MCAM Human

What is MCAM/CD146 and what is its basic molecular structure?

MCAM (Melanoma Cell Adhesion Molecule), also known as MUC18 or CD146, is a putative adhesion molecule that belongs to the immunoglobulin superfamily (IgSF) . It is a transmembrane glycoprotein with a molecular weight that varies between 108-140 kDa depending on post-translational modifications and experimental conditions . The protein consists of an extracellular domain spanning from Val24 to Gly559 (as referenced in the recombinant forms), a transmembrane domain, and a cytoplasmic tail . When analyzed by SDS-PAGE under reducing conditions, MCAM/CD146 typically appears at approximately 140 kDa in cell lysates and between 108-124 kDa for recombinant forms .

What binding partners has MCAM/CD146 been shown to interact with?

MCAM/CD146 engages in specific protein-protein interactions that contribute to its biological functions. One well-characterized interaction partner is Galectin-3. In functional ELISA experiments, when Recombinant Human MCAM/CD146 Fc Chimera is immobilized at 1 μg/mL (100 μL/well), it binds Recombinant Human Galectin-3 with an ED50 of 0.25-1.25 μg/mL . This interaction may contribute to MCAM/CD146's roles in cell adhesion and signaling pathways. Beyond direct binding partners, MCAM/CD146 participates in complex cellular networks involving adhesion molecules, signaling receptors, and extracellular matrix components that collectively regulate processes like angiogenesis, cell migration, and immune responses.

What are the optimal conditions for detecting MCAM/CD146 in Western blotting experiments?

For optimal Western blot detection of MCAM/CD146, researchers should implement the following protocol:

• Sample preparation: Prepare cell lysates under reducing conditions using appropriate lysis buffers containing protease inhibitors

• Gel separation: Use 8-10% SDS-PAGE gels to adequately resolve the 140 kDa MCAM/CD146 protein

• Transfer: PVDF membranes are recommended for optimal protein binding

• Blocking: Use 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Incubate with 1 μg/mL of anti-MCAM/CD146 antibody (e.g., Mouse Anti-Human MCAM/CD146 Monoclonal Antibody, Clone #128018) overnight at 4°C

• Secondary antibody: Use HRP-conjugated Anti-Mouse IgG Secondary Antibody

• Detection: Apply Immunoblot Buffer Group 1 for optimal results

• Expected results: A specific band should be detected at approximately 140 kDa

This protocol has been validated using HeLa human cervical epithelial carcinoma cell line lysates, where a specific band for MCAM/CD146 was detected at approximately 140 kDa .

How can flow cytometry be optimized for detecting MCAM/CD146 expression?

For effective flow cytometric analysis of MCAM/CD146 expression, follow these methodological guidelines:

• Cell preparation: Prepare single-cell suspensions at 1×10^6 cells/100 μL in flow cytometry buffer (PBS with 1-2% FBS)

• Antibody selection: Use validated anti-MCAM/CD146 antibodies (e.g., Mouse Anti-Human MCAM/CD146 Monoclonal Antibody, Clone #128018)

• Control inclusion: Include appropriate isotype controls (e.g., MAB002 has been used as an isotype control in validated protocols)

• Staining procedure: Incubate cells with primary antibody, followed by fluorophore-conjugated secondary antibody (e.g., anti-Mouse PE-conjugated Secondary Antibody)

• Analysis parameters: Set appropriate gates based on forward/side scatter and fluorescence intensity

This approach has been successfully implemented for detecting MCAM/CD146 in HeLa human cervical epithelial carcinoma cell line, demonstrating specific staining compared to isotype controls .

What strategies exist for generating and validating anti-MCAM/CD146 antibodies for research?

Generating and validating anti-MCAM/CD146 antibodies requires a systematic approach:

• Antigen design: Based on available structural data, human MCAM/CD146 from Val24-Gly559 has been used successfully as an immunogen

• Antibody formats: Various formats have been developed including:

  • Monoclonal antibodies (e.g., Clone #128018)

  • Single chain (scFv) antibodies selected from phage antibody display libraries

  • Fc chimera proteins combining MCAM/CD146 with human IgG domains

• Validation methods:

  • Direct ELISA binding assays

  • Western blot analysis showing specific bands at expected molecular weight

  • Flow cytometry on positive control cell lines like HeLa

  • Cross-reactivity testing against related proteins (e.g., ALCAM, BCAM)

  • Immunocytochemistry showing appropriate cellular localization

Phage antibody display libraries have been successfully used to generate internalizing human single chain (scFv) antibodies targeting MCAM/CD146, with specificity validated through yeast surface human proteome display .

What approaches are recommended for studying MCAM/CD146 in various in vitro and ex vivo models?

For comprehensive MCAM/CD146 research, several complementary experimental models are recommended:

• Cell line models: HeLa human cervical epithelial carcinoma cells have been validated for MCAM/CD146 expression . BPH-1 cells transfected with pCMV-MCAM can serve as a controlled expression system .

• Primary cell cultures: BG01V human embryonic stem cells express MCAM/CD146 and can be used for studying its role in stemness and differentiation .

• Ex vivo tissue models: Tumor fragment spheroids cultured ex vivo provide a three-dimensional model that better preserves the in vivo tissue architecture and heterogeneity. These have been successfully targeted with quantum dot-labeled anti-MCAM scFv antibodies .

• Recombinant protein systems: NS0-derived human MCAM/CD146 Fc chimera proteins can be used for biochemical and binding studies . These recombinant proteins maintain functional binding to known interaction partners like Galectin-3 .

Each model system offers distinct advantages, and researchers should select the appropriate approach based on their specific research questions and available resources.

How can MCAM/CD146 be utilized as a target for therapeutic development in cancer?

MCAM/CD146 represents a promising therapeutic target in cancer, particularly for malignancies where it is overexpressed such as mesothelioma. Several approaches have demonstrated potential:

• Antibody-based therapies: Internalizing single chain (scFv) antibodies targeting MCAM/CD146 have been specifically selected for mesothelioma and can deliver lethal doses of liposome-encapsulated small molecule drugs to both epithelioid and sarcomatous subtypes of mesothelioma cells . This approach leverages the differential expression of MCAM/CD146 in tumor versus normal mesothelial cells.

• Diagnostic imaging: Technetium (99mTc)-labeled anti-MCAM scFv has been used in single-photon emission computed tomography (SPECT/CT) studies to successfully detect mesothelioma organotypic xenografts in vivo . This demonstrates the potential for MCAM/CD146-targeted approaches in both diagnostic and therapeutic applications.

• Molecular targeting considerations: When developing MCAM/CD146-targeted therapies, researchers should consider:

  • Specificity for tumor versus normal tissues

  • Internalization properties of targeting antibodies

  • Binding affinity and epitope accessibility

  • Potential for immunogenicity

  • Combination with other therapeutic modalities

The development of therapeutic strategies targeting MCAM/CD146 represents an active area of research with promising preclinical results, particularly in mesothelioma models.

What methodologies are recommended for studying the role of MCAM/CD146 in angiogenesis and metastasis?

Investigating MCAM/CD146's role in angiogenesis and metastasis requires specialized methodological approaches:

• In vitro angiogenesis models:

  • Endothelial tube formation assays with MCAM/CD146 manipulation

  • Co-culture systems with tumor and endothelial cells

  • Spheroid sprouting assays in 3D matrices

• Metastasis models:

  • Transwell migration and invasion assays

  • 3D organotypic models mimicking tissue architecture

  • Ex vivo tissue fragment cultures that maintain tumor microenvironment interactions

  • In vivo xenograft models, including mesothelioma organotypic xenografts that have been successfully used to study MCAM/CD146-targeting

• Molecular interaction studies:

  • Functional ELISA assays to study binding between MCAM/CD146 and potential partners like Galectin-3

  • Co-immunoprecipitation studies to identify binding partners in relevant cellular contexts

  • Live cell imaging to track MCAM/CD146 dynamics during cellular migration and invasion

These complementary approaches provide a comprehensive toolkit for dissecting the complex roles of MCAM/CD146 in angiogenesis and metastasis processes.

How can functional genomics approaches be applied to study MCAM/CD146 biology?

Functional genomics provides powerful tools for investigating MCAM/CD146 biology at multiple levels:

• Gene expression modulation:

  • Overexpression systems using plasmids containing full-length human MCAM cDNA (e.g., pCMV-MCAM) under control of the CMV promoter have been successfully used in transfection experiments with lipofectamine

  • RNA interference (siRNA, shRNA) for temporary knockdown

  • CRISPR/Cas9 for permanent gene knockout or precise editing

• Functional readouts:

  • Phage antibody binding assays to assess surface expression

  • Biotin-labeled anti-fd bacteriophage followed by SA-PE for detection in transfected cells

  • Phenotypic assays measuring adhesion, migration, and invasion

• Systematic interaction studies:

  • Yeast surface human proteome display has been successfully used to identify MCAM as a target antigen recognized by mesothelioma-targeting phage antibodies

  • This approach represents an innovative strategy for mapping tumor cell surface epitope space

These methodologies allow researchers to systematically investigate the functional consequences of MCAM/CD146 expression modulation and identify its interaction partners in various cellular contexts.

What are common challenges in detecting MCAM/CD146 and how can they be addressed?

Researchers frequently encounter several challenges when working with MCAM/CD146:

• Molecular weight variability:

  • Problem: MCAM/CD146 appears at different molecular weights (108-140 kDa) depending on glycosylation and experimental conditions

  • Solution: Include positive control lysates with known MCAM/CD146 expression (e.g., HeLa cells); use gradient gels (6-12%) to better resolve proteins in this range

• Antibody specificity:

  • Problem: Non-specific binding or cross-reactivity with related adhesion molecules

  • Solution: Use validated antibodies that have been tested for cross-reactivity with related proteins like ALCAM and BCAM ; include appropriate negative controls

• Protein aggregation:

  • Problem: MCAM/CD146 can form aggregates affecting detection

  • Solution: Optimize sample preparation conditions; for recombinant proteins, reconstitute at recommended concentrations (e.g., 500 μg/mL in PBS)

• Experimental variability:

  • Problem: Inconsistent results across experiments

  • Solution: Standardize protocols; determine optimal antibody dilutions for each application; maintain consistent handling of biological materials

• Low expression levels:

  • Problem: Difficulty detecting MCAM/CD146 in certain cell types

  • Solution: Optimize detection methods; consider using signal amplification approaches; concentrate samples when necessary

Addressing these challenges requires careful optimization and standardization of experimental protocols.

What quality control procedures should be implemented when working with recombinant MCAM/CD146 proteins?

When working with recombinant MCAM/CD146 proteins, researchers should implement the following quality control procedures:

• Purity assessment:

  • SDS-PAGE with silver staining and quantitative densitometry by Coomassie Blue Staining (aim for >95% purity)

  • Size exclusion chromatography to confirm homogeneity

• Structural validation:

  • Confirm expected molecular weight (predicted mass of 87 kDa for the core protein, 108-124 kDa with post-translational modifications)

  • Verify disulfide-linked homodimer formation for Fc chimera constructs

  • N-terminal sequence analysis to confirm correct processing (e.g., starting at Val24)

• Functional testing:

  • Binding assays with known interaction partners (e.g., Galectin-3)

  • Determining ED50 values in functional ELISAs (expected range: 0.25-1.25 μg/mL for Galectin-3 binding)

• Contaminant testing:

  • Endotoxin testing using LAL method (target: <0.10 EU per 1 μg protein)

  • Mycoplasma testing for proteins produced in mammalian expression systems

• Storage stability:

  • Assessing activity retention after freeze-thaw cycles

  • Validating shelf-life under recommended storage conditions

These quality control measures ensure the reliability and reproducibility of experiments using recombinant MCAM/CD146 proteins.

What considerations are important when designing experiments to study MCAM/CD146 in different cancer types?

When designing experiments to study MCAM/CD146 in cancer research contexts, several key considerations should be addressed:

• Expression heterogeneity:

  • MCAM/CD146 expression varies significantly between cancer types and even within the same tumor

  • Immunohistochemical analysis of mesothelioma tissue microarrays has shown MCAM is widely expressed by both epithelioid and sarcomatous types of mesothelioma (>80% of cases) but not by normal mesothelial cells

  • Include appropriate sampling strategies to account for this heterogeneity

• Model system selection:

  • Cell line models: Validate MCAM/CD146 expression in selected cell lines

  • Ex vivo models: Tumor fragment spheroids cultured ex vivo provide a system that better preserves tumor heterogeneity

  • In vivo models: Mesothelioma organotypic xenografts have been successfully used for MCAM/CD146-targeting studies

• Functional readouts:

  • Select assays relevant to the cancer type under investigation

  • Consider using multiple complementary assays to assess different aspects of cancer biology

  • Include appropriate controls for each experimental system

• Translational relevance:

  • Correlate experimental findings with clinical data when possible

  • Consider how findings might translate to diagnostic or therapeutic applications

  • Evaluate potential as a biomarker or therapeutic target based on expression patterns

These considerations help ensure that MCAM/CD146 research yields relevant and translatable insights across different cancer types.

How is MCAM/CD146 being investigated as a potential target for immunotherapy approaches?

MCAM/CD146 represents an emerging target for immunotherapy approaches, with several innovative strategies under investigation:

• Antibody-based therapies:

  • Single chain (scFv) antibodies targeting MCAM/CD146 have been selected from phage antibody display libraries and shown to internalize into mesothelioma cells

  • These antibodies have been exploited to deliver lethal doses of liposome-encapsulated small molecule drugs to both epithelioid and sarcomatous subtypes of mesothelioma cells

• Theranostic applications:

  • Anti-MCAM scFv has been labeled with technetium (99mTc) for single-photon emission computed tomography (SPECT/CT) imaging of mesothelioma organotypic xenografts in vivo

  • This dual diagnostic/therapeutic potential allows for patient selection and treatment monitoring

• Target validation approaches:

  • Confirmation of differential expression between tumor and normal tissues is critical

  • Immunohistochemical analysis of tissue microarrays confirms MCAM/CD146 is expressed in >80% of mesothelioma samples but not in normal mesothelium

  • Expression in multiple cancer subtypes (both epithelioid and sarcomatous mesothelioma) broadens potential applications

• Delivery system optimization:

  • Quantum dot-labeled anti-MCAM scFv has been used to target primary mesothelioma cells in tumor fragment spheroids cultured ex vivo

  • Various conjugation strategies can be explored to optimize tumor targeting and therapeutic payload delivery

The development of MCAM/CD146-targeted immunotherapies represents a promising direction for cancers with limited treatment options, such as mesothelioma.

What novel technologies are being developed to study MCAM/CD146 interactions in the tumor microenvironment?

Cutting-edge technologies are advancing our understanding of MCAM/CD146 interactions within the complex tumor microenvironment:

• Advanced screening platforms:

  • Yeast surface human proteome display has been used to identify MCAM/CD146 as the target antigen bound by mesothelioma-targeting phage antibodies

  • This represents a novel cloning strategy for mapping the targetable tumor cell surface epitope space

• Ex vivo culture systems:

  • Tumor fragment spheroids cultured ex vivo provide a platform that preserves tissue architecture and cellular heterogeneity

  • These systems allow for targeting studies using quantum dot-labeled anti-MCAM scFv to primary mesothelioma cells

• In vivo imaging technologies:

  • Single-photon emission computed tomography combined with computed tomography (SPECT/CT) using technetium (99mTc)-labeled anti-MCAM scFv has been used to detect mesothelioma organotypic xenografts

  • These approaches enable real-time visualization of MCAM/CD146-expressing cells in living systems

• Functional binding assays:

  • When Recombinant Human MCAM/CD146 Fc Chimera is immobilized at 1 μg/mL (100 μL/well), it binds Recombinant Human Galectin-3 with a measurable ED50 of 0.25-1.25 μg/mL

  • These assays provide quantitative measurements of binding interactions

These emerging technologies offer unprecedented insights into MCAM/CD146 biology within the tumor microenvironment, potentially leading to new therapeutic strategies.

What are the promising directions for combining MCAM/CD146 targeting with other therapeutic modalities?

The future of MCAM/CD146-targeted therapies likely lies in combinatorial approaches:

• Antibody-drug conjugates (ADCs):

  • Internalizing antibodies against MCAM/CD146, such as the scFvs selected from phage antibody display libraries , represent ideal vehicles for delivering cytotoxic payloads

  • The demonstrated ability to deliver liposome-encapsulated small molecule drugs to mesothelioma cells provides proof-of-concept for this approach

• Combination with immunotherapy:

  • MCAM/CD146-targeted approaches could be combined with immune checkpoint inhibitors to enhance anti-tumor immunity

  • Targeting MCAM/CD146 on tumor vasculature could improve immune cell infiltration into tumors

• Theranostic applications:

  • Combining diagnostic imaging with therapeutic delivery

  • The successful use of technetium (99mTc)-labeled anti-MCAM scFv for SPECT/CT imaging of mesothelioma xenografts demonstrates feasibility

• Rational combination based on biology:

  • Targeting MCAM/CD146 alongside its binding partners (e.g., Galectin-3)

  • Combining with therapies targeting complementary pathways in angiogenesis or metastasis

• Personalized approaches:

  • Patient selection based on MCAM/CD146 expression profiles

  • Tailoring combination strategies based on molecular characteristics of individual tumors

These combinatorial strategies may overcome resistance mechanisms and improve therapeutic outcomes in MCAM/CD146-positive malignancies.