HMMR Antibody, FITC conjugated

What is HMMR and what cellular functions does it perform?

HMMR (Hyaluronan Mediated Motility Receptor) is a 162-175 kDa type I membrane protein also known as RHAMM, Intracellular hyaluronic acid-binding protein, or CD antigen CD168. It functions as a microtubule-associated spindle assembly factor and hyaluronan-mediated motility receptor . HMMR is predominantly a coiled-coil protein with microtubule binding domains at the N-terminus and a bZip motif at the C-terminus . The protein plays critical roles in multiple cellular processes including cell motility, mitotic spindle formation, and cell division.

At the molecular level, HMMR can form complexes with proteins such as FAK/SRC in the cytoplasm independent of membrane-expressed CD44. This interaction occurs through HMMR's C-terminal region and activates downstream signaling pathways that regulate cell migration, invasion, and immune evasion mechanisms . In cancer research, HMMR has gained significant attention for its role in facilitating antiphagocytic efficiency and promoting immune evasion in hepatocellular carcinoma.

How does the FITC conjugation process affect antibody performance?

FITC (Fluorescein isothiocyanate) conjugation has significant effects on antibody performance that researchers must consider when designing experiments. The conjugation process chemically links FITC molecules to the antibody structure, which enables fluorescent detection but can alter binding characteristics. Research has demonstrated that the FITC-labeling index in antibodies is negatively correlated with binding affinity for target antigens . This means higher FITC labeling can reduce the antibody's ability to recognize and bind to HMMR.

Immunohistochemically, antibodies with higher FITC-labeling indices tend to demonstrate increased sensitivity but are also more prone to producing non-specific staining . This creates an important experimental trade-off: higher sensitivity versus reduced specificity. When using FITC-conjugated HMMR antibodies, researchers should carefully balance these factors by selecting preparations with appropriate labeling densities for their specific application to minimize binding affinity decreases while maintaining adequate detection sensitivity.

What are the primary applications for HMMR Antibody, FITC conjugated?

HMMR Antibody, FITC conjugated has multiple validated research applications across different methodologies:

The antibody has been successfully tested on multiple cell lines including HepG2, K-562, C6, and T-47D cells . For tissue cross-reactivity studies, FITC-labeled HMMR antibodies are particularly valuable as they allow direct visualization without secondary antibodies . This antibody is primarily used in cancer research, immunology, and cell biology studies investigating HMMR's role in cellular motility, tumor progression, and immune evasion mechanisms.

How should researchers optimize staining protocols for HMMR detection in different sample types?

Optimizing HMMR detection protocols requires systematic adjustment of multiple parameters based on sample type and research objectives. For cellular samples, fixation method significantly impacts antibody accessibility to HMMR. Paraformaldehyde (4%) fixation for 15-20 minutes at room temperature is generally effective for preserving HMMR epitopes while maintaining cellular architecture.

For tissue sections, antigen retrieval is critical. Evidence suggests that TE buffer at pH 9.0 provides optimal antigen retrieval for HMMR detection, though citrate buffer at pH 6.0 may serve as an alternative . The incubation time and temperature should be adjusted based on tissue type, with thicker sections requiring longer incubation periods. Blocking procedures should include both protein blocking (3-5% BSA) and endogenous peroxidase blocking if using enzymatic detection systems.

When optimizing dilutions, titration experiments are essential. Start with manufacturer-recommended dilutions (such as 1:200-1:800 for IF applications) and prepare a dilution series to determine optimal signal-to-noise ratio for each sample type . Always include appropriate positive controls (HepG2 or K-562 cells have confirmed HMMR expression) and negative controls (primary antibody omission and isotype controls) to validate staining specificity.

What controls are essential when working with FITC-conjugated HMMR antibodies?

A robust experimental design with appropriate controls is critical when working with FITC-conjugated HMMR antibodies:

• Isotype Control: Use a matched FITC-conjugated mouse IgG1, κ isotype control (for monoclonal antibodies) or FITC-conjugated rabbit IgG (for polyclonal antibodies) at the same concentration as the primary antibody to assess non-specific binding .

• Autofluorescence Control: Unstained samples to determine baseline autofluorescence, particularly important in tissues with high endogenous fluorescence (like liver or kidney).

• Blocking Controls: Samples with primary antibody omitted but including all blocking reagents to assess effectiveness of blocking procedures.

• Positive Expression Controls: Include cell lines with known HMMR expression (HepG2, K-562, C6, T-47D) to confirm antibody functionality .

• Negative Expression Controls: Include cell lines with minimal HMMR expression or HMMR-knockout cells to verify specificity.

• Absorption Controls: Pre-incubate the antibody with recombinant HMMR protein before staining to confirm binding specificity. Significant signal reduction indicates specific binding.

When using flow cytometry, compensation controls are additionally required to account for spectral overlap when using multiple fluorophores. FITC is designed to be excited by the 488 nm laser and detected using an optical filter centered near 530 nm (typically a 525/40 nm bandpass filter) .

What is the optimal protocol for preserving FITC-conjugated antibody activity?

Preserving FITC-conjugated HMMR antibody activity requires careful handling and storage. FITC conjugates are particularly sensitive to light exposure, pH changes, and repeated freeze-thaw cycles. For long-term preservation:

• Storage Conditions: Store at -20°C in aliquots to prevent repeated freeze-thaw cycles. FITC-conjugated antibodies remain stable for approximately 12 months when properly stored . Refrigeration at 2-8°C is suitable for short-term storage (up to 2 weeks).

• Protection from Light: Always wrap containers in aluminum foil or use amber vials to protect from light exposure, which causes photobleaching of the FITC fluorophore.

• Buffer Conditions: The optimal pH for FITC-conjugated antibodies is 7.2-7.4. Most commercial preparations include phosphate buffered solutions with stabilizers and protein protectants to maintain antibody integrity .

• Working Solution Preparation: When preparing working dilutions, use freshly prepared buffer immediately before the experiment. Keep working solutions on ice and protected from light throughout the procedure.

• Concentration: The typical working concentration is 5 μL per test for flow cytometry applications . For other applications, follow the manufacturer's recommended dilutions and optimize through titration experiments.

No antioxidants or preservatives should be added post-manufacture as these may interfere with antibody-antigen binding or cause fluorophore quenching. After each use, return the stock immediately to appropriate storage conditions.

How does HMMR contribute to cancer immune evasion mechanisms?

HMMR plays a sophisticated role in cancer immune evasion, particularly in hepatocellular carcinoma (HCC), through several mechanistic pathways. Research has revealed that HMMR facilitates antiphagocytic efficiency via the HMMR-CD47 axis of liver cancer cells . This mechanism involves the formation of protein complexes that trigger downstream signaling cascades affecting immune surveillance.

At the molecular level, HMMR can form complexes with FAK/SRC proteins in two distinct cellular compartments. In the cell membrane, HMMR-CD44-FAK-SRC forms a four-protein complex, while in the cytoplasm, HMMR-FAK-SRC creates a three-protein complex that functions independently of CD44 . Domain-mapping experiments have demonstrated that the C-terminal region of HMMR is required for FAK interaction, with direct binding confirmed through GST pull-down assays in cell-free conditions .

The downstream effects of these interactions include activation of FAK/SRC signaling pathway and nuclear translocation of the p50/p65 heterodimer, which is essential for NF-κB signaling activation. This creates a pro-inflammatory microenvironment that induces CCL2 and TNF-α production, sustaining CD47 expression ("don't eat me" signal) and facilitating immune escape mechanisms . Importantly, loss of HMMR could potentially break this "don't eat me" signal and sensitize tumors to immune checkpoint blockade therapies, suggesting HMMR as a promising therapeutic target for enhancing immunotherapy efficacy in HCC patients.

What techniques can differentiate between membrane and cytoplasmic HMMR localization?

Differentiating between membrane and cytoplasmic HMMR localization requires specialized techniques that preserve spatial information while providing sufficient resolution:

• Subcellular Fractionation with Western Blotting: This biochemical approach involves separating membrane and cytoplasmic components before immunoblotting. Research protocols have successfully separated these fractions to detect HMMR-protein complexes in different cellular compartments . The method requires careful validation using compartment-specific markers (Na+/K+ ATPase for membrane, GAPDH for cytoplasm).

• Confocal Microscopy with Z-stack Analysis: High-resolution confocal microscopy with optical sectioning provides detailed visualization of HMMR distribution. Co-staining with membrane markers (such as CD44) and cytoplasmic markers helps delineate these compartments. Z-stack acquisition with deconvolution enhances spatial resolution.

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions (such as HMMR-FAK) with subcellular resolution. It generates fluorescent signals only when proteins are within 40 nm of each other, allowing visualization of compartment-specific interactions.

• Immunoelectron Microscopy: For the highest resolution analysis, immunogold labeling with electron microscopy precisely localizes HMMR within cellular ultrastructure, clearly distinguishing membrane-associated from cytoplasmic pools.

• FRET Analysis: Fluorescence resonance energy transfer using differentially labeled antibodies can measure proximity between HMMR and known membrane or cytoplasmic markers, providing functional information about protein interactions in distinct compartments.

These approaches have revealed that HMMR can function through distinct signaling mechanisms depending on its subcellular localization, with cytoplasmic HMMR activating FAK/SRC signaling independently of membrane-expressed CD44 .

How do FITC-labeling indices affect HMMR antibody binding characteristics?

The FITC-labeling index (number of FITC molecules per antibody) significantly impacts HMMR antibody performance through several mechanisms. Research demonstrates a negative correlation between FITC-labeling index and antigen binding affinity . This effect occurs because extensive FITC conjugation can:

• Modify Critical Binding Sites: FITC molecules attach to lysine residues, potentially altering the three-dimensional structure of the antibody's variable regions.

• Create Steric Hindrance: Higher labeling densities introduce bulky fluorophore molecules that physically interfere with antigen-antibody interactions.

• Alter Electrostatic Properties: Each FITC molecule changes the net charge of the antibody, potentially disrupting ionic interactions with the antigen.

Immunohistochemical studies have demonstrated that antibodies with higher FITC-labeling indices tend to produce more sensitive staining but are also more prone to non-specific binding . This creates a critical trade-off that researchers must navigate. For optimal research outcomes, it is recommended that FITC-labeled antibodies used in tissue cross-reactivity studies be carefully selected from several differently labeled preparations to:

• Minimize decreases in binding affinity

• Achieve appropriate detection sensitivity

• Avoid non-specific staining that could lead to misinterpretation of results

When designing experiments with FITC-conjugated HMMR antibodies, researchers should consider using preparations with moderate labeling indices (typically 3-5 FITC molecules per antibody) for optimal balance between sensitivity and specificity.

How can researchers address non-specific staining issues with FITC-conjugated HMMR antibodies?

Non-specific staining is a common challenge when working with FITC-conjugated HMMR antibodies, particularly those with high labeling indices . Multiple strategies can be implemented to minimize this issue:

• Optimize Antibody Dilution: Titrate the antibody concentration to determine the optimal signal-to-noise ratio. Higher dilutions often reduce background while maintaining specific signal.

• Enhanced Blocking Protocols: Implement multi-step blocking procedures including serum (5-10%) from the same species as the secondary antibody (if used), plus bovine serum albumin (1-3%) to block non-specific binding sites. For tissues with high endogenous biotin, add an avidin-biotin blocking step.

• Pre-absorption Controls: Pre-incubate the antibody with recombinant HMMR protein to confirm binding specificity. This process removes antibodies that specifically bind to HMMR, leaving only non-specific binders.

• Selection of Antibody Preparation: Choose FITC-conjugated antibodies with moderate labeling indices to balance sensitivity and specificity . Antibodies with excessive FITC conjugation tend to produce more non-specific staining.

• Buffer Optimization: Adjust salt concentration in washing buffers (0.3-0.5M NaCl) to disrupt low-affinity, non-specific binding. Adding non-ionic detergents (0.1-0.3% Triton X-100) can reduce hydrophobic interactions causing background.

• Autofluorescence Reduction: Treat samples with sodium borohydride (0.1-1%) or commercial autofluorescence quenchers to reduce tissue autofluorescence, particularly in formalin-fixed tissues.

• Alternative Detection Systems: If persistent non-specific staining occurs, consider alternative detection systems such as tyramide signal amplification or quantum dot conjugation, which may provide better signal-to-noise ratios.

By systematically implementing these approaches, researchers can significantly improve staining specificity while maintaining sufficient sensitivity for detecting HMMR expression.

What experimental approaches can investigate HMMR-FAK/SRC interactions in cancer models?

Investigating HMMR-FAK/SRC interactions requires sophisticated experimental approaches spanning molecular, cellular, and in vivo techniques:

• Co-Immunoprecipitation (Co-IP): This technique has successfully identified HMMR-FAK-SRC complexes in both membrane and cytoplasmic fractions . When performing Co-IP, separate cellular compartments (membrane vs. cytoplasm) to examine location-specific interactions. Validate with reciprocal Co-IP using antibodies against each complex component.

• Domain Mapping Experiments: Create truncated HMMR proteins to identify specific interaction domains. Research has demonstrated that the C-terminal region of HMMR is required for FAK interaction . Express these constructs in relevant cell lines and examine their ability to activate FAK/SRC signaling.

• GST Pull-down Assays: This cell-free approach confirms direct protein-protein interactions. GST-tagged HMMR protein can be used to pull down purified FAK, demonstrating direct binding independent of other cellular factors .

• Phosphorylation Analysis: Measure phosphorylation levels of FAK (Tyr397) and SRC (Tyr416) using phospho-specific antibodies after HMMR manipulation. Western blotting or phospho-flow cytometry can quantify these activation markers.

• NF-κB Activation Assays: As a downstream effect of HMMR-FAK-SRC signaling, monitor nuclear translocation of p50/p65 heterodimers using subcellular fractionation or imaging techniques .

• CRISPR/Cas9 Genome Editing: Generate HMMR-knockout or domain-specific mutant cell lines to evaluate the functional significance of these interactions on phenotypes such as cell migration, immune evasion, and tumor progression.

• In Vivo Models: Utilize mouse models with manipulated HMMR expression to assess effects on tumor growth, immune infiltration, and response to checkpoint inhibitor therapy. These models can validate findings from cellular systems in a physiologically relevant context .

These complementary approaches provide comprehensive insights into the molecular mechanisms by which HMMR-FAK-SRC complexes promote cancer progression and immune evasion.

How can HMMR expression be quantified using FITC-conjugated antibodies?

Accurate quantification of HMMR expression using FITC-conjugated antibodies requires standardized methodologies and appropriate controls:

• Flow Cytometry Quantification: This technique allows precise measurement of HMMR expression at the single-cell level. For reliable quantification:

  • Use calibration beads with known quantities of FITC molecules to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • Apply a standardized protocol with 0.20 μg antibody per 10^6 cells

  • Include matched isotype controls to determine positive staining thresholds

  • Measure median fluorescence intensity (MFI) rather than percent positive cells for more accurate expression level assessment

• Quantitative Immunofluorescence Microscopy:

  • Perform image acquisition using standardized exposure settings

  • Include internal control samples in each experiment to normalize between batches

  • Use image analysis software to measure integrated fluorescence intensity

  • Correct for background fluorescence using isotype controls

  • When possible, include calibration slides with known quantities of fluorophore

• Tissue Microarray Analysis:

  • Apply H-score methodology (staining intensity × percentage of positive cells)

  • Use automated image analysis systems with machine learning algorithms for reproducible quantification

  • Segment subcellular compartments to quantify membrane versus cytoplasmic expression

• Correlation with Gene Expression:

  • Validate protein expression measurements with corresponding mRNA levels

  • Use multiplex approaches to correlate HMMR expression with related markers (CD44, CD47)

  • Apply multivariate analysis to identify expression patterns in different cell populations

When interpreting HMMR expression data, researchers should consider that high-HMMR/low-CD44 expression patterns may indicate predominant cytoplasmic HMMR activity, which contributes to immune evasion through CD47 upregulation . This expression profile may serve as a prognostic biomarker and predict response to immunotherapy in cancer patients.

How does HMMR expression correlate with immunotherapy response in cancer patients?

HMMR expression levels have emerged as potential predictive biomarkers for immunotherapy response, particularly in hepatocellular carcinoma. Research indicates that HMMR expression status is associated with distinct patterns of immune evasion and therapy outcomes. Patients with HMMR-low expression profiles may demonstrate better responses to anti-PD-1 immunotherapy . This correlation stems from HMMR's role in maintaining CD47 expression (the "don't eat me" signal) through FAK/SRC signaling pathway activation.

The relationship between HMMR and immunotherapy efficacy involves several mechanistic pathways:

• HMMR loss enhances CD8+ T cell infiltration in tumor tissues, potentially improving T cell-mediated anti-tumor immunity .

• Low HMMR expression may reduce CD47 levels on cancer cells, making them more susceptible to phagocytosis by macrophages and dendritic cells.

• The cytoplasmic HMMR-FAK-SRC complex activates NF-κB signaling, creating a pro-inflammatory environment that sustains CD47 expression and facilitates immune escape .

Clinically, these findings suggest that HMMR status could serve as a stratification marker for patient selection in immunotherapy trials. Patients with HMMR-low expression profiles might be more likely to benefit from immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway . Additionally, the development of therapeutic strategies targeting HMMR could potentially enhance the efficacy of existing immunotherapies by disrupting this immune evasion mechanism.

What are the technical considerations for developing HMMR-targeted therapeutic strategies?

Developing HMMR-targeted therapeutic strategies requires addressing several technical considerations spanning from target validation to delivery optimization:

• Target Specificity: HMMR performs different functions based on its subcellular localization. Therapeutic approaches must consider whether to target membrane-associated HMMR (CD44-dependent pathway) or cytoplasmic HMMR (CD44-independent pathway) . The C-terminal region of HMMR, which interacts with FAK, represents a particularly promising target for disrupting immune evasion mechanisms .

• Therapeutic Modalities:

  • Monoclonal antibodies targeting HMMR extracellular domains

  • Small molecule inhibitors disrupting HMMR-FAK interactions

  • Peptide-based approaches targeting the C-terminal region

  • siRNA or antisense oligonucleotides for HMMR knockdown

  • Proteolysis-targeting chimeras (PROTACs) for HMMR degradation

• Delivery Challenges: Targeting cytoplasmic HMMR requires developing delivery systems capable of crossing the plasma membrane. Potential approaches include:

  • Nanoparticle-based delivery systems

  • Cell-penetrating peptide conjugates

  • Exosome-based delivery platforms

  • Antibody-drug conjugates for targeted delivery

• Combination Strategies: HMMR targeting may be most effective when combined with established immunotherapies. Research suggests that disrupting the HMMR-CD47 axis could synergistically improve the efficacy of PD-1/PD-L1 blockade therapies . This presents opportunities for rational combination approaches that simultaneously target multiple immune evasion mechanisms.

• Biomarker Development: Companion diagnostics using FITC-conjugated HMMR antibodies could help identify patients most likely to benefit from HMMR-targeted therapies. Flow cytometry or immunohistochemistry approaches using standardized protocols would be essential for accurate patient stratification.

These considerations highlight the complexity of developing HMMR-targeted therapies but also underscore the significant potential of this approach for enhancing cancer immunotherapy efficacy.

What are emerging techniques for studying HMMR-mediated signaling pathways?

The investigation of HMMR-mediated signaling pathways is advancing through several cutting-edge technologies that provide unprecedented insights into molecular mechanisms:

• Spatial Transcriptomics and Proteomics: These techniques enable simultaneous visualization of HMMR protein expression and downstream signaling molecules with subcellular resolution. By preserving spatial context, researchers can identify compartment-specific signaling events and correlate them with tissue microenvironments.

• CRISPR-based Screening: Genome-wide or targeted CRISPR screens can identify novel components of HMMR signaling networks. CRISPR activation (CRISPRa) or interference (CRISPRi) approaches allow for nuanced manipulation of HMMR expression levels to study dosage effects on downstream pathways.

• Protein-Protein Interaction Mapping: BioID or APEX2 proximity labeling approaches can identify proteins that interact with HMMR in living cells, potentially revealing previously unknown components of HMMR-mediated signaling complexes beyond the established FAK/SRC interaction .

• Live-Cell Imaging with Optogenetics: These approaches enable real-time visualization of HMMR dynamics and signaling activation with precise spatiotemporal control. Light-inducible dimerization of HMMR domains can trigger specific signaling events to dissect pathway kinetics and dependencies.

• Single-Cell Multi-omics: Integrating single-cell RNA sequencing, ATAC-seq, and proteomics provides comprehensive views of how HMMR signaling influences cellular states and heterogeneity within tumor microenvironments.

• Phosphoproteomics: Quantitative phosphoproteomics before and after HMMR manipulation can identify the complete spectrum of phosphorylation events downstream of HMMR activation, providing a systems-level view of signaling networks.

These emerging technologies will help resolve outstanding questions about how HMMR signaling varies between normal and malignant cells, how cytoplasmic versus membrane-bound HMMR activate distinct signaling cascades, and how these pathways can be therapeutically targeted.

What is the relationship between HMMR and CD47 in determining cancer immune surveillance?

The relationship between HMMR and CD47 represents a critical axis in cancer immune surveillance, with significant implications for immunotherapy development. Recent research has uncovered a sophisticated regulatory network:

HMMR functions as an upstream regulator of CD47 expression through activation of the FAK/SRC signaling pathway . The cytoplasmic HMMR-FAK-SRC complex triggers nuclear translocation of the p50/p65 heterodimer, activating NF-κB signaling. This activation induces production of pro-inflammatory cytokines including CCL2 and TNF-α, which sustain CD47 expression on cancer cell surfaces .

CD47, known as the "don't eat me" signal, binds to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells, inhibiting phagocytosis of cancer cells. This immune evasion mechanism operates through several interconnected pathways:

• In hepatocellular carcinoma, HMMR maintains CD47 expression through both CD44-dependent and CD44-independent mechanisms . This dual regulatory pathway explains why patients with HMMR-high/CD44-low expression profiles still maintain elevated CD47 levels and exhibit poor prognosis.

• Loss of HMMR through genetic manipulation reduces CD47 expression, enhancing macrophage-mediated phagocytosis of cancer cells and improving CD8+ T cell infiltration .

• Unlike direct CD47 blockade, which causes systemic adverse effects like anemia and thrombocytopenia due to CD47's expression on normal cells, targeting HMMR may provide tumor-specific downregulation of CD47, potentially avoiding these toxicities .