MST1R Antibody

What is MST1R and why is it a significant target for antibody-based research?

MST1R (Macrophage Stimulating 1 Receptor), also known as RON (Recepteur d'Origine Nantais), is a transmembrane receptor tyrosine kinase belonging to the MET proto-oncogene family. It plays crucial roles in regulating cell proliferation, motility, adhesion, and apoptosis through multiple signaling cascades. MST1R is highly conserved across species and is expressed predominantly on epithelial cells, macrophages, and recently identified in certain T-cell subsets .

How do I select the appropriate MST1R antibody for my specific research application?

Selection of an appropriate MST1R antibody requires careful consideration of several experimental parameters:

• Target epitope specificity: MST1R antibodies target different regions of the receptor, including the extracellular domain, phosphorylation sites (e.g., pTyr1238, pSer1394), or specific amino acid sequences (e.g., AA 89-275, AA 1092-1316) . Choose based on your research focus:

  • Phospho-specific antibodies (e.g., ABIN710336 targeting pTyr1238) for activation studies

  • Domain-specific antibodies for structural or functional investigations

  • N-terminal vs. C-terminal targeting for different isoform detection

• Validated applications: Verify that the antibody has been validated for your intended application (WB, ELISA, IHC, ICC, IP) .

• Species reactivity: Confirm cross-reactivity with your experimental model (human, mouse, rat, monkey) .

• Clonality:

  • Polyclonal antibodies offer broader epitope recognition but potential batch variability

  • Monoclonal antibodies provide consistent specificity but may have limited epitope detection

• Validation data: Review available validation data, particularly for your specific application and cell/tissue type .

For optimal results in signaling studies, consider antibodies targeting phosphorylation sites like pTyr1238, as these can directly monitor activation status of the receptor in response to its ligand MSP (Macrophage Stimulating Protein) .

What is the structure and processing of MST1R, and how does this impact antibody selection?

MST1R is synthesized as a single transmembrane pro-protein that undergoes proteolytic processing to form its mature receptor structure. Understanding this processing is crucial for antibody selection:

• Pro-protein cleavage: The initial pro-protein is cleaved by proteases in its extracellular portion, resulting in:

  • A 40 kDa alpha (α) chain that is solely extracellular

  • A 150 kDa beta (β) chain containing the transmembrane and intracellular domains

• Mature receptor domains:

  • Extracellular sema domain (involved in ligand binding)

  • Transmembrane domain

  • Intracellular portion containing the kinase domain

• Glycosylation: MST1R is glycosylated, which affects its molecular weight and potentially antibody recognition

This structural complexity necessitates strategic antibody selection:

• Antibodies targeting the α-chain detect only the extracellular component

• Antibodies against the β-chain detect the functional receptor

• Phospho-specific antibodies (such as those targeting pTyr1238) detect activated forms of the receptor

For comprehensive studies, consider using multiple antibodies targeting different domains to distinguish between precursor and processed forms of MST1R.

How can MST1R antibodies be utilized to investigate its role in cancer progression?

MST1R antibodies enable multifaceted investigation of this receptor's role in oncogenesis through several methodological approaches:

• Expression profiling: Using MST1R antibodies for immunohistochemistry (IHC) or Western blotting (WB) to:

  • Quantify expression levels across cancer types and correlate with clinical outcomes

  • Track expression changes during cancer progression

  • Evaluate heterogeneity of expression within tumor samples

• Activation state assessment: Phospho-specific antibodies (e.g., targeting pTyr1238) allow researchers to:

  • Determine the proportion of activated MST1R in tumor samples

  • Monitor changes in activation following treatment interventions

  • Correlate activation patterns with downstream signaling events

• Therapeutic targeting approaches:

  • Neutralizing antibodies can block MST1R signaling in functional studies

  • Antibody-based screening assays help identify potential small molecule inhibitors

  • Dual targeting of MST1R and related receptors (e.g., MET) with blocking antibodies has shown enhanced growth inhibition compared to single receptor targeting

• Mechanism investigation:

  • Co-immunoprecipitation using MST1R antibodies to identify novel interaction partners

  • Characterize MST1R signaling in different cellular contexts using cell-type specific analyses

  • Investigate cross-talk with other oncogenic pathways like STAT3 signaling

Research has demonstrated that MST1R activation contributes to cancer progression through multiple mechanisms, including immune modulation within the tumor microenvironment, activation of oncogenic pathways, and conferring protection under cellular stress conditions . Therefore, using MST1R antibodies to monitor both expression and activation state provides valuable insights into cancer biology and potential therapeutic strategies.

What methodological approaches can be used to study MST1R mutations and gene copy number alterations?

Investigation of MST1R genetic alterations requires sophisticated methodological approaches that can be complemented with antibody-based validation:

• Gene copy number analysis:

  • Quantitative PCR (qPCR) serves as an initial screening method to detect MST1R gene amplification

  • Fluorescence in situ hybridization (FISH) provides cellular-level confirmation of gene copy number changes

  • Array Comparative Genomic Hybridization (aCGH) offers genome-wide context for MST1R alterations

• Mutation detection and characterization:

  • Targeted sequencing of MST1R exons to identify mutations (e.g., R1018G juxtamembrane mutation found in 11% of gastric cancer samples)

  • Whole exome/genome sequencing for comprehensive mutation profiling

  • Functional validation of mutations using site-directed mutagenesis and expression systems

• Correlation with protein expression:

  • Western blotting with MST1R antibodies to determine if genetic alterations translate to protein level changes

  • Immunohistochemistry to visualize spatial distribution of MST1R in tissue samples with known genetic alterations

  • Phospho-specific antibodies to assess if mutations affect activation status

• Functional consequences assessment:

  • Cell viability assays following MST1R stimulation with MSP in cell lines harboring genetic alterations

  • In vitro kinase assays to measure enzymatic activity of mutant MST1R proteins

  • Co-stimulation assays with related receptors (e.g., MET) to evaluate synergistic effects

Research has shown that high MST1R gene copy number correlates with poor survival (p = 0.01) and is associated with high MET and ERBB2 gene copy number in gastric and esophageal cancers. Additionally, novel mutations like R1018G in the juxtamembrane domain have been identified in about 11% of samples, underscoring the importance of genetic screening alongside protein-level analyses .

How can MST1R antibodies be used to investigate the cross-talk between MST1R and other receptor tyrosine kinases?

Investigating receptor cross-talk requires sophisticated methodological approaches where MST1R antibodies play a central role:

• Co-immunoprecipitation studies:

  • Use MST1R antibodies to pull down receptor complexes and probe for associated RTKs (particularly MET)

  • Reverse co-IP with antibodies against other RTKs to confirm interactions

  • Analysis under different stimulation conditions (MSP alone, dual ligands, inhibitors)

• Phosphorylation dynamics:

  • Employ phospho-specific antibodies (e.g., pTyr1238) to track MST1R activation patterns when other RTKs are activated or inhibited

  • Time-course experiments to determine sequential activation patterns

  • Multiplex Western blotting to simultaneously detect phosphorylation of multiple receptors and downstream effectors

• Functional assays with selective inhibition/activation:

  • Cell migration, invasion, and survival assays following:

    • MSP stimulation alone

    • Dual stimulation (e.g., MSP + HGF for MST1R + MET)

    • Selective inhibition of one pathway while activating another

  • Quantify differences between single and dual receptor activation to assess synergistic effects

• Downstream signaling convergence:

  • Track activation of shared effectors (e.g., STAT3) using phospho-specific antibodies

  • Compare signaling profiles when receptors are activated individually versus simultaneously

  • Identify unique versus redundant signaling outcomes

Research has demonstrated that co-stimulation of MST1R and MET leads to enhanced malignant phenotypes compared to stimulation of either receptor alone. This suggests functional synergy between these related RTKs, potentially explaining why dual targeting approaches might be more effective than single-receptor inhibition strategies . Understanding these interactions is critical for developing effective therapeutic strategies that address potential resistance mechanisms arising from compensatory signaling.

What are the optimal conditions for using MST1R antibodies in Western blotting applications?

Achieving optimal results with MST1R antibodies in Western blotting requires attention to several methodological details:

• Sample preparation:

  • Cell lysis buffer: Use RIPA or NP-40 based buffers containing phosphatase inhibitors (crucial for phospho-specific antibodies like pTyr1238)

  • Protein loading: 20-50 μg total protein per lane, depending on expression level

  • Denaturation: Heat samples at 95°C for 5 minutes in reducing buffer containing SDS and β-mercaptoethanol

• Gel electrophoresis and transfer parameters:

  • Use 7-8% gels to optimally resolve the 150 kDa β-chain of MST1R

  • Consider gradient gels (4-15%) when attempting to visualize both α (40 kDa) and β chains

  • Transfer to PVDF membranes (rather than nitrocellulose) for improved protein retention

  • Employ wet transfer at 30V overnight at 4°C for complete transfer of high molecular weight proteins

• Antibody incubation conditions:

  • Primary antibody dilution: 1:500 to 1:2000, optimized for each specific antibody

  • Incubation: Overnight at 4°C for optimal sensitivity

  • Blocking: 5% BSA in TBST (preferred over milk for phospho-specific antibodies)

  • Secondary antibody: Anti-rabbit HRP for most MST1R antibodies (e.g., ABIN710336)

• Signal detection considerations:

  • Enhanced chemiluminescence (ECL) detection systems

  • Exposure time optimization based on expression levels

  • Consider using lower-sensitivity secondary antibodies for highly expressed targets to avoid saturation

• Controls and validation:

  • Positive control: Cell lines with known MST1R expression (e.g., gastric or pancreatic cancer cell lines)

  • Negative control: MST1R-negative cell lines or siRNA-treated samples

  • Phospho-controls: Include samples from MSP-stimulated cells (shows increased signal with phospho-specific antibodies) and phosphatase-treated lysates (shows decreased signal)

For optimal results with phospho-specific MST1R antibodies (e.g., pTyr1238), stimulate cells with MSP (100 ng/ml for 10-30 minutes) prior to lysis to maximize phosphorylation signal detection .

How should researchers design experiments to study MST1R activation and signaling using antibody-based approaches?

Designing rigorous experiments to study MST1R activation and downstream signaling requires careful planning and appropriate controls:

• Stimulation protocol optimization:

  • Ligand selection: Recombinant MSP (Macrophage Stimulating Protein) at 50-200 ng/ml

  • Time course: Short intervals (5, 15, 30, 60 minutes) to capture rapid phosphorylation events

  • Serum starvation: 12-24 hours prior to stimulation to reduce baseline activation

  • Dose-response experiments to determine minimal effective concentration

• Phosphorylation detection strategy:

  • Western blotting with phospho-specific antibodies (e.g., pTyr1238, pSer1394)

  • Strip and reprobe membranes with total MST1R antibodies to normalize phospho-signal

  • Include phosphatase inhibitors (sodium orthovanadate, NaF) in all buffers

  • For quantification, calculate phospho-MST1R/total MST1R ratio

• Downstream signaling assessment:

  • Monitor key pathways activated by MST1R:

    • STAT3 phosphorylation

    • RAS/MAPK pathway activation

    • PI3K/AKT signaling

    • β-catenin nuclear translocation

  • Time-staggered analysis to map signaling cascade kinetics

  • Comparison between cell types to identify context-dependent signaling differences

• Inhibitor studies:

  • Use HSP90 inhibitors (e.g., EC154) to disrupt MST1R stability

  • Apply selective kinase inhibitors to distinguish between direct and indirect effects

  • Employ blocking antibodies to prevent ligand-receptor interaction

  • Genetic approaches (siRNA, CRISPR) to validate antibody-based findings

• Functional readouts:

  • Cell migration assays in Boyden chambers

  • Cell survival under stress conditions

  • Apoptosis resistance measurement

  • Correlate signaling intensity with functional outcomes

Research has demonstrated that MST1R activation leads to STAT3 phosphorylation and increased cell viability. Importantly, co-activation with related receptor MET enhances these phenotypes beyond what is observed with single receptor stimulation, suggesting important cross-talk mechanisms that should be included in experimental designs .

What are the critical considerations when using MST1R antibodies for immunohistochemistry or immunocytochemistry?

Successful application of MST1R antibodies in immunohistochemistry (IHC) or immunocytochemistry (ICC) requires optimized protocols that account for the specific characteristics of MST1R:

• Sample preparation considerations:

  • Fixation: 10% neutral buffered formalin for 24-48 hours for tissue samples

  • For phospho-specific antibodies (e.g., pTyr1238), immediate fixation is crucial to preserve phosphorylation status

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), optimized for each specific antibody

  • Section thickness: 4-5 μm for optimal antibody penetration and signal detection

• Antibody optimization:

  • Titration: Test multiple dilutions (1:50 to 1:500) to determine optimal concentration

  • Incubation conditions: Overnight at 4°C typically yields best results for MST1R antibodies

  • Detection systems: Consider amplification systems (e.g., tyramide signal amplification) for low-abundance targets

  • Background reduction: Use appropriate blocking reagents (e.g., 5% normal serum from secondary antibody species)

• Controls and validation:

  • Positive tissue controls: Use tissues with known MST1R expression (e.g., normal colon, prostate; cancer samples from colon, prostate, stomach)

  • Negative controls: Omit primary antibody or use isotype control

  • Peptide blocking: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Correlation with other methods: Compare IHC results with Western blot or RNA expression data

• Scoring and interpretation approaches:

  • Membranous versus cytoplasmic staining: MST1R can localize to both compartments

  • Semi-quantitative scoring: Consider intensity (0-3+) and percentage of positive cells

  • Digital image analysis: For more objective quantification when available

  • Subcellular localization assessment: May provide insights into activation status or isoform expression

• Special considerations for phospho-specific antibodies:

  • Use phosphatase inhibitors in all buffers

  • Compare serial sections stained with phospho-specific versus total MST1R antibodies

  • Include samples with known activation status (e.g., MSP-stimulated cells for ICC)

Research indicates that MST1R expression patterns in tissues correlate with clinical outcomes, particularly in gastric, esophageal, and pancreatic cancers, making accurate and consistent IHC methodology critical for translational studies .

How can researchers address inconsistent or unexpected results when using MST1R antibodies?

When encountering inconsistent or unexpected results with MST1R antibodies, systematic troubleshooting is essential:

• Antibody validation and selection issues:

  • Confirm antibody specificity: Test on positive and negative control samples

  • Epitope mapping: Ensure the antibody recognizes the appropriate region for your application

  • Check for batch-to-batch variation: Compare lot numbers and request validation data from supplier

  • Consider testing multiple antibodies targeting different epitopes

• Technical considerations by application:

  • Western blotting troubleshooting:

    • No signal: Check protein loading, transfer efficiency, antibody dilution

    • Multiple bands: Evaluate for isoforms, degradation products, or non-specific binding

    • Unexpected molecular weight: Consider post-translational modifications, proteolytic processing

    • High background: Optimize blocking, washing, and antibody concentration

  • IHC/ICC troubleshooting:

    • Weak staining: Optimize antigen retrieval, antibody concentration, incubation time

    • Non-specific staining: Improve blocking, reduce primary antibody concentration

    • Inconsistent staining: Standardize fixation time, processing conditions

• Biological variability considerations:

  • Expression heterogeneity: MST1R expression varies across:

    • Cell types (epithelial cells, macrophages, T-cell subsets)

    • Cancer subtypes and differentiation status

    • Microenvironmental factors that may regulate expression

  • Alternative splicing and isoforms: Several MST1R splice variants exist:

    • RON30, RON62, and others with altered molecular weights

    • E2E3 variant with modified signaling properties

    • Consider using antibodies that can distinguish between isoforms

• Experimental design refinements:

  • Include time-course analysis for activation studies

  • Use appropriate positive controls (MSP-stimulated samples)

  • Document experimental conditions thoroughly for reproducibility

  • Consider the effects of cell confluence, passage number, and culture conditions

If phospho-specific antibodies yield unexpected results, ensure phosphatase inhibitors are present in all buffers and verify the stimulation protocol is effective by including established downstream targets (e.g., STAT3 phosphorylation) as readouts of MST1R activation .

How should researchers interpret complex patterns of MST1R expression and activation in cancer samples?

Interpreting complex patterns of MST1R expression and activation in cancer samples requires sophisticated analysis approaches:

• Expression pattern heterogeneity:

  • Intratumoral heterogeneity: MST1R expression often varies within a single tumor:

    • Map expression patterns in relation to tumor architecture (e.g., invasive front vs. tumor core)

    • Correlate with markers of differentiation, proliferation, and metastatic potential

    • Consider single-cell approaches for highly heterogeneous samples

  • Subcellular localization interpretation:

    • Membrane localization: Potential for ligand-dependent activation

    • Cytoplasmic accumulation: Possible internalization following activation or altered trafficking

    • Nuclear localization: Reported for certain MST1R variants with distinct functions

• Activation state assessment:

  • Phosphorylation pattern analysis:

    • Compare phospho-MST1R (pTyr1238) with total MST1R distribution

    • Evaluate co-localization with downstream signaling markers (pSTAT3, pERK)

    • Consider multiplex IHC approaches to simultaneously visualize multiple components

  • Correlation with ligand availability:

    • Assess MSP expression in tumor or stromal cells

    • Evaluate potential for autocrine vs. paracrine activation mechanisms

    • Consider alternative activation mechanisms (ligand-independent, cross-activation)

• Genetic-protein correlation analysis:

  • Copy number alterations:

    • High MST1R gene copy number (35.5% of gastroesophageal cancers) correlates with poor survival (p = 0.01)

    • Compare protein expression levels with gene copy number status

    • Evaluate concordance vs. discordance between genetic and protein data

  • Mutational impact assessment:

    • R1018G juxtamembrane mutation (11% of samples) may alter signaling properties

    • Determine if mutations affect antibody recognition, particularly for epitope-specific antibodies

    • Correlate mutational status with activation patterns

• Multiparameter integration:

  • Co-expression analysis:

    • Evaluate MST1R in context of related receptors (MET, ERBB2)

    • Consider pathway redundancy and compensatory mechanisms

    • Develop composite scoring systems incorporating multiple markers

  • Clinical correlation approaches:

    • Correlate expression/activation patterns with patient outcomes

    • Stratify analysis by tumor subtype, stage, and treatment history

    • Consider multivariate models to isolate MST1R-specific effects

Research has demonstrated that MST1R overexpression in cancers correlates with worse clinical outcomes, and that co-expression with related receptors like MET may have synergistic effects on tumor progression. Additionally, specific genetic alterations in MST1R may have functional consequences that are detectable at the protein level with appropriate antibody-based approaches .

How are MST1R antibodies being used to develop novel therapeutic approaches for cancer?

MST1R antibodies are instrumental in the development of novel therapeutic strategies through several innovative approaches:

• Therapeutic antibody development:

  • Blocking antibodies: Design and testing of antibodies that directly inhibit MST1R activation by:

    • Preventing MSP ligand binding to the receptor

    • Inhibiting receptor dimerization

    • Neutralizing circulating MSP ligand

  • Dual-targeting strategies:

    • Combined blocking of MST1R and MET shows enhanced anti-tumor effects over single-receptor targeting

    • Simultaneous targeting overcomes potential compensatory mechanisms

    • Studies show this approach results in increased apoptosis and reduced viability in cancer models

• Antibody-drug conjugates (ADCs):

  • Utilizing MST1R antibodies as targeting vehicles for cytotoxic payloads

  • Selection of antibodies with optimal internalization properties

  • Evaluation of target density requirements for effective ADC delivery

  • Testing various linker and payload combinations for maximum efficacy

• Combination therapy optimization:

  • HSP90 inhibitor combinations:

    • HSP90 inhibitors (e.g., EC154) disrupt MST1R stability and signaling

    • MST1R antibodies can monitor target engagement during treatment

    • Studies show HSP90 inhibition reduces MST1R expression, disrupts signaling, and inhibits cancer cell migration

  • Pathway-specific combinations:

    • Using MST1R antibodies to identify optimal downstream pathway inhibitor combinations

    • Targeting convergent signaling nodes (e.g., STAT3) in combination with direct MST1R inhibition

    • Personalized approaches based on activation status in individual tumors

• Predictive biomarker development:

  • Phospho-specific MST1R antibodies as companion diagnostics

  • Expression level assessment to identify patients most likely to benefit from MST1R-targeted therapies

  • Multi-receptor profiling to guide combination treatment strategies

The development of antibodies specifically targeting MST1R, as described in patent US9403909B2, represents an important advance in this field. These engineered antibodies are being evaluated for their ability to block MST1R signaling and potentially serve as therapeutic agents, underscoring the importance of ongoing research in this area .

What recent technological advances have improved the development and application of MST1R antibodies in research?

Recent technological advances have significantly enhanced the development and application of MST1R antibodies:

• Advanced antibody engineering technologies:

  • In silico design approaches:

    • Computational antibody design based on structural insights

    • Patent US9403909B2 describes antibodies derived from recombinant libraries based on in silico designed amino acid sequences

    • Rational epitope selection for optimal target engagement

  • Display technologies:

    • Phage, yeast, and mammalian display platforms for antibody selection

    • Directed evolution of antibodies with improved affinity and specificity

    • Selection under conditions that mimic physiological environments

• Improved validation methodologies:

  • CRISPR-based validation:

    • Knockout cell lines as definitive negative controls

    • Domain-specific modifications to test epitope specificity

    • Isogenic cell line panels with defined MST1R alterations

  • Multiplexed detection platforms:

    • Simultaneous assessment of multiple RTKs and downstream effectors

    • Single-cell analysis of MST1R expression and activation

    • Spatial transcriptomic correlation with protein expression patterns

• Phospho-specific antibody innovations:

  • Site-specific phosphorylation detection:

    • Antibodies targeting specific phosphorylation sites (e.g., pTyr1238, pSer1394)

    • Enhanced sensitivity through improved production and purification methods

    • Validation using phosphatase treatments and site-directed mutagenesis

  • Temporal dynamics assessment:

    • Tools for tracking phosphorylation kinetics in live cells

    • Antibody-based biosensors for real-time activation monitoring

    • Improved fixation methods to preserve phosphorylation status

• Advanced imaging applications:

  • Super-resolution microscopy:

    • Nanoscale visualization of MST1R clustering and interactions

    • Co-localization studies with improved spatial resolution

    • Quantitative analysis of receptor distribution and trafficking

  • Multiplex immunohistochemistry/immunofluorescence:

    • Simultaneous detection of MST1R and related receptors

    • Combined assessment of expression and activation status

    • Correlation with tumor microenvironment features

• Therapeutic development platforms:

  • High-throughput screening:

    • Antibody-based assays for identifying small molecule inhibitors

    • Functional readouts for large-scale compound testing

    • Phenotypic screening approaches with MST1R activation as readout

  • Improved delivery technologies:

    • Antibody formulation advances for enhanced stability

    • Novel conjugation chemistries for ADC development

    • Tumor-penetrating antibody designs

These technological advances have collectively accelerated the development of more specific, sensitive, and versatile MST1R antibodies, enabling increasingly sophisticated investigations of this receptor's biology and therapeutic targeting .