lhfpl6 Antibody

What is LHFPL6 and why is it significant for cancer research?

LHFPL6 (Lipoma HMGIC Fusion Partner-Like 6) is a member of the LHFP subfamily, which belongs to the four-transmembrane protein-encoding gene superfamily. It was first identified as a translocation partner of the high mobility group A2 (HMGA2) gene in benign lipomas. LHFPL6 has gained significant interest in cancer research, particularly in gastric cancer (GC), where its overexpression is associated with poor prognosis. Recent studies indicate that LHFPL6 may be involved in activating epithelial-mesenchymal transition (EMT) and shows positive correlation with M2 macrophage abundance, which are potent immunosuppressors in the tumor microenvironment. These findings suggest LHFPL6 could serve as both a prognostic biomarker and potential therapeutic target for GC .

What are the standard methods for detecting LHFPL6 protein expression in research samples?

Several standard methods can be employed to detect LHFPL6 protein expression:

• Western Blotting: The most common method for quantifying LHFPL6 protein levels in cell or tissue lysates. Protocols typically involve running samples on SDS-PAGE gels, transferring to membranes, blocking, and incubating with LHFPL6 primary antibody followed by secondary antibody detection. Target/β-actin bands can be quantified using gel image processing systems to calculate relative protein levels .

• Immunohistochemistry (IHC): Used to examine LHFPL6 expression in tissue sections. Images are typically obtained at 400× magnification, and H-SCORE (range 0-300) is calculated to quantify staining intensity .

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for examining LHFPL6 levels in cell supernatants or body fluids. Optical density readings at 450 nm are measured using a microplate reader .

• Immunofluorescence: Allows visualization of LHFPL6 localization within cells and can be used in co-localization studies with other proteins .

What considerations are important when selecting an LHFPL6 antibody for research applications?

When selecting an LHFPL6 antibody for research applications, consider the following factors:

• Antibody Specificity: Choose antibodies validated to specifically recognize LHFPL6 without cross-reactivity to other LHFPL family members (LHFPL1-5). Review validation data including western blots showing single bands at the expected molecular weight.

• Antibody Type: Consider whether monoclonal or polyclonal antibodies are more suitable for your application. Monoclonal antibodies offer higher specificity but may be less sensitive than polyclonal antibodies.

• Species Reactivity: Ensure the antibody recognizes LHFPL6 in your species of interest (human, mouse, rat, etc.).

• Application Compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IF, ELISA, etc.).

• Epitope Information: Understanding which region of LHFPL6 the antibody recognizes can be important, especially if studying specific domains or potential splice variants.

• Positive Controls: Identify appropriate positive control samples (such as HGC27 gastric cancer cells, which show high LHFPL6 expression) .

How can LHFPL6 antibodies be utilized to investigate the role of LHFPL6 in epithelial-mesenchymal transition?

To investigate LHFPL6's role in epithelial-mesenchymal transition (EMT), researchers can employ several advanced approaches using LHFPL6 antibodies:

• Co-immunoprecipitation (Co-IP) Studies: Use LHFPL6 antibodies to pull down protein complexes and analyze binding partners involved in EMT pathways. This helps identify direct protein-protein interactions that may mediate LHFPL6's effect on EMT.

• Dual Immunofluorescence Staining: Perform co-staining of LHFPL6 with established EMT markers (E-cadherin, N-cadherin, vimentin, Snail, Slug, etc.) to assess correlation between LHFPL6 expression and EMT marker localization in tissue samples or cell lines.

• Chromatin Immunoprecipitation (ChIP): If LHFPL6 is suspected to interact with transcription factors regulating EMT, ChIP assays using LHFPL6 antibodies can help identify potential DNA binding sites.

• Functional Studies with Gene Manipulation: As demonstrated in previous research, LHFPL6 knockdown or overexpression systems combined with LHFPL6 antibody-based detection can be used to assess how modulating LHFPL6 levels affects EMT marker expression and cellular phenotypes. Research indicates that LHFPL6 may be involved in EMT activation in gastric cancer .

• Migration and Invasion Assays: Transwell assays coupled with LHFPL6 antibody-based detection can assess how LHFPL6 expression correlates with cell migration and invasion capabilities, key functional readouts of EMT .

What approaches can be used to study the relationship between LHFPL6 and tumor-associated macrophage polarization?

Several sophisticated approaches can be employed to study the relationship between LHFPL6 and tumor-associated macrophage polarization:

• Co-culture Systems: Establish co-culture systems between gastric cancer cells with varying LHFPL6 expression levels and THP-1-derived macrophages. This allows modeling of tumor-macrophage interactions in vitro. After co-culture, assess macrophage polarization markers like CD206 and CD163 (M2 markers) using immunofluorescence assays .

• Conditioned Media Experiments: Collect conditioned media from LHFPL6-overexpressing or LHFPL6-knockdown cancer cells and treat macrophages to determine if secreted factors from cancer cells with different LHFPL6 expression affect macrophage polarization.

• Multiplex Cytokine Analysis: Use LHFPL6 antibodies to isolate LHFPL6-high and LHFPL6-low cancer cells, then analyze their cytokine/chemokine secretion profiles to identify factors that might influence macrophage polarization.

• In vivo Models: Develop mouse models with LHFPL6-overexpressing or LHFPL6-knockdown tumors and use LHFPL6 antibodies along with macrophage markers for immunohistochemical analysis of tumor sections to assess macrophage infiltration and polarization in vivo.

• Bioinformatic Analysis: Use algorithms like CIBERSORT to analyze the relationship between LHFPL6 expression levels and macrophage polarization in publicly available datasets. Research has demonstrated a positive correlation between LHFPL6 expression and M2 macrophage abundance .

What methodological challenges exist when using LHFPL6 antibodies for prognostic biomarker validation in clinical samples?

Several methodological challenges must be addressed when validating LHFPL6 as a prognostic biomarker using antibodies:

• Antibody Standardization: Ensuring consistent antibody performance across different batches and laboratories requires rigorous validation. Establish standard protocols with detailed antibody dilutions, incubation times, and detection methods.

• Sample Processing Variability: Fixation time, tissue processing methods, and antigen retrieval techniques can affect antibody binding and staining intensity. Standardize these parameters to ensure comparable results across clinical samples.

• Scoring System Standardization: Develop and validate a standardized scoring system for LHFPL6 expression in tissue samples. The H-SCORE method (range 0-300) has been used in previous studies, with higher scores indicating stronger positive staining .

• Threshold Determination: Carefully determine the cutoff value that defines "high" versus "low" LHFPL6 expression. Previous studies have used median expression as a cutoff, but optimal thresholds should be determined based on survival outcomes .

• Multi-institutional Validation: For robust biomarker validation, testing across multiple institutions with different patient cohorts is essential to confirm reproducibility and broad applicability.

• Integration with Other Biomarkers: Assess how LHFPL6 expression correlates with established prognostic markers and whether combined assessment improves prognostic value.

How can LHFPL6 antibodies be incorporated into studies exploring the relationship between LHFPL6 methylation and expression?

LHFPL6 antibodies can be strategically incorporated into studies investigating the relationship between LHFPL6 methylation and expression through several approaches:

• Paired Methylation-Expression Analysis: Analyze the same patient samples for both LHFPL6 promoter methylation (using bisulfite sequencing or methylation-specific PCR) and protein expression (using LHFPL6 antibodies in IHC or Western blot). This allows direct correlation between methylation status and protein levels on a per-sample basis.

• Cell Line Models: Treat gastric cancer cell lines with demethylating agents (such as 5-azacytidine) and measure changes in LHFPL6 protein expression using LHFPL6 antibodies to establish causality between methylation and expression.

• Chromatin Immunoprecipitation (ChIP) Analysis: Use antibodies against methylation-related proteins (DNMTs, MBDs) and histone modifications to examine the chromatin state at the LHFPL6 promoter, correlating these findings with LHFPL6 expression.

• Integration with Public Databases: Correlate LHFPL6 methylation data from databases like TCGA, MethSurv, and MEXPRESS with experimental protein expression data generated using LHFPL6 antibodies. Previous research has demonstrated approaches for analyzing the relationship between LHFPL6 promoter methylation levels and expression levels using TCGA-STAD data .

• Survival Analysis Based on Methylation Status: Compare survival outcomes in patient groups stratified by both LHFPL6 methylation status and protein expression to determine whether methylation adds prognostic value beyond protein expression alone.

What are the optimal conditions for using LHFPL6 antibodies in Western blotting experiments?

For optimal Western blotting results with LHFPL6 antibodies, consider the following technical recommendations:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors for cell/tissue lysis

  • Determine optimal protein concentration (typically 20-50 μg per lane)

  • Denature samples at 95°C for 5 minutes in reducing sample buffer

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Include positive controls (HGC27 gastric cancer cells show high LHFPL6 expression)

  • Include molecular weight markers to confirm band size

• Transfer Conditions:

  • Transfer to PVDF membrane (preferred over nitrocellulose for LHFPL6)

  • Use standard transfer buffer with 20% methanol

  • Transfer at 100V for 1-1.5 hours or 30V overnight at 4°C

• Antibody Incubation:

  • Block membrane in 5% non-fat milk in TBST for 1 hour

  • Incubate with primary LHFPL6 antibody (typically 1:1000 dilution) overnight at 4°C

  • Wash 3x with TBST, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 4x with TBST, 5 minutes each

• Detection:

  • Use ECL substrate appropriate for expected expression level

  • Image using a gel documentation system (e.g., ChemiDoc XRS+)

  • Calculate relative protein levels by normalizing to loading controls like β-actin

What protocols are recommended for LHFPL6 immunohistochemistry in cancer tissue samples?

The following protocol recommendations are based on previously successful LHFPL6 immunohistochemistry in cancer tissue samples:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Section tissues at 4-5 μm thickness

• Deparaffinization and Rehydration:

  • Xylene: 2 changes, 10 minutes each

  • 100% ethanol: 2 changes, 5 minutes each

  • 95% ethanol: 5 minutes

  • 70% ethanol: 5 minutes

  • Distilled water: rinse

• Antigen Retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Microwave or pressure cooker method: 15-20 minutes

  • Cool to room temperature: 20 minutes

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase: 3% H₂O₂ for 10 minutes

  • Block non-specific binding: 5% normal serum for 30 minutes

  • Primary LHFPL6 antibody: Optimized dilution (typically 1:100-1:200), overnight at 4°C

  • Wash: PBS, 3 changes, 5 minutes each

  • Secondary antibody: 30-60 minutes at room temperature

  • Wash: PBS, 3 changes, 5 minutes each

• Detection and Counterstaining:

  • DAB substrate: 2-10 minutes (monitor under microscope)

  • Counterstain: Hematoxylin for 30 seconds

  • Blue in running tap water: 5 minutes

  • Dehydrate through alcohol series

  • Clear in xylene and mount

• Scoring:

  • Image at 400× magnification using a microscope (e.g., NIKON Eclipse Ni-E)

  • Calculate H-SCORE (range 0-300) to quantify staining intensity

  • Higher scores indicate stronger positive staining

• Controls:

  • Include positive control (gastric cancer tissue with known LHFPL6 expression)

  • Include negative control (omit primary antibody)

  • Consider including normal gastric tissue for comparison

How can LHFPL6 antibodies be utilized effectively in co-culture experiments studying tumor-immune cell interactions?

LHFPL6 antibodies can be effectively utilized in co-culture experiments studying tumor-immune cell interactions through the following methodological approaches:

• Co-culture System Setup:

  • Differentiate THP-1 monocytes into macrophages using PMA (10 ng/mL for 48 hours) to obtain M0 macrophages

  • Seed gastric cancer cells (with varying LHFPL6 expression) in upper chamber and macrophages in lower chamber of a transwell apparatus with 0.4-μm pore size

  • Co-culture for 48 hours to allow secreted factors to exchange between chambers

• LHFPL6 Expression Manipulation:

  • Establish gastric cancer cell lines with stable LHFPL6 knockdown (using shRNA) or overexpression

  • Verify knockdown/overexpression efficiency using Western blot with LHFPL6 antibodies before co-culture

  • HGC27 cells are recommended as they show high LHFPL6 expression naturally

• Analysis of Macrophage Polarization:

  • After co-culture, collect macrophages from the lower chamber

  • Perform immunofluorescence staining for M2 macrophage markers (CD206, CD163)

  • Use fluorescence microscopy (400× magnification) to visualize and quantify marker expression

• Secreted Factor Analysis:

  • Collect co-culture supernatants and measure secreted LHFPL6 using ELISA

  • Analyze secreted cytokines/chemokines that may mediate LHFPL6's effects on macrophage polarization

• Functional Assays:

  • Use LHFPL6 antibody to neutralize secreted LHFPL6 in the co-culture system and assess its impact on macrophage polarization

  • Compare results with control antibody (isotype-matched) treatment

• Validation Through siRNA Rescue Experiments:

  • Perform rescue experiments by re-introducing LHFPL6 in knockdown cells to confirm specificity

  • Verify LHFPL6 re-expression using antibody-based detection methods

How should researchers interpret discrepancies between LHFPL6 mRNA and protein expression data?

When researchers encounter discrepancies between LHFPL6 mRNA and protein expression data, several analytical approaches and considerations should be applied:

• Post-transcriptional Regulation Assessment:

  • MicroRNA regulation: Investigate whether miRNAs targeting LHFPL6 may be causing discrepancies. The ceRNA network construction based on LHFPL6 expression can provide insights into miRNA-mediated regulation .

  • RNA stability: Assess mRNA half-life using actinomycin D chase experiments paired with RT-qPCR to determine if differences in mRNA stability contribute to expression discrepancies.

• Protein Turnover Analysis:

  • Perform cycloheximide chase experiments to determine LHFPL6 protein half-life.

  • Investigate ubiquitination or other post-translational modifications that might affect protein stability.

• Technical Considerations:

  • Antibody specificity: Verify LHFPL6 antibody specificity using positive and negative controls, including knockdown validation .

  • Isoform detection: Determine if your mRNA detection method captures all relevant isoforms compared to the protein detection method.

  • Sample preparation differences: Consider whether sample collection methods for protein vs. RNA analysis might introduce systematic biases.

• Methodological Integration:

  • Perform concurrent analysis of matched samples for both mRNA (RT-qPCR) and protein (Western blot/IHC) from the same specimens.

  • Consider single-cell approaches to determine if cell population heterogeneity explains bulk measurement discrepancies.

• Biological Interpretation:

  • Time-course analysis: Examine whether temporal delays between transcription and translation could explain observed discrepancies.

  • Tissue/cellular context: Consider whether the tumor microenvironment influences post-transcriptional regulation of LHFPL6.

What factors should be considered when interpreting LHFPL6 expression changes in the context of cancer progression?

When interpreting LHFPL6 expression changes in cancer progression, researchers should consider several contextual factors:

• Tumor Heterogeneity Considerations:

  • Intratumoral heterogeneity: Assess LHFPL6 expression across different regions of the same tumor using IHC to understand spatial variation.

  • Temporal heterogeneity: Compare LHFPL6 expression in paired primary and metastatic samples or during treatment to understand dynamic changes.

• Clinical Correlation Analysis:

  • Stage-specific patterns: LHFPL6 expression has been shown to correlate with pathological stage in gastric cancer (p = 0.00266) .

  • Survival impact: Analyze relationships between LHFPL6 expression and multiple survival parameters (OS, DSS, DFI, PFI) using Kaplan-Meier analyses and Cox regression models .

  • Multivariate analysis: Determine whether LHFPL6 is an independent prognostic factor when accounting for other clinicopathological variables.

• Molecular Context Integration:

  • EMT association: Interpret LHFPL6 expression in the context of EMT marker expression, as LHFPL6 may be involved in EMT activation .

  • Immune contexture: Consider LHFPL6's relationship with immune cell infiltration, particularly M2 macrophages, which showed positive correlation with LHFPL6 expression .

  • Methylation status: Interpret expression in context of epigenetic regulation, as methylation analysis can provide additional insights .

• Functional Impact Assessment:

  • Phenotypic effects: Correlate LHFPL6 expression with functional readouts like migration, invasion, and colony formation in experimental models .

  • Mechanistic insights: Consider LHFPL6's position in signaling networks and potential downstream effects on cellular behavior.

• Technical Validation:

  • Multi-platform confirmation: Validate expression changes using complementary techniques (IHC, Western blot, ELISA) .

  • Multiple cohort validation: Confirm findings across independent patient cohorts (TCGA-STAD, GSE118919, GSE29272, GSE13861) .

What troubleshooting approaches are recommended for weak or non-specific LHFPL6 antibody signals?

When encountering weak or non-specific LHFPL6 antibody signals, researchers should implement the following systematic troubleshooting approaches:

• Antibody-Related Troubleshooting:

  • Titration optimization: Test a range of antibody dilutions (1:100, 1:500, 1:1000, 1:2000) to identify optimal concentration.

  • Fresh antibody preparation: Ensure antibody hasn't deteriorated due to improper storage or repeated freeze-thaw cycles.

  • Alternative antibody evaluation: Test antibodies from different suppliers or those targeting different epitopes of LHFPL6.

  • Validation controls: Use positive control samples (HGC27 cells) with known high LHFPL6 expression .

• Sample Preparation Optimization:

  • Protein extraction method: Compare different lysis buffers (RIPA, NP-40, Triton X-100) to determine optimal extraction efficiency.

  • Fixation parameters: For IHC/IF, optimize fixation time and conditions as overfixation can mask epitopes.

  • Antigen retrieval: Test different antigen retrieval methods (heat-induced vs. enzymatic) and buffers (citrate, EDTA, Tris) to maximize epitope accessibility.

• Protocol Modifications:

  • Western blot:

    • Increase protein loading (50-100 μg)

    • Extend primary antibody incubation (overnight at 4°C)

    • Use more sensitive detection systems (ECL Plus/Prime)

    • Optimize transfer conditions for membrane-bound proteins

  • IHC/IF:

    • Increase antibody incubation time or temperature

    • Use amplification systems (biotin-streptavidin)

    • Reduce background with additional blocking steps

• Specificity Verification:

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

  • Knockout/knockdown validation: Compare signal between LHFPL6 knockdown cells and controls

  • Molecular weight verification: Ensure detected bands match expected molecular weight

• Technical Considerations:

  • Fresh reagents: Prepare fresh buffers and blocking solutions

  • Optimize blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers)

  • Reduce background: Increase washing steps duration and frequency

How might LHFPL6 antibodies be utilized in developing targeted therapies for gastric cancer?

LHFPL6 antibodies could play crucial roles in developing targeted therapies for gastric cancer through several innovative approaches:

• Therapeutic Antibody Development:

  • Design and test function-blocking antibodies targeting LHFPL6's extracellular domains to inhibit its activity in cancer cells

  • Develop antibody-drug conjugates (ADCs) using LHFPL6 antibodies to deliver cytotoxic agents specifically to LHFPL6-overexpressing cancer cells

  • Create bispecific antibodies targeting both LHFPL6 and immune effector cells to enhance anti-tumor immune responses

• Patient Stratification for Clinical Trials:

  • Use validated LHFPL6 IHC protocols to select patients with LHFPL6-overexpressing tumors for targeted therapy trials

  • Establish optimal cutoff values for "high" LHFPL6 expression that correlate with therapeutic response

  • Develop companion diagnostic assays using standardized LHFPL6 antibody protocols

• Combination Therapy Research:

  • Investigate LHFPL6 expression changes in response to standard chemotherapies using antibody-based detection

  • Test combinations of LHFPL6-targeting approaches with immune checkpoint inhibitors, given LHFPL6's correlation with M2 macrophages

  • Explore synergistic effects with drugs targeting the EMT pathway, as LHFPL6 may be involved in EMT activation

• Mechanism-Based Drug Discovery:

  • Use LHFPL6 antibodies in pull-down assays to identify binding partners that could serve as alternative therapeutic targets

  • Employ proximity ligation assays with LHFPL6 antibodies to verify drug-induced disruption of key protein-protein interactions

  • Develop screening assays using LHFPL6 antibodies to identify small molecules that modulate LHFPL6 expression or function

• Monitoring Treatment Response:

  • Use LHFPL6 antibodies to assess changes in LHFPL6 expression in liquid biopsies or circulating tumor cells during treatment

  • Develop minimally invasive LHFPL6 detection methods for longitudinal patient monitoring

What novel research directions might emerge from studying the relationship between LHFPL6 and other oncogenic pathways?

Several promising research directions could emerge from studying the relationship between LHFPL6 and other oncogenic pathways:

• LHFPL6-HMGA2 Interaction Studies:

  • LHFPL6 was first identified as a translocation partner of HMGA2 in benign lipomas

  • Investigate whether LHFPL6 and HMGA2 interact functionally in gastric cancer through co-immunoprecipitation studies using LHFPL6 antibodies

  • Explore whether LHFPL6-HMGA2 cooperation drives specific oncogenic programs in different cancer types

• EMT Regulatory Network Mapping:

  • Use LHFPL6 antibodies in ChIP-seq experiments to identify genomic regions where LHFPL6 or its partners might regulate EMT-related genes

  • Perform proteomics analysis of LHFPL6-interacting proteins in EMT-induced versus epithelial cancer cells

  • Investigate whether LHFPL6 acts upstream or downstream of known EMT master regulators like SNAIL, TWIST, and ZEB1

• Tumor Microenvironment Communication:

  • Study how LHFPL6-expressing cancer cells communicate with various stromal components, especially given its correlation with M2 macrophages

  • Investigate whether LHFPL6 affects exosome composition and function in intercellular communication

  • Explore LHFPL6's potential role in cancer-associated fibroblast activation

• Epigenetic Regulation Mechanisms:

  • Study how DNA methylation and histone modifications regulate LHFPL6 expression in different cancer contexts

  • Investigate whether LHFPL6 itself influences epigenetic programming in cancer cells

  • Explore potential feedback loops between LHFPL6 expression and epigenetic regulators

• Cancer Stem Cell Connection:

  • Given LHFPL6's association with EMT, which often promotes stemness, investigate its role in cancer stem cell maintenance

  • Use LHFPL6 antibodies to isolate and characterize potential LHFPL6-high cancer stem cell populations

  • Study whether LHFPL6 expression correlates with established cancer stem cell markers in patient samples

• Therapeutic Resistance Mechanisms:

  • Investigate whether LHFPL6 expression confers resistance to conventional therapies through EMT or immune evasion

  • Study changes in LHFPL6 expression in matched pre-treatment and post-relapse patient samples

  • Explore whether targeting LHFPL6 might overcome specific therapy resistance mechanisms

Recommended LHFPL6 Antibody Dilutions for Various Applications

Positive Control Samples for LHFPL6 Expression Validation

Expected Results from LHFPL6 Functional Studies

Key considerations when working with LHFPL6 antibodies in cancer research

LHFPL6 antibodies represent valuable tools for investigating the role of this protein in cancer, particularly in gastric cancer where it appears to have significant prognostic implications. When working with LHFPL6 antibodies, researchers should carefully consider antibody specificity, optimal application-specific protocols, appropriate positive controls, and consistent scoring methods. The promising findings regarding LHFPL6's involvement in EMT and tumor-associated macrophage polarization suggest it could serve as both a prognostic biomarker and potential therapeutic target .