FLRT3 Antibody

Basic Research Questions

• What is FLRT3 and what are its primary biological functions?

FLRT3 is a leucine-rich transmembrane protein that functions in cell-cell adhesion, cell migration, and axon guidance, exerting either attractive or repulsive roles depending on its interaction partners. It plays a crucial role in embryonic development, particularly in ventral closure, headfold fusion, and definitive endoderm migration . In the nervous system, FLRT3 contributes to spatial organization of brain neurons and promotes neurite outgrowth .

Methodologically, FLRT3's functions are typically studied through knockout/knockdown experiments, protein-protein interaction assays, and functional cellular assays. The protein's multifunctionality stems from its ability to interact with different partners including ADGRL3 (latrophilin), ROBO1, and UNC5B receptors, each mediating distinct biological processes .

• What types of FLRT3 antibodies are available for research applications?

Several types of FLRT3 antibodies are currently available for research:

When selecting an antibody, researchers should consider the specific epitope recognition, validated applications, and target species to ensure experimental success. Validation data from manufacturers should be carefully reviewed to ensure specificity and sensitivity for the intended application.

• How can researchers validate FLRT3 antibody specificity in experimental systems?

Methodological approach to FLRT3 antibody validation:

• Multiple validation techniques should be employed:

  • siRNA knockdown experiments to confirm signal reduction following FLRT3 downregulation

  • Tagged GFP-FLRT3 cell lines to assess co-localization with antibody staining

  • Independent antibodies targeting different epitopes to confirm consistency in staining patterns

• Control inclusion for meaningful interpretation:

  • Positive controls: tissues with known FLRT3 expression (e.g., renal cell carcinoma)

  • Negative controls: tissues with minimal expression or primary antibody omission

  • Isotype controls to assess non-specific binding

• Western blot analysis to confirm band specificity at expected molecular weight

• Cross-reactivity assessment against other FLRT family members (FLRT1, FLRT2) using ELISA or other binding assays

The Human Protein Atlas employs standardized validation protocols and assigns reliability scores such as "Enhanced," "Supported," "Approved," or "Uncertain" based on comparison with external evidence for protein localization .

Intermediate Research Questions

• What are the optimal experimental conditions for using FLRT3 antibodies in immunohistochemistry?

For successful FLRT3 immunohistochemistry, researchers should consider the following methodological approach:

• Sample preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues have been successfully used for FLRT3 detection in cancer samples

  • Antigen retrieval protocols should be optimized (typically heat-induced epitope retrieval)

  • Section thickness of 4-5μm is generally recommended

• Staining protocol optimization:

  • Antibody dilution should be empirically determined for each antibody lot

  • Extended incubation times (overnight at 4°C) may improve signal-to-noise ratio

  • Blocking with 3-5% normal serum matching the secondary antibody host species

• Signal detection considerations:

  • DAB (3,3'-diaminobenzidine) provides good contrast for membranous FLRT3 staining

  • Fluorescent detection allows for multiplex staining with immune cell markers

  • Appropriate counterstains to visualize cellular architecture

• Interpretation guidelines:

  • Assess membrane FLRT3 expression with particular attention to cell borders

  • Score intensity on standardized scale (0, 1+, 2+, 3+)

  • Evaluate percentage of positive cells in representative fields

Recent studies have shown papillary renal cell cancer and clear cell RCC exhibit highest membrane FLRT3 expression and can serve as positive controls .

• How can FLRT3 antibodies be used to investigate FLRT3's role in T cell inhibition?

To investigate FLRT3's role in T cell inhibition, researchers can employ several methodological approaches:

• T cell functional assays:

  • Proliferation assays: Compare T cell proliferation (using CFSE dilution) when co-cultured with FLRT3-expressing cells versus controls

  • Cytokine production: Measure IFN-γ and TNF-α production by ELISA or intracellular cytokine staining

  • Use FLRT3 Fc fusion proteins at titrated concentrations with plate-coated OKT3 to assess direct effects on T cells

• Receptor analysis:

  • Flow cytometry to analyze UNC5B expression on activated T cells

  • Assess co-expression of UNC5B with other checkpoint receptors (LAG-3, TIM-3, PD-1)

  • Use FLRT3 binding assays to confirm functional receptor expression

• Blocking studies:

  • Apply FLRT3-blocking antibodies (e.g., NP591) that specifically disrupt FLRT3-UNC5B interactions

  • Compare with control antibodies that block alternate interactions (e.g., NP592 for FLRT3-LPHN3)

  • Measure restoration of T cell function following antibody treatment

• Mixed lymphocyte reaction (MLR) assays:

  • Activate PBMCs in the presence of FLRT3-expressing cancer cell lines (e.g., SKOV3, A549)

  • Add FLRT3-blocking antibodies and measure enhanced cytokine production

Recent studies demonstrated that FLRT3 inhibits T cell proliferation and suppresses IFN-γ production similar to PD-L1 across multiple donors, and that blocking the FLRT3-UNC5B interaction can reverse these effects .

• What technical considerations should be addressed when using FLRT3 antibodies in flow cytometry?

When using FLRT3 antibodies for flow cytometry, researchers should address these technical considerations:

• Sample preparation optimization:

  • Cell fixation method affects epitope accessibility (fresh versus fixed cells)

  • Permeabilization requirements depend on whether detecting surface or intracellular FLRT3

  • Single-cell suspensions from tissues require gentle dissociation to preserve membrane proteins

• Antibody selection and titration:

  • Verify flow cytometry validation for specific antibody clone

  • Perform titration experiments to determine optimal concentration

  • Consider directly conjugated antibodies to reduce background and simplify protocols

• Controls for accurate interpretation:

  • Fluorescence-minus-one (FMO) controls to set gating boundaries

  • Isotype controls matched to primary antibody

  • FLRT3-positive and negative cell lines as biological controls

• Multi-parameter considerations:

  • Select compatible fluorochromes when analyzing FLRT3 alongside other markers

  • Compensation controls to correct spectral overlap

  • Sequential gating strategy to identify specific cell populations expressing FLRT3

• Data analysis approach:

  • Report both percentage positive cells and mean/median fluorescence intensity

  • Consider density plots rather than histograms for heterogeneous populations

  • Compare FLRT3 expression levels on different cell subsets within the same sample

Flow cytometry has been successfully used to analyze FLRT3 receptor expression (UNC5B) on activated T cells, revealing upregulation following stimulation with anti-CD3/CD28 or OKT3 .

Advanced Research Questions

• How can researchers investigate the FLRT3-UNC5B interaction in cancer immunotherapy models?

For investigating FLRT3-UNC5B interactions in cancer immunotherapy contexts, researchers should employ multiple sophisticated approaches:

• Protein-protein interaction characterization:

  • ELISA-based binding assays using FLRT3-Fc and UNC5B-Fc fusion proteins

  • Surface plasmon resonance to measure binding kinetics and affinity

  • Co-immunoprecipitation to confirm interaction in cell lysates

  • Antibody epitope mapping to identify critical binding regions

• Advanced cellular models:

  • CAR-T cell models with FLRT3-expressing tumor targets (e.g., EpCAM+ OVCAR-5 model)

  • BiTE therapy models (e.g., EGFR+ RD model with EGFR/CD3 BiTEs)

  • Mixed lymphocyte reaction assays with FLRT3-expressing cancer cells

  • 3D tumor spheroid models to assess T cell infiltration and killing

• In vivo humanized models:

  • Establish FLRT3-expressing tumor xenografts in immunodeficient mice

  • Adoptive transfer of human T cells with or without CAR-T/BiTE therapy

  • Administer FLRT3-blocking antibodies (NP591) to disrupt FLRT3-UNC5B interactions

  • Analyze:

    • Tumor growth kinetics

    • T cell infiltration into tumor microenvironment

    • T cell-tumor cell engagement metrics

    • Cytokine production in tumor milieu

• Multiplex analysis of the tumor microenvironment:

  • Immunofluorescence to simultaneously detect FLRT3, UNC5B, and T cell markers

  • Spatial analysis of T cell distribution relative to FLRT3-expressing regions

  • Single-cell RNA sequencing to correlate FLRT3/UNC5B expression with T cell functional states

Recent studies demonstrated that FLRT3 expression on tumors significantly reduced T cell-mediated antitumor immunity, inhibited T cell recruitment/infiltration, and reduced T cell engagement with tumor cells, which could be reversed by FLRT3-UNC5B blocking antibodies .

• What methodological approaches can resolve contradictory FLRT3 expression data across different studies?

To reconcile contradictory FLRT3 expression data, researchers should implement these methodological approaches:

• Standardized antibody validation protocols:

  • Use multiple independently validated antibodies targeting different epitopes

  • Implement enhanced validation techniques (siRNA knockdown, GFP-tagged expression)

  • Document complete antibody information (clone, lot, dilution, incubation conditions)

• Multi-platform expression analysis:

  • Compare protein detection (IHC, WB, flow cytometry) with transcript analysis (qPCR, RNA-seq)

  • Use multiple antibody-independent methods (mass spectrometry, CRISPR screens)

  • Assess membrane versus total cellular expression

• Systematic analysis of biological variables:

  • Tissue/tumor heterogeneity: analyze multiple regions within samples

  • Cancer type stratification: document significant expression differences between cancer types (e.g., highest in renal cell carcinoma)

  • Patient characteristics: account for treatment history, disease stage, and demographic factors

  • Cell line authentication and passage number documentation

• Quantitative scoring and reporting:

  • Implement automated image analysis with standardized thresholds

  • Report both staining intensity and percentage of positive cells

  • Use H-score or other established quantitative metrics

  • Include representative images with all publications

• Meta-analysis approach:

  • Systematically compare methodology across studies

  • Identify patterns in discrepancies related to techniques or sample types

  • Use statistical methods to account for inter-study variability

Researchers should particularly note that membrane FLRT3 expression varies significantly across tumor types, with highest expression observed in papillary and clear cell renal cell carcinomas .

• How can FLRT3 antibodies be utilized to develop and validate novel cancer immunotherapies?

FLRT3 antibodies play crucial roles in developing novel cancer immunotherapies through these methodological approaches:

• Therapeutic antibody development pipeline:

  • Engineering of humanized monoclonal antibodies with optimal binding properties

  • Development of antibodies with IgG4P Fc backbone (reduced Fc receptor interaction)

  • Function-selective antibodies:

    • FLRT3-UNC5B blocking antibodies (e.g., NP591)

    • FLRT3-LPHN3 blocking antibodies (e.g., NP592)

  • Antibody optimization for stability, half-life, and tissue penetration

• Preclinical efficacy assessment:

  • In vitro T cell rescue assays:

    • Measure restoration of T cell proliferation

    • Analyze recovery of cytokine production (IFN-γ, TNF-α)

    • Assess enhanced killing of FLRT3+ tumor cells

  • Ex vivo tumor slice cultures to evaluate antibody penetration and T cell activation

  • Humanized mouse models with patient-derived xenografts

• Combination therapy evaluation:

  • FLRT3 blockade with established checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

  • Integration with adoptive cell therapies (CAR-T, TIL)

  • Combination with bispecific T cell engagers (BiTEs)

  • Analysis of synergistic versus additive effects

• Biomarker development:

  • Correlation of FLRT3 expression with response to immunotherapy

  • UNC5B expression on tumor-infiltrating lymphocytes as predictive marker

  • Multiplex analysis of FLRT3 with other checkpoint molecules

Recent research has demonstrated that FLRT3-blocking antibodies can reverse T cell inhibition and enhance anti-tumor responses in humanized cancer models, supporting the concept that axon guidance proteins can function as T cell checkpoints and represent promising targets for cancer immunotherapy .

• What are the most significant challenges in interpreting FLRT3 antibody staining patterns in complex tissues?

Interpreting FLRT3 antibody staining in complex tissues presents several methodological challenges:

• Distinguishing specific cellular expression patterns:

  • Membrane versus cytoplasmic localization requires high-resolution imaging

  • Differentiation between FLRT3 expression in tumor cells versus stromal components

  • Cell type-specific expression requires co-staining with lineage markers

  • Intracellular trafficking may affect staining patterns at different cellular stages

• Technical artifacts and their mitigation:

  • Edge effects in tissue sections can create false positive membrane staining

  • Necrotic tissue areas often show non-specific antibody binding

  • Endogenous peroxidase activity must be adequately blocked

  • Tissue processing variables (fixation time, processing protocols) affect staining intensity

  • Serial sectioning to confirm consistent staining patterns

• Quantification challenges:

  • Heterogeneous expression requires assessment of multiple representative fields

  • Objective scoring systems must account for both intensity and distribution

  • Digital image analysis parameters require validation against pathologist scoring

  • Defining positive/negative thresholds impacts reported expression rates

• Biological interpretation complexities:

  • Correlation of FLRT3 expression with functional consequences requires additional assays

  • Expression may change during disease progression

  • Treatment effects on expression patterns must be considered

  • Interaction with the tumor microenvironment may regulate expression

• Validation approaches:

  • RNA-scope or in situ hybridization to confirm transcript localization

  • Laser capture microdissection followed by Western blot or mass spectrometry

  • Multiple antibodies targeting different epitopes to confirm patterns

Recent research identified highest membrane FLRT3 expression on papillary renal cell cancer and clear cell RCC, with variable expression across other tumor types, highlighting the importance of tissue-specific validation and interpretation .