MS4A12 Antibody

What is MS4A12 and what is its cellular distribution in normal and cancerous tissues?

MS4A12 is a membrane spanning 4-domains protein that functions as a colon-selective store-operated calcium channel. Expression analysis and immunohistochemistry have revealed that MS4A12 is a colonic epithelial cell lineage gene confined to the apical membrane of colonocytes with strict transcriptional repression in all other normal tissue types . This highly specific expression pattern makes it particularly valuable for colorectal cancer research. In terms of malignancy, expression of MS4A12 is maintained upon malignant transformation in approximately 63% of colon cancers . This selective expression profile suggests MS4A12 could serve as both a diagnostic marker and potential therapeutic target for colorectal cancer.

What are the recommended applications and conditions for using MS4A12 antibodies?

MS4A12 antibodies, such as the mouse monoclonal antibody clone OTI1H8, are primarily validated for Western blot (WB) applications with a recommended dilution of 1:2000 . When working with MS4A12 antibodies, researchers should consider the following technical parameters:

• Reactivity: Specifically designed for human samples

• Antibody host: Mouse-derived antibodies are commonly used

• Isotype: Typically IgG1

• Immunogen: Full-length human recombinant protein of MS4A12 (NP_060186) produced in HEK293T cells

• Storage conditions: Store at -20°C as received

• Stability: Generally stable for 12 months from receipt

For optimal results, researchers should include appropriate positive controls (colon epithelial tissue or colon cancer cell lines) and negative controls (non-colonic tissues) to verify antibody specificity.

How does MS4A12 function as a calcium channel and what experimental approaches verify this activity?

MS4A12 has been identified as a novel component of store-operated calcium entry (SOCE) in intestinal cells . Calcium flux analyses have revealed that MS4A12 plays a critical role in regulating calcium signaling in colonic epithelial cells. When designing experiments to study MS4A12's calcium channel function, researchers should consider:

• Calcium flux analysis using fluorescent calcium indicators to measure intracellular calcium levels

• Store depletion assays using agents like thapsigargin to activate SOCE followed by calcium readdition

• Patch-clamp electrophysiology to directly measure calcium currents

• Molecular manipulation of MS4A12 expression (RNAi or overexpression) to correlate with changes in calcium flux

• Pharmacological intervention with calcium channel blockers to assess their effects on MS4A12 function

These approaches provide complementary evidence for MS4A12's function as a store-operated calcium channel specifically in colonic cells.

What is the relationship between MS4A12 expression and EGFR signaling in colon cancer?

Research has demonstrated a significant functional relationship between MS4A12 and epidermal growth factor receptor (EGFR) signaling in colon cancer cells. Using RNAi-mediated gene silencing, studies have shown that loss of MS4A12 in LoVo colon cancer cells attenuates EGFR-mediated effects . Specifically:

• Cell proliferation is significantly impaired

• Cell motility is reduced

• Chemotactic invasion capability is decreased

Conversely, cancer cells expressing MS4A12 are sensitized and respond to lower concentrations of epidermal growth factor . This suggests that MS4A12 functions as a positive regulator of EGFR signaling, potentially amplifying its effects on cancer cell behavior. This relationship has important implications for understanding therapy resistance in colorectal cancer, as EGFR is a major therapeutic target in this disease.

How does MS4A12 influence cell differentiation in colon cancer and what experimental models demonstrate this?

MS4A12 appears to play a critical role in colon cancer cell differentiation. Research using sodium butyrate (BS) as a differentiation inducer in the LoVo colon cancer cell line has provided valuable insights:

• When LoVo cells are treated with 4 mmol/L BS for over 48 hours, MS4A12 variant-1 (a specific transcript of the MS4A12 gene) expression increases significantly alongside established differentiation markers alkaline phosphatase (ALP) and E-cadherin .

• When MS4A12 variant-1 is silenced, the elevation of these differentiation markers (ALP and E-cadherin) in BS-treated cells is inhibited .

• Silencing MS4A12 variant-1 also leads to significant resistance to BS-induced cell cycle arrest and apoptosis .

These findings suggest that MS4A12 is not merely associated with differentiation but actively participates in the differentiation process of colon epithelial cells. The sodium butyrate-induced differentiation of LoVo cells serves as an effective experimental model for studying MS4A12's role in colon cancer differentiation.

What is the prognostic value of MS4A12 expression in colorectal cancer patients?

Survival analysis using GEO datasets (GSE39582 and GSE38832) encompassing 681 distinct colon cancer samples has revealed stage-specific prognostic significance of MS4A12 expression:

• In early-stage colon cancer: Patients with low MS4A12 expression demonstrate poor survival (Hazard Ratio = 1.72; p = 0.036) .

• In advanced-stage colon cancer: MS4A12 expression shows little prognostic value (Hazard Ratio = 0.89; p = 0.601) .

This stage-dependent prognostic pattern suggests that MS4A12 could serve as a risk classification marker specifically for early-stage colon cancer. The differential prognostic significance between early and advanced disease stages indicates that MS4A12's functional impact on tumor biology may evolve during disease progression. Researchers investigating MS4A12 as a biomarker should therefore carefully consider cancer stage when designing studies and interpreting results.

What approaches are recommended for studying the mechanisms of MS4A12-dependent calcium signaling in colon cancer?

To effectively investigate MS4A12's role in calcium signaling within colon cancer cells, researchers should consider these methodological approaches:

• Real-time calcium imaging using fluorescent indicators (Fura-2, Fluo-4) to visualize spatial and temporal changes in calcium concentration within individual cells

• SOCE-specific assays involving store depletion with agents like thapsigargin followed by calcium readdition to specifically examine store-operated calcium entry

• Simultaneous monitoring of MS4A12 localization and calcium flux using fluorescently-tagged MS4A12 constructs combined with calcium indicators

• Molecular perturbation studies comparing calcium dynamics between:

  • Wild-type cells

  • MS4A12-silenced cells (using RNAi approaches)

  • MS4A12-overexpressing cells

• Pharmacological intervention using calcium channel blockers to assess their effects on MS4A12-mediated calcium signaling

• Integration with EGFR signaling studies to determine how calcium flux via MS4A12 influences downstream EGFR pathway activation

These approaches would help elucidate the specific mechanisms by which MS4A12 contributes to calcium homeostasis in colonic epithelial cells and how this function relates to its roles in differentiation and cancer progression.

How can researchers distinguish between MS4A12 variants and what is their functional significance?

Research has identified MS4A12 variant-1 as a specific transcript of the MS4A12 gene that plays a role in colon cancer cell differentiation . To effectively study MS4A12 variants:

• Transcript-specific PCR primers should be designed to target unique regions of each variant, allowing specific amplification and quantification

• Variant-specific antibodies (when available) can be used to distinguish protein isoforms in immunoblotting or immunohistochemistry

• Expression constructs for individual variants can be created for overexpression studies to determine isoform-specific functions

• Variant-specific siRNA or shRNA can be designed for selective knockdown studies

• RNA-seq analysis can quantify different transcript variants in various conditions

The functional significance of MS4A12 variants should be investigated through comparative studies examining their:

• Subcellular localization

• Calcium channel properties

• Interaction with EGFR signaling

• Effects on differentiation markers

• Impact on cell proliferation, motility, and invasion

Current evidence suggests that MS4A12 variant-1 in particular is associated with differentiation status in colon cancer cells, as its expression increases during BS-induced differentiation .

What strategies can address the challenges of reproducibility in MS4A12 immunohistochemistry across different tissue samples?

Ensuring reproducibility in MS4A12 immunohistochemistry across tissue samples presents several challenges that can be addressed through the following strategies:

• Antibody validation:

  • Verify antibody specificity using positive controls (colon tissue) and negative controls (non-colonic tissues)

  • Compare results with MS4A12 mRNA expression data

  • Use MS4A12-silenced cells as negative controls

• Standardized protocols:

  • Optimize and standardize fixation methods

  • Establish consistent antigen retrieval procedures

  • Standardize antibody dilutions (based on lot-specific titration)

  • Use automated staining platforms when possible

• Scoring systems:

  • Develop clear, objective scoring criteria for MS4A12 expression

  • Consider digital image analysis for quantification

  • Implement multi-observer scoring to minimize subjective interpretation

• Sample preparation considerations:

  • Account for pre-analytical variables (time to fixation, fixation duration)

  • Consider tissue microarrays for simultaneous processing of multiple samples

  • Include standard reference samples in each staining batch

• Context-specific interpretation:

  • Account for MS4A12's apical membrane localization in colonocytes

  • Consider differentiation status of the tissue

  • Note relationship to other markers (e.g., differentiation markers like E-cadherin)

Implementation of these standardized approaches will enhance the reliability and reproducibility of MS4A12 immunohistochemistry, particularly important given its potential role as a diagnostic and prognostic marker.

How might MS4A12's homology to CD20 inform potential therapeutic development strategies?

MS4A12 has been identified as a sequence homologue of CD20 , which has significant implications for therapeutic development:

Given CD20's successful targeting in B-cell lymphomas with antibodies like rituximab, the structural homology suggests several potential approaches for MS4A12-targeted therapies:

• Development of MS4A12-specific monoclonal antibodies with mechanisms similar to anti-CD20 antibodies (complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity)

• Exploration of membrane topology to identify accessible epitopes for antibody binding

• Development of antibody-drug conjugates leveraging MS4A12's selective expression

• Investigation of bispecific antibodies targeting both MS4A12 and other relevant targets

• Modulation of calcium signaling through MS4A12 targeting as a therapeutic strategy

The success of CD20-targeted therapies provides a valuable framework for developing MS4A12-directed approaches, while accounting for the unique biology and expression pattern of MS4A12 in colorectal tissue.

What is the relationship between MS4A12 expression changes and genetic/epigenetic alterations in colorectal cancer?

The relationship between MS4A12 expression and genetic/epigenetic alterations in colorectal cancer appears complex and warrants further investigation. Current research suggests:

• Genetic alterations involving MS4A12 occur at relatively low frequencies in colorectal cancer. In comparison to other genes, MS4A12 shows a lower incidence of genetic alterations compared to genes like AGT (2.2%) and GNB1 (1.4%) .

• Epigenetic regulation, particularly DNA methylation, may play a more significant role in controlling MS4A12 expression. This hypothesis is supported by observations of other genes in the same context, such as CXCL12, where promoter hypermethylation appears to cause downregulation in colorectal cancer .

• The maintenance of MS4A12 expression in 63% of colon cancers suggests that complete silencing is not a universal feature of malignant transformation . This variable expression pattern might reflect the heterogeneity of genetic and epigenetic alterations in colorectal cancer.

Future research directions should include:

• Comprehensive methylation analysis of the MS4A12 promoter in normal and cancerous colon tissues

• Correlation studies between MS4A12 expression and specific genetic alterations

• Investigation of transcription factors regulating MS4A12 expression

• Analysis of histone modifications at the MS4A12 locus

Understanding these relationships could provide insights into the mechanisms of MS4A12 dysregulation in colorectal cancer and potentially reveal new therapeutic opportunities.

How can researchers integrate MS4A12 status with other molecular markers to improve colorectal cancer classification?

Integrating MS4A12 status with other molecular markers could significantly enhance colorectal cancer classification, particularly for early-stage disease where MS4A12 has demonstrated prognostic value . Researchers should consider:

• Multimarker panels:

  • Combine MS4A12 with established molecular markers (microsatellite instability, KRAS/BRAF mutation status, CpG island methylator phenotype)

  • Develop tissue-specific panels incorporating other colonocyte-specific markers

  • Include markers of differentiation status (E-cadherin, ALP) given MS4A12's role in differentiation

• Integrated classification systems:

  • Develop algorithms that weight MS4A12 appropriately based on disease stage

  • Create decision trees that incorporate MS4A12 status at specific nodes

  • Validate integrated classification in independent cohorts

• Functional pathway integration:

  • Group MS4A12 with other calcium signaling mediators

  • Combine with EGFR pathway markers given their functional relationship

  • Incorporate with markers of epithelial differentiation

• Clinical-molecular integration:

  • Develop models that combine MS4A12 status with clinical parameters

  • Create stage-specific integration approaches

  • Validate with survival outcomes and treatment response data

A proposed integration framework might include:

This integrated approach would leverage MS4A12's stage-specific prognostic value while acknowledging its limitations in advanced disease.

What are common challenges when working with MS4A12 antibodies and how can they be addressed?

Researchers working with MS4A12 antibodies may encounter several challenges that can be addressed through specific methodological approaches:

• Specificity concerns:

  • Challenge: Cross-reactivity with other MS4A family members

  • Solution: Use monoclonal antibodies raised against unique MS4A12 epitopes; validate specificity using MS4A12-silenced cells; include appropriate tissue controls

• Sensitivity limitations:

  • Challenge: Detecting low expression levels of MS4A12

  • Solution: Optimize signal amplification steps; use higher antibody concentrations (while monitoring background); employ more sensitive detection systems

• Membrane protein detection issues:

  • Challenge: Adequate extraction and detection of membrane-bound MS4A12

  • Solution: Use appropriate membrane protein extraction buffers; optimize sample preparation to preserve membrane integrity; consider non-denaturing conditions for certain applications

• Variability between antibody lots:

  • Challenge: Performance differences between manufacturing lots

  • Solution: Validate each new lot against previous standards; maintain consistent positive controls; consider purchasing larger quantities of effective lots

• Application-specific optimization:

  • Challenge: Different optimal conditions for various applications (WB, IHC, etc.)

  • Solution: Perform application-specific titration; optimize buffers and incubation times for each application; follow manufacturer recommendations for starting conditions (e.g., 1:2000 dilution for WB)

These challenges highlight the importance of thorough validation and optimization when working with MS4A12 antibodies to ensure reliable and reproducible results across different experimental contexts.

How can contradictory findings regarding MS4A12 expression in colon cancer be reconciled?

Research on MS4A12 in colon cancer has occasionally produced seemingly contradictory findings. These discrepancies can be addressed through careful analysis and methodological considerations:

• Stage-dependent effects:

  • Contradictory finding: Different prognostic significance in early versus advanced disease

  • Reconciliation: Always separate analyses by stage; recognize that MS4A12's biological role may evolve during disease progression

• Heterogeneity within samples:

  • Contradictory finding: Variable expression in different regions of the same tumor

  • Reconciliation: Use multiple sampling within tumors; quantify heterogeneity; consider average versus focal expression patterns

• Methodological differences:

  • Contradictory finding: Different results between protein and mRNA detection methods

  • Reconciliation: Use complementary approaches (IHC, WB, qPCR); compare results between methods; consider post-transcriptional regulation

• Variant-specific effects:

  • Contradictory finding: Different functions attributed to MS4A12

  • Reconciliation: Specify which variant is being studied; use variant-specific detection methods; consider that variants may have distinct functions

• Context-dependent regulation:

  • Contradictory finding: Different expression patterns in cell lines versus patient samples

  • Reconciliation: Always compare findings between models; validate cell line findings in primary tissues; consider microenvironmental influences

By carefully addressing these potential sources of contradiction through improved experimental design and data interpretation, researchers can develop a more coherent understanding of MS4A12's role in colorectal cancer.