NTRK3 Antibody

What is NTRK3 and what role does it play in cellular function?

NTRK3 (neurotrophic receptor tyrosine kinase 3) encodes a membrane-bound receptor protein also known as TrkC. It is a 94.4 kilodalton protein that functions as a receptor for neurotrophin-3 (NT-3) . Upon ligand binding, NTRK3 autophosphorylates and activates members of the MAPK pathway, regulating cellular growth and differentiation . The receptor consists of three distinct domains: an extracellular domain that binds NT-3, a transmembrane domain, and a cytoplasmic domain with tyrosine kinase activity . NTRK3 plays critical roles in neuronal development but has also been implicated in various cancer types, exhibiting either oncogenic or tumor suppressive functions depending on the tissue context.

What applications are NTRK3 antibodies commonly used for in research?

NTRK3 antibodies are utilized in multiple experimental applications, with the most common being:

• Western blotting (WB) for protein expression analysis

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

• Immunohistochemistry (IHC) for tissue localization

• Immunoprecipitation (IP) for protein complex studies

• Immunofluorescence labeling for subcellular localization

Most commercially available antibodies are validated for at least two of these applications, with WB and ELISA being the most consistently validated across suppliers .

What are the different types of NTRK3 antibodies available and how should they be selected?

NTRK3 antibodies are available in several formats:

Selection should be based on the specific experimental context, keeping in mind that antibodies targeting the C-terminus are preferred for detecting fusion proteins, as this region is typically retained in NTRK gene fusions .

How can NTRK3 antibodies be optimized for immunohistochemical detection of fusion proteins?

Optimizing NTRK3 antibody detection for fusion proteins requires careful consideration of several factors:

• Antibody selection: Use antibodies targeting the C-terminal portion of NTRK3, as this region is retained in fusion proteins . The clone EPR17341 has been validated for detecting NTRK fusions .

• Staining protocol optimization:

  • Establish proper antigen retrieval conditions (heat-induced epitope retrieval is often preferred)

  • Optimize antibody concentration through titration experiments

  • Determine optimal incubation time and temperature

  • Include proper positive and negative controls

• Scoring criteria: Define positive results as staining above background in at least 1% of tumor cells in any pattern (membranous, cytoplasmic, perinuclear, or nuclear) .

• Interpretation considerations:

  • NTRK3 fusion detection by IHC has lower sensitivity (approximately 79.4%) compared to NTRK1 and NTRK2 fusions (96.2-100%)

  • Be aware that tumors with neural or smooth muscle differentiation may show false-positive staining

  • Consider histologic tumor type during interpretation

For tumors highly suspected of harboring ETV6-NTRK3 fusions, confirmation with FISH targeting NTRK3 is recommended due to the lower sensitivity of IHC for NTRK3 fusions .

What are the comparative advantages and limitations of different methods for detecting NTRK3 gene fusions?

Several techniques can detect NTRK3 fusions, each with distinct advantages and limitations:

For research requiring high sensitivity for NTRK3 fusions, NGS or a combination approach (IHC screening followed by confirmatory FISH or NGS) is recommended .

How does NTRK3 function as a conditional tumor suppressor and what implications does this have for research?

NTRK3 demonstrates context-dependent roles in cancer, functioning as:

• Oncogene: In breast cancer and hepatocellular carcinoma through fusion events

• Tumor suppressor: In colorectal cancer through various mechanisms:

  • NTRK3 is frequently methylated in colorectal adenomas and cancers but not in normal colon

  • Induced NTRK3 expression (without its ligand NT-3) induces apoptosis by increasing caspase activity 2-3 fold compared to controls

  • NTRK3 expression suppresses anchorage-independent colony formation and in vivo tumor growth

  • Addition of NT-3 (100 ng/mL) suppresses NTRK3-induced apoptosis, demonstrating dependence receptor behavior

  • Somatic mutations of NTRK3 observed in colorectal cancers can inactivate its tumor suppressor functions

Research implications:

• Tissue context must be considered when studying NTRK3 function

• Both genetic (mutation) and epigenetic (methylation) mechanisms should be evaluated

• NT-3 presence significantly alters NTRK3 signaling outcomes

• Experimental designs should account for NTRK3's dual nature as both a potential oncogene (via fusion events) and tumor suppressor

This conditional behavior explains seemingly contradictory research findings and highlights the importance of comprehensive analysis of both NTRK3 expression and its signaling context.

What is the relationship between NTRK3 expression, tumor mutation burden, and immune infiltration?

NTRK3 has been identified as a potential prognostic biomarker in bladder cancer (BLCA) with significant connections to tumor mutation burden (TMB) and immune infiltration:

• Expression patterns:

  • NTRK3 expression is typically higher in low TMB tumors compared to high TMB tumors

  • High NTRK3 expression correlates with higher immune and stromal scores calculated by the ESTIMATE algorithm

• Immune infiltration correlations:

  • NTRK3 expression positively correlates with infiltrating levels of multiple immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells)

  • Low TMB samples (which tend to have higher NTRK3 expression) show higher fractions of memory resting CD4+ T cells and resting mast cells

  • High TMB samples have higher fractions of CD8+ T cells, memory activated CD4+ T cells, and T follicular helper cells

• Immunomodulatory interactions:

  • NTRK3 shows significant positive correlation with:

    • Chemokine CCL14

    • Immunoinhibitor ADORA2A

    • Immunostimulator CXCL12

These findings suggest NTRK3 may serve as a TMB-related biomarker that influences immune infiltration patterns and potentially affects immunotherapy response, making it an important target for immunotherapeutic research.

How can NTRK3 mutations be functionally validated in cancer research models?

Functional validation of NTRK3 mutations requires multiple approaches:

• Expression studies:

  • Clone wild-type and mutant NTRK3 into expression vectors

  • Transfect into appropriate cell lines lacking endogenous NTRK3 expression (e.g., HCT116, RKO, HT29 for colorectal cancer studies)

  • Compare phenotypic effects on proliferation, apoptosis, colony formation, and migration

• Apoptosis assessment:

  • Measure caspase activity using luminescent/fluorescent caspase substrates

  • Confirm with complementary apoptosis assays (e.g., DNA:histone complex detection)

  • Test with and without NT-3 ligand (100 ng/mL) to evaluate dependence receptor function

• In vivo tumor formation:

  • Implant cells expressing wild-type or mutant NTRK3 in animal models

  • Monitor tumor growth rates, invasion, and metastasis

  • Analyze tumor microenvironment changes, particularly immune cell infiltration

• Signaling pathway analysis:

  • Assess phosphorylation status of MAPK and PI3K/AKT pathway components

  • Evaluate interaction with other signaling pathways relevant to the cancer type

These methodologies can determine whether specific NTRK3 mutations represent loss-of-function alterations that compromise tumor suppressor activity or gain-of-function changes that promote oncogenic signaling.

How can researchers troubleshoot weak or inconsistent NTRK3 antibody staining?

When encountering weak or inconsistent NTRK3 staining, consider these systematic troubleshooting approaches:

• Sample preparation issues:

  • Fixation time: Overfixation can mask epitopes; standardize fixation time (24-48 hours recommended)

  • Tissue processing: Improper processing can denature proteins; ensure consistent protocols

  • Storage conditions: Long-term slide storage may reduce antigenicity; use freshly cut sections

• Antigen retrieval optimization:

  • Test multiple methods: Compare heat-induced (citrate buffer pH 6.0 vs. EDTA pH 9.0) and enzymatic retrieval

  • Adjust retrieval time: Extend heat treatment for challenging samples

  • Retrieval temperature: Ensure consistent temperature throughout the treatment

• Antibody-specific factors:

  • Concentration: Perform titration experiments (typically 1:50 to 1:500 dilutions)

  • Incubation conditions: Test extended incubation times (overnight at 4°C vs. 1-2 hours at room temperature)

  • Detection systems: Switch to more sensitive detection systems (polymer-based vs. avidin-biotin)

  • Clone selection: NTRK3 fusion detection has lower sensitivity (~79.4%); consider alternative clones or approaches

• NTRK3 expression patterns:

  • Expression can be focal in NTRK3 fusion-positive tumors

  • Consider positive if ≥1% of tumor cells show staining above background

  • Evaluate multiple patterns (membranous, cytoplasmic, perinuclear, nuclear)

• Controls:

  • Include known positive controls (tissues with confirmed NTRK3 expression)

  • Use cell line controls with different NTRK3 expression levels

  • Consider tissue-specific controls, as expression patterns vary by tissue type

For persistent issues with NTRK3 fusion detection, consider complementary methods like FISH or NGS, particularly for samples suspected of harboring NTRK3 fusions .

What considerations should be made when selecting NTRK3 antibodies for specific experimental purposes?

Selecting the optimal NTRK3 antibody requires careful consideration of several factors:

• Experimental application:

  • Western blot: Select antibodies validated for denatured proteins, typically targeting linear epitopes

  • IHC/IF: Choose antibodies compatible with fixed tissues that recognize accessible epitopes

  • IP: Select antibodies with high affinity for native protein conformation

  • ELISA: Consider pre-validated ELISA-specific antibodies or matched antibody pairs

• Target recognition:

  • Isoform specificity: NTRK3 has multiple isoforms; select antibodies recognizing relevant isoforms (e.g., TrkCT1 is a non-catalytic isoform)

  • Domain targeting: For fusion detection, use C-terminal targeting antibodies (e.g., clone EPR17341)

  • Species reactivity: Confirm cross-reactivity with target species (human, mouse, rat)

• Technical specifications:

  • Clonality: Monoclonal for specific epitope recognition; polyclonal for broader epitope detection

  • Host species: Consider secondary antibody compatibility and potential cross-reactivity

  • Conjugation: Direct conjugates (fluorophores, enzymes) simplify protocols but may have lower sensitivity

  • Format: Purified IgG, ascites fluid, or culture supernatant (purified preferred for quantitative applications)

• Validation data:

  • Review literature citations demonstrating successful antibody use in similar applications

  • Examine supplier validation data (western blots, IHC images)

  • Consider antibody validation score or certification status

• Application-specific considerations:

  • For NTRK fusion detection: Pan-TRK antibodies targeting conserved C-terminus show better sensitivity for NTRK1/2 (96.2-100%) than NTRK3 (79.4%)

  • For mechanistic studies: Antibodies distinguishing between phosphorylated and non-phosphorylated forms

A systematic evaluation of these factors will help identify the most appropriate NTRK3 antibody for specific research objectives.

How is NTRK3 being investigated as a potential biomarker and therapeutic target?

NTRK3 is emerging as a significant biomarker and therapeutic target across multiple cancer types:

• Prognostic biomarker applications:

  • In bladder cancer, NTRK3 has been identified as a TMB-related prognostic biomarker with the highest hazard ratio among 36 differentially expressed genes

  • NTRK3 expression correlates with immune infiltration patterns, suggesting potential immunotherapy response prediction applications

  • Methylation status of NTRK3 in colorectal cancers may serve as an early detection biomarker

• Therapeutic targeting approaches:

  • NTRK gene fusion detection informs eligibility for TRK inhibitor therapies

  • Context-dependent function requires tailored therapeutic strategies:

    • In fusion-positive cancers: TRK inhibition

    • In cancers where NTRK3 acts as a tumor suppressor: Approaches to restore expression or function

• Combination therapy strategies:

  • NTRK3's positive correlation with immunomodulators (CCL14, ADORA2A, CXCL12) suggests potential synergy with immunotherapies

  • The dual role of NTRK3 as dependence receptor indicates NT-3/NTRK3 axis modulation could enhance traditional therapies

• Detection method development:

  • Improved NTRK3 fusion detection methods combining the accessibility of IHC with the specificity of molecular techniques

  • Development of companion diagnostics for NTRK-targeted therapies

Future research directions include developing more sensitive detection methods for NTRK3 fusions, elucidating tissue-specific roles of NTRK3 in cancer progression, and expanding therapeutic strategies to target both fusion and non-fusion NTRK3 alterations.