LRRC3B Antibody

What is LRRC3B and why is it important in research?

LRRC3B (Leucine Rich Repeat Containing 3B) is a tumor suppressor protein with significant implications in cancer biology and immunotherapy. The canonical human protein consists of 259 amino acid residues with a molecular mass of 29.3 kDa and is primarily localized to the cell membrane . It is predominantly expressed in the testis, skeletal muscle, and cerebral cortex, and has been identified as a critical component in anti-tumor immune responses . Recent studies have established LRRC3B as an important biomarker for predicting response to anti-PD-1 therapy, making its detection crucial for cancer research and potential therapeutic applications .

What are the principal applications for LRRC3B antibodies in research?

LRRC3B antibodies are versatile research tools employed across multiple immunodetection techniques:

These applications are essential for investigating LRRC3B's role in tumor progression, metastasis, and immune response modulation .

What species reactivity should be considered when selecting LRRC3B antibodies?

When selecting LRRC3B antibodies, researchers should consider cross-reactivity profiles based on experimental models. Most commercially available antibodies demonstrate reactivity to human LRRC3B, while some also cross-react with mouse and rat orthologs . LRRC3B gene orthologs have been identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . For comparative studies across species, it is advisable to verify the antibody's cross-reactivity spectrum through literature validation or preliminary testing, particularly when working with non-human experimental models .

What are the optimal conditions for LRRC3B antibody validation in research applications?

Rigorous validation of LRRC3B antibodies is essential for experimental reliability. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines with known LRRC3B expression levels (e.g., testis, skeletal muscle, and cerebral cortex tissue for positive controls) .

• Knockout/knockdown verification: Employ LRRC3B-knockout cells or siRNA knockdown to confirm specificity.

• Epitope mapping: Verify recognition of the intended epitope region, such as amino acids 34-204 as used in some commercial antibodies .

• Multiple detection methods: Cross-validate findings using at least two independent techniques (e.g., Western blot and immunofluorescence).

• Batch consistency testing: When changing antibody lots, perform parallel experiments to ensure consistent results.

For immunofluorescence applications, researchers should optimize fixation protocols, permeabilization conditions, and blocking solutions to minimize background while maximizing specific signal detection .

How should multiplex immunofluorescence experiments with LRRC3B be designed?

For multiplex immunofluorescence involving LRRC3B, careful experimental design is critical:

• Antibody selection: Choose antibodies raised in different host species to avoid cross-reactivity. For LRRC3B multiplex with immune markers like CD4 and CD8, researchers have successfully used rabbit polyclonal anti-LRRC3B antibodies (1:200 dilution) alongside mouse monoclonal antibodies for immune markers .

• Staining sequence optimization: Perform pre-experimental optimization of individual immunohistochemistry protocols before attempting multiplexing.

• Signal separation: Use spectrally distinct fluorophores with minimal overlap. Commercial kits like Opal 7-color fluorescent IHC kits have been successfully employed for co-staining LRRC3B with CD4 and CD8 .

• Control samples: Include single-stained controls and unstained controls in each experiment.

• Signal amplification: Consider tyramide signal amplification for low-abundance targets.

The protocol should include a 1-hour room temperature incubation with primary antibodies followed by appropriate secondary antibody incubation, with DAPI counterstaining for nuclear visualization .

What are the recommended protocols for detecting LRRC3B expression in cancer tissues?

For optimal detection of LRRC3B in cancer tissues:

• Tissue preparation: Use freshly fixed (10% neutral buffered formalin, 24 hours) and paraffin-embedded tissues, sectioned at 3-5 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes.

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and non-specific binding with 5% normal serum.

• Primary antibody: Incubate with anti-LRRC3B antibody (typically 1:100-1:200 dilution) for 1 hour at room temperature or overnight at 4°C .

• Detection system: Use appropriate detection systems based on the primary antibody host and experimental design.

• Counterstaining: Counterstain with hematoxylin for brightfield applications or DAPI for fluorescence.

• Scoring system: Implement a standardized scoring system based on staining intensity and percentage of positive cells when quantifying LRRC3B expression.

This protocol has been successfully used in studies examining LRRC3B expression in relation to cancer progression and immune cell infiltration .

How can LRRC3B expression analysis be integrated with methylation studies in cancer research?

Integrating LRRC3B expression analysis with methylation studies requires a multi-omics approach:

This integrated approach provides deeper insights into the regulatory mechanisms of LRRC3B in cancer progression and can identify potential biomarkers for immunotherapy response prediction .

What strategies can address the challenge of detecting low LRRC3B expression in cancer tissues?

Detecting low LRRC3B expression presents significant challenges, particularly in cancer tissues where tumor suppressor expression may be downregulated. Researchers can employ several strategies to overcome this limitation:

• Signal amplification techniques:

  • Use tyramide signal amplification (TSA) systems, which can increase sensitivity by 10-100 fold

  • Consider polymer-based detection systems for enhanced sensitivity in IHC applications

  • Employ cooled CCD cameras with extended exposure times for immunofluorescence imaging

• Antibody optimization:

  • Test multiple antibody clones that target different epitopes of LRRC3B

  • Optimize concentration through titration experiments (typically 1:50-1:500 range)

  • Consider using antibody cocktails targeting multiple epitopes simultaneously

• Sample preparation enhancements:

  • Optimize antigen retrieval conditions specific to LRRC3B epitopes

  • Use freshly collected samples when possible to minimize protein degradation

  • Consider thicker tissue sections (5-7 μm) for IHC applications

• Alternative detection approaches:

  • Complement protein detection with mRNA analysis (ISH or qRT-PCR)

  • Consider digital pathology tools for quantitative analysis of weak signals

These approaches have been successfully applied in studies investigating LRRC3B's role in bladder cancer and other malignancies .

How can LRRC3B antibodies be utilized to investigate tumor immune microenvironment interactions?

LRRC3B antibodies offer powerful tools for investigating tumor-immune interactions:

• Multiplex immunophenotyping: Combine LRRC3B staining with immune cell markers (CD4, CD8, CD68, etc.) to assess correlations between LRRC3B expression and immune cell infiltration patterns. This approach has revealed associations between LRRC3B expression and immunosuppressive cell populations, including MDSCs, CAFs, M2-TAMs, M1-TAMs, and Treg cells .

• Spatial analysis: Employ digital pathology and spatial transcriptomics to analyze the geographic relationship between LRRC3B-expressing cells and immune cell populations within the tumor microenvironment.

• Functional assays: Use LRRC3B antibodies in cell sorting applications to isolate LRRC3B-expressing populations for functional studies, including co-culture experiments with immune cells.

• Longitudinal assessment: Apply LRRC3B immunostaining in sequential biopsies during immunotherapy to track changes in expression and correlate with treatment response.

• 3D cell culture models: Implement LRRC3B antibody staining in tumor spheroid or organoid models to assess spatial relationships in three-dimensional contexts.

These approaches provide insights into how LRRC3B influences the anti-tumor immune response and may help identify patients likely to benefit from immunotherapy .

What are common pitfalls in LRRC3B antibody-based experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with LRRC3B antibodies:

• Non-specific binding:

  • Problem: Background staining obscuring specific LRRC3B signals

  • Solution: Optimize blocking conditions using 5-10% normal serum matching the host of the secondary antibody; consider adding 0.1-0.3% Triton X-100 for membrane permeabilization; use protein-free blocking buffers for phospho-specific detection

• Inconsistent results across experiments:

  • Problem: Variable staining intensity between experiments

  • Solution: Standardize fixation times, antibody concentrations, and incubation conditions; include positive control samples in each experiment; prepare larger volumes of working antibody dilutions to use across multiple experiments

• Cross-reactivity with other LRR-containing proteins:

  • Problem: False positive signals from related proteins

  • Solution: Validate antibody specificity using knockout/knockdown controls; compare results with multiple antibodies targeting different epitopes; perform peptide competition assays

• Epitope masking:

  • Problem: Inability to detect LRRC3B due to protein interactions or conformational changes

  • Solution: Test multiple antigen retrieval methods; try different fixation protocols; consider native versus denatured conditions depending on the application

• Low signal intensity:

  • Problem: Weak or undetectable LRRC3B staining

  • Solution: Implement signal amplification systems; increase antibody concentration; extend incubation times; optimize detection systems based on expression levels

Maintaining detailed laboratory records of optimization experiments can help systematically address these challenges .

How should researchers quantify and interpret LRRC3B expression in relation to tumor progression?

Quantification and interpretation of LRRC3B expression requires rigorous methodology:

These approaches enable robust analysis of LRRC3B's role in tumor progression and its potential as a biomarker for prognosis and treatment response .

How can LRRC3B antibodies contribute to immunotherapy response prediction?

LRRC3B antibodies are becoming valuable tools for immunotherapy response prediction:

• Predictive biomarker development:

  • LRRC3B expression and promoter methylation status have demonstrated potential as predictive biomarkers for anti-PD-1 therapy response in non-small cell lung cancer (NSCLC) and breast cancer (BRCA)

  • Immunohistochemical assessment of LRRC3B in pre-treatment biopsies can be integrated into multiparametric prediction models

  • Combined analysis of LRRC3B protein expression and promoter methylation provides superior predictive power compared to either parameter alone

• Implementation methodology:

  • Standardize LRRC3B antibody-based assays for clinical application

  • Develop companion diagnostic approaches using validated antibody clones

  • Integrate with multiplex IHC panels including PD-L1, CD8, and other immune markers

  • Correlate with established biomarkers such as tumor mutational burden and microsatellite instability

• Translational applications:

  • Use LRRC3B antibodies to monitor dynamic changes in expression during treatment

  • Develop threshold values for clinical decision-making

  • Create integrated scoring systems combining LRRC3B with other immune parameters

This emerging application of LRRC3B antibodies represents a promising approach to enhancing precision medicine in cancer immunotherapy .

What role might LRRC3B play in the development of new cancer therapeutic approaches?

Understanding LRRC3B's functions through antibody-based research is opening new therapeutic avenues:

• Therapeutic target identification:

  • LRRC3B has demonstrated tumor suppressor functions by inhibiting cancer cell proliferation and invasion, particularly in bladder cancer

  • Its role in β-catenin pathway regulation suggests potential for targeted intervention in Wnt signaling-dependent cancers

  • The association between LRRC3B expression and immune cell function points to opportunities for combination approaches with existing immunotherapies

• Epigenetic therapy approaches:

  • LRRC3B promoter hypermethylation serves as a potential target for demethylating agents

  • Combining demethylating drugs with immunotherapy could synergistically enhance anti-tumor responses by restoring LRRC3B expression

  • Monitoring treatment efficacy through LRRC3B antibody-based assays provides a mechanistic biomarker

• Novel therapeutic modalities:

  • Development of proteolysis-targeting chimeras (PROTACs) or molecular glues to modulate LRRC3B activity

  • Engineered immune cells designed to recognize tumor environments with altered LRRC3B expression

  • Nanoparticle-based delivery systems targeting cells with specific LRRC3B expression patterns

These approaches represent promising directions for translating LRRC3B research into clinical applications, with antibody-based detection serving as critical tools for development and validation .