V7-positive cases that show higher risk of progression .

• Clinical trial stratification: Employing standardized AR-V7 detection methods to stratify patients in clinical trials testing novel agents, facilitating the identification of therapies specifically effective in AR-V7-positive or AR-V7-negative populations .

• Multi-omic integration: Combining AR-V7 detection with broader genomic, transcriptomic, and proteomic profiling to create comprehensive tumor signatures that more precisely predict treatment responses and resistance patterns .

The successful integration of AR-V7 testing into precision medicine workflows will require continued refinement of detection methodologies, prospective validation in diverse clinical contexts, and development of clear guidelines for clinical implementation based on robust evidence.