TSPAN4 Antibody

What is TSPAN4 and why is it significant for research?

TSPAN4 (tetraspanin 4) is a member of the transmembrane 4 superfamily, characterized by four hydrophobic structural domains. This 26.1 kDa protein (238 amino acids) is involved in crucial cellular processes including adhesion, migration, membrane-remodeling, and signal transduction. TSPAN4 has emerged as a significant research target due to its roles in the immune system, nervous system, cancer pathogenesis, and infection. Most notably, TSPAN4 serves as a valuable marker for visualizing migrasomes in migrating cells, with its overexpression markedly enhancing migrasome formation . Research has demonstrated TSPAN4's role in driving glioblastoma progression through EGFR stability regulation, making it an important target for cancer research .

What are the most effective applications for TSPAN4 antibodies?

TSPAN4 antibodies have demonstrated effectiveness across multiple applications, with particular utility in Western Blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . For migrasome visualization, immunofluorescence techniques using TSPAN4 antibodies provide exceptional results as TSPAN4 overexpression enhances migrasome formation. When investigating tumor microenvironments, IHC applications reveal infiltration patterns of immunosuppressive cells that correlate with TSPAN4 expression. For quantitative protein analysis, Western blotting provides reliable detection of the 26.1 kDa TSPAN4 protein .

How do I select the appropriate TSPAN4 antibody for my experimental model?

Selection should be guided by your experimental model and specific research questions. Consider these factors:

• Species reactivity: Confirm the antibody's reactivity with your model organism. While human TSPAN4 is well-characterized, orthologous proteins exist in canine, porcine, monkey, mouse, and rat models .

• Application compatibility: Verify the antibody has been validated for your specific application. Some antibodies perform optimally in WB but may be suboptimal for IHC or IF .

• Clonality considerations: Monoclonal antibodies (like the mouse IgM antibody described) offer high specificity but potentially limited epitope recognition, while polyclonal antibodies provide broader epitope recognition but potentially more cross-reactivity .

• Conjugation requirements: For multiplex assays, flow cytometry, or imaging applications, consider whether you need conjugated antibodies or conjugation-ready formats .

• Validation evidence: Review literature and technical documentation showing successful application in similar experimental contexts, particularly in tumor microenvironment or migrasome research if relevant to your study .

How can I effectively design experiments to study TSPAN4's role in tumor microenvironment regulation?

Designing experiments to investigate TSPAN4's role in tumor microenvironment regulation requires a multifaceted approach:

• Cell type-specific analysis: Implement single-cell RNA sequencing to characterize TSPAN4 expression across different cell populations within the tumor microenvironment. This approach has successfully identified high TSPAN4 expression in specific malignant clusters and revealed significant cell-cell interactions between TSPAN4-high expressing malignant cells and CD8Tex, microglia, and endothelial cells .

• Loss and gain of function experiments: Design parallel knockdown and overexpression experiments in relevant cell lines (e.g., glioma cell lines U87 MG and LN229) to assess TSPAN4's direct effects on tumor cell phenotypes and surrounding immune cells .

• Microenvironment characterization: Combine TSPAN4 manipulation with comprehensive immune profiling using techniques like CIBERSORT algorithm analysis for immune cell infiltration or flow cytometry to directly measure M1/M2 macrophage polarization .

• Functional assays: Include proliferation assays (EdU, CCK8), migration assays, and co-culture systems to assess TSPAN4's impact on both tumor cell behavior and immune cell function .

• In vivo validation: Extend findings to mouse models using orthotopic implantation of TSPAN4-manipulated cells, followed by comprehensive tumor microenvironment profiling.

What methodological approaches are effective for studying TSPAN4-mediated migrasome formation?

To effectively study TSPAN4-mediated migrasome formation:

• Live-cell imaging: Implement time-lapse microscopy of fluorescently-tagged TSPAN4 to visualize migrasome dynamics in real-time. This approach allows monitoring of migrasome formation, release, and uptake by recipient cells .

• Genetic manipulation: Use CRISPR/Cas9 or RNAi approaches to modulate TSPAN4 expression levels. As demonstrated in U87 MG and LN229 glioma cells, TSPAN4 knockdown significantly impacts migrasome formation while overexpression enhances it .

• Quantitative analysis: Employ automated image analysis software to quantify migrasome number, size, and distribution under different experimental conditions. Representative images of migrasomes in TSPAN4 knockdown versus control cells show clear phenotypic differences that can be quantified .

• Biochemical characterization: Isolate migrasomes through differential centrifugation followed by proteomic and lipidomic analysis to determine their molecular composition and how it changes with TSPAN4 manipulation.

• Functional studies: Assess the biological effects of TSPAN4-derived migrasomes on recipient cells through co-culture experiments, analyzing changes in recipient cell phenotype, signaling, and gene expression.

What are the critical considerations when using TSPAN4 antibodies in multiplex immunostaining?

When using TSPAN4 antibodies in multiplex immunostaining, consider these critical factors:

• Antibody compatibility: Ensure that the TSPAN4 antibody's host species and isotype do not conflict with other antibodies in your panel. For example, if using the mouse IgM monoclonal antibody (60638-4-PBS), avoid other mouse IgM antibodies in the panel to prevent cross-reactivity .

• Sequential staining optimization: Determine the optimal staining sequence, especially when studying TSPAN4 alongside immune checkpoints (PD-L1, CTLA-4, LAG-3) that show correlation with TSPAN4 expression .

• Signal amplification requirements: Due to variable TSPAN4 expression across different cell types, determine whether signal amplification methods are needed for detection of low-expressing populations.

• Spectral overlap mitigation: When using fluorescently-labeled antibodies, carefully select fluorophores to minimize spectral overlap, particularly important when analyzing TSPAN4 alongside multiple immunomodulators .

• Validation controls: Include single-stained controls, isotype controls, and biological controls (TSPAN4 knockdown/overexpression samples) to confirm staining specificity, especially important given TSPAN4's complex expression pattern across tumor and immune cells .

How does TSPAN4 expression correlate with cancer progression across different tumor types?

TSPAN4 expression shows significant correlations with cancer progression across multiple tumor types:

What methodological approaches should be used to investigate TSPAN4's role in glioblastoma progression?

To investigate TSPAN4's role in glioblastoma progression:

• Expression profiling in patient cohorts: Analyze TSPAN4 expression across different glioma grades, molecular subtypes (IDH mutant vs. wild-type, MGMT methylated vs. unmethylated), and correlate with patient survival data using techniques like immunohistochemistry, RNA-seq, and protein quantification .

• Cell line manipulation studies: Conduct parallel knockdown and overexpression experiments in multiple glioma cell lines (as demonstrated with U87 MG and LN229 cells) to assess the direct impact on cell proliferation using EdU and CCK8 assays .

• Migrasome analysis: Visualize and quantify migrasome formation in TSPAN4-manipulated glioma cells using fluorescence microscopy, as migrasomes may alleviate cellular stress in glioblastoma .

• EGFR regulation assessment: Investigate TSPAN4's impact on EGFR stability and downstream signaling pathways, as previous research indicates TSPAN4 drives GBM progression through EGFR regulation .

• Single-cell analysis: Apply single-cell RNA sequencing to identify specific cellular populations with high TSPAN4 expression, as demonstrated in the GSE141460 dataset where TSPAN4 showed notably high expression in malignant cluster 10 .

• In vivo models: Develop orthotopic xenograft models using TSPAN4-manipulated glioma cells to assess tumor growth, invasion, and survival outcomes.

How does TSPAN4 interact with immune checkpoint molecules in the tumor microenvironment?

TSPAN4 shows significant interactions with immune checkpoint molecules in the tumor microenvironment:

• Correlation with key immune checkpoints: Analysis reveals significant positive correlations between TSPAN4 expression and well-known immune checkpoint molecules including PD-L1 (programmed death-ligand 1), PD-1 (programmed cell death protein 1), CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), and LAG-3 (lymphocyte-activation gene 3) in multiple cancer types including BLCA, GBMLGG, KIPAN, and LUSC .

• Immunomodulator associations: TSPAN4 expression correlates with numerous immunoregulatory signatures including chemokines, receptors, MHC molecules, immunoinhibitors, and immunostimulators in BLCA, GBMLGG, KIPAN, LUSC, and STAD, suggesting a broad role in immune regulation .

• Immunosuppressive cell correlation: TSPAN4 expression positively correlates with immunosuppressive cell signatures, including exhausted T-cells, effector Treg T-cells, resting Treg T-cells, and Th1-like cells in glioma, indicating its role in shaping an immunosuppressive microenvironment .

• M2 macrophage polarization: Experimental evidence from both U87 MG and LN229 glioma cell lines demonstrates that TSPAN4 overexpression promotes M2 macrophage polarization, while knockdown reverses this effect, as confirmed by both qRT-PCR and flow cytometry analyses .

• Cell-cell interaction networks: Single-cell analysis reveals significant interactions between TSPAN4-high expressing malignant cells and immune populations including CD8Tex cells and microglia, suggesting direct communication between TSPAN4-expressing tumor cells and immune checkpoint-expressing immune cells .

How can I optimize Western blot protocols for reliable TSPAN4 detection?

Optimizing Western blot protocols for reliable TSPAN4 detection requires attention to several technical considerations:

• Sample preparation: Since TSPAN4 is a membrane protein with four transmembrane domains, use lysis buffers containing non-ionic detergents (e.g., Triton X-100 or NP-40) to effectively solubilize membrane proteins without denaturing them. Avoid boiling samples for extended periods to prevent aggregation of transmembrane proteins .

• Protein loading concentration: Begin with higher protein concentrations (50-80 μg/lane) as TSPAN4 may be expressed at relatively low levels in some cell types. The expected molecular weight is approximately 26.1 kDa .

• Transfer optimization: Use a semi-dry transfer system with PVDF membrane (rather than nitrocellulose) for improved binding of hydrophobic proteins. Transfer at lower voltage for longer duration (e.g., 25V for 2 hours) for efficient transfer of membrane proteins .

• Blocking optimization: Test different blocking solutions (5% non-fat milk vs. 5% BSA) to determine which provides optimal signal-to-noise ratio for your specific TSPAN4 antibody .

• Antibody dilution and incubation: For primary antibody, begin with a 1:500 to 1:1000 dilution and incubate overnight at 4°C. For detection of mouse monoclonal antibodies like 60638-4-PBS, use appropriate anti-mouse IgM secondary antibodies that minimize cross-reactivity with other immunoglobulin classes .

• Positive controls: Include cell lines known to express TSPAN4 at high levels, such as specific malignant glioma cell lines (particularly from cluster 10 identified in single-cell studies) .

What are common pitfalls when performing immunohistochemistry with TSPAN4 antibodies?

When performing immunohistochemistry with TSPAN4 antibodies, researchers should be aware of these common pitfalls:

• Inadequate antigen retrieval: TSPAN4 is a membrane protein that may require aggressive antigen retrieval methods. Test both heat-induced epitope retrieval (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0) and enzymatic retrieval methods to determine optimal conditions .

• Non-specific membrane staining: The hydrophobic nature of TSPAN4 can lead to non-specific membrane staining. Include appropriate isotype controls and perform antibody validation using TSPAN4 knockdown tissues/cells as negative controls .

• Variable expression across cell types: TSPAN4 expression varies significantly across different cell types within a tumor. Single-cell analysis has shown high expression in specific malignant clusters but not others, which can lead to misinterpretation of negative results if analyzing bulk tissue .

• Co-expression analysis challenges: When studying co-expression with immune markers (e.g., M2 macrophage markers or immune checkpoints), signal overlap may be difficult to interpret without multiplex staining or sequential section analysis .

• Methylation impact on expression: TSPAN4 expression is significantly influenced by DNA methylation status. In tumors with high TSPAN4 methylation, protein expression may be undetectable despite biological relevance of the TSPAN4 pathway to the disease progression .

• Cross-reactivity with other tetraspanins: Due to structural similarities with other tetraspanin family members, some antibodies may exhibit cross-reactivity. Verify antibody specificity through appropriate controls and RNA-based validation methods when possible .

How should I approach troubleshooting inconsistent results in TSPAN4 functional studies?

When encountering inconsistent results in TSPAN4 functional studies, employ a systematic troubleshooting approach:

• Verify knockdown/overexpression efficiency: Inconsistent functional outcomes often stem from variable knockdown or overexpression efficiency. Validate TSPAN4 manipulation at both mRNA (qRT-PCR) and protein levels (Western blot), establishing minimum thresholds for inclusion in functional analyses (e.g., >70% knockdown or >3-fold overexpression) .

• Cell line heterogeneity assessment: Perform single-cell analysis to determine if your cell population contains heterogeneous subpopulations with different TSPAN4 expression patterns. As shown in GBM single-cell data, TSPAN4 expression can be highly enriched in specific malignant clusters (e.g., cluster 10) .

• Migrasome formation quantification: Since TSPAN4 is a key regulator of migrasome formation, inconsistent migrasome visualization can indicate technical issues with your TSPAN4 manipulation. Standardize migrasome imaging protocols using positive controls .

• Microenvironment influences: For macrophage polarization experiments, standardize macrophage preparation methods and culture conditions, as variability in starting macrophage populations can significantly impact polarization outcomes .

• Technical validation across methods: Validate key findings using complementary methodologies. For instance, confirm M2 macrophage polarization using both qRT-PCR of marker genes and flow cytometry analysis of surface markers as demonstrated in the literature .

• Time-course analyses: Perform time-course experiments to account for temporal variations in TSPAN4-mediated effects, as cellular responses to TSPAN4 manipulation may vary significantly over time.

How should data from TSPAN4 expression studies be analyzed in relation to tumor microenvironment components?

Data analysis for TSPAN4 expression in relation to tumor microenvironment components should follow these methodological approaches:

What statistical approaches are most appropriate for analyzing TSPAN4 correlation with patient survival outcomes?

When analyzing TSPAN4 correlation with patient survival outcomes, these statistical approaches are most appropriate:

How can I effectively interpret TSPAN4 immunostaining patterns in heterogeneous tumor samples?

Effective interpretation of TSPAN4 immunostaining in heterogeneous tumor samples requires systematic evaluation:

• Pattern recognition: Distinguish between different TSPAN4 staining patterns: membrane-localized (consistent with its tetraspanin nature), cytoplasmic, or punctate (potentially representing migrasomes). Document the predominant pattern in different regions and cell types within the tumor .

• Semi-quantitative scoring: Implement a standardized scoring system assessing both staining intensity (0-3+) and percentage of positive cells to generate H-scores or Allred scores that account for heterogeneous expression .

• Spatial context analysis: Analyze TSPAN4 expression in relation to tumor architecture - particularly at tumor margins, invasive fronts, necrotic regions, and perivascular areas - as expression may vary in these microenvironments .

• Cell type identification: Use sequential sections or multiplex immunostaining to identify cell types expressing TSPAN4. As single-cell data has shown, TSPAN4 expression can be highly enriched in specific malignant clusters while absent in others .

• Correlation with molecular characteristics: Integrate TSPAN4 immunostaining data with molecular characteristics (e.g., IDH mutation status, MGMT methylation status in gliomas) to identify pattern associations with established molecular subtypes .

• Digital pathology approaches: When available, utilize digital pathology and artificial intelligence-based image analysis to quantify TSPAN4 expression across entire tumor sections, overcoming visual assessment limitations and reducing inter-observer variability.

How does TSPAN4's role in migrasome formation relate to cancer progression and therapeutic resistance?

The relationship between TSPAN4-mediated migrasome formation and cancer progression presents an emerging research direction:

• Stress response mechanism: Migrasomes appear to serve as a cellular stress response mechanism in glioblastoma, with evidence suggesting they alleviate cellular stress. TSPAN4's role as a migrasome marker and enhancer of migrasome formation may therefore contribute to cancer cell survival under stress conditions .

• Intercellular communication: Migrasomes can mediate intercellular communication, potentially transferring proteins, lipids, and nucleic acids between tumor cells or between tumor and stromal cells. TSPAN4-enhanced migrasome formation may facilitate this communication, promoting tumor progression .

• Therapeutic implications: The visualization of migrasomes in TSPAN4 knockdown versus control cells in both U87 MG and LN229 glioma models suggests that targeting TSPAN4 could disrupt migrasome-mediated processes, representing a potential therapeutic strategy .

• Resistance mechanisms: Migrasomes may contribute to therapeutic resistance by removing cellular toxins or damaged organelles. TSPAN4's enhancement of migrasome formation could therefore promote resistance to chemotherapy or targeted therapies .

• Experimental approaches: Investigating this relationship requires methodologies like transmission electron microscopy for ultrastructural analysis of migrasomes, tracking experiments to follow migrasome fate, and therapeutic response studies in TSPAN4-manipulated cells to determine if migrasome disruption enhances treatment efficacy .

What are the most promising approaches for targeting TSPAN4 in cancer immunotherapy research?

Promising approaches for targeting TSPAN4 in cancer immunotherapy research include:

• Combination with immune checkpoint inhibitors: Given TSPAN4's positive correlation with immune checkpoints including PD-L1, PD-1, CTLA-4, and LAG-3, combining TSPAN4 targeting with established checkpoint inhibitors may enhance immunotherapy efficacy, particularly in BLCA, GBMLGG, KIPAN, and LUSC where these correlations are strongest .

• Modulation of macrophage polarization: Experimental evidence demonstrates TSPAN4's role in promoting M2 macrophage polarization. Targeting TSPAN4 could potentially redirect macrophages toward an anti-tumoral M1 phenotype, as confirmed by both qRT-PCR and flow cytometry analyses in multiple glioma cell lines .

• Disruption of immunosuppressive networks: TSPAN4 expression correlates with immunosuppressive cell signatures including exhausted T-cells and regulatory T-cells. Targeting TSPAN4 may disrupt these immunosuppressive networks, potentially reinvigorating anti-tumor immune responses .

• Antibody-drug conjugates: Developing TSPAN4-targeted antibody-drug conjugates could deliver cytotoxic payloads specifically to TSPAN4-expressing tumor cells, such as the malignant cluster 10 identified in GBM single-cell analysis .

• CAR-T cell approaches: Engineering chimeric antigen receptor T cells targeting TSPAN4 could provide specific recognition of TSPAN4-high tumor cells while sparing normal tissues with lower expression.

• Small molecule inhibitors: Developing small molecules that disrupt TSPAN4's interactions with partner proteins (like EGFR in glioblastoma) could impair its function in promoting tumor progression .

How might advances in single-cell analysis techniques enhance our understanding of TSPAN4 biology in the tumor microenvironment?

Advanced single-cell analysis techniques offer significant potential for enhancing our understanding of TSPAN4 biology:

• Cellular heterogeneity resolution: Single-cell RNA sequencing has already revealed that TSPAN4 expression is not uniform across tumor cells but highly enriched in specific malignant clusters (e.g., cluster 10 in GBM). Further application of these techniques could identify additional TSPAN4-high cell populations with unique biological properties .

• Spatial context integration: Combining single-cell transcriptomics with spatial transcriptomics or multiplexed immunofluorescence could map the physical location of TSPAN4-expressing cells within the tumor microenvironment, revealing spatial relationships with immune cells and stromal components .

• Cell-cell interaction networks: Cell-cell interaction analysis using computational tools has identified significant interactions between TSPAN4-high malignant cells and immune populations including CD8Tex cells, microglia, and endothelial cells. Further development of these tools could uncover additional interaction partners and signaling mechanisms .

• Single-cell multi-omics integration: Integrating single-cell transcriptomics with proteomics, epigenomics, or metabolomics could provide multi-dimensional insights into how TSPAN4 expression affects cellular phenotype at multiple levels .

• Lineage tracing applications: Applying single-cell lineage tracing in TSPAN4-manipulated tumor models could determine whether TSPAN4-high cells represent a distinct evolutionary lineage within tumors and trace their developmental trajectories .

• Therapeutic response prediction: Single-cell analysis of TSPAN4 expression patterns before and after treatment could identify cellular populations that persist following therapy, potentially contributing to recurrence through TSPAN4-mediated mechanisms .