HK3 Antibody

What is HK3 and why is it important in research?

Hexokinase 3 (HK3) is an enzyme that phosphorylates glucose to produce glucose-6-phosphate (G6P), representing the first critical step in most glucose metabolism pathways . As a member of the hexokinase family, HK3 is allosterically inhibited by its product (G6P) . The importance of HK3 in research stems from its emerging roles in:

• Cancer biology and immune evasion mechanisms

• Correlation with immune infiltrates in tumor microenvironments

• Prediction of immunotherapy responses, particularly in NSCLC patients

• Metabolic regulation in specialized cell types, especially in immune and cancer cells

Recent research has revealed novel functions beyond glucose metabolism, including HK3-mediated O-GlcNAcylation of EP300, which plays a role in tumor immune evasion mechanisms . This finding suggests HK3 may serve as a promising therapeutic target for enhancing cancer immunotherapy efficacy.

What types of HK3 antibodies are available for research applications?

Based on the search results, there are multiple types of HK3 antibodies available for research applications:

Each antibody offers different advantages depending on the research application. Monoclonal antibodies provide high specificity and reproducibility, while polyclonal antibodies may offer broader epitope recognition and potentially stronger signals in certain applications .

How does HK3 expression correlate with clinical and molecular characteristics in cancer?

Analysis of The Cancer Genome Atlas (TCGA) data has revealed significant correlations between HK3 expression and both clinical and molecular characteristics in cancer, particularly in non-small cell lung cancer (NSCLC) .

Key findings include:

• Cases with low HK3 expression tend to be more malignant entities with frequent genomic aberrations of driver oncogenes

• HK3 expression is linked to immune responses and inflammatory activities based on gene ontology analysis

• HK3 expression shows a significant trend in predicting efficacy of immunotherapy for patients receiving PD-1 inhibitor treatment (Keytruda)

These correlations suggest HK3 expression may serve as a potential biomarker for predicting immunotherapy response and understanding tumor immune microenvironments.

What are the optimal conditions for using HK3 antibodies in Western Blot applications?

For optimal Western Blot results with HK3 antibodies, researchers should follow these evidence-based protocols:

• Sample preparation:

  • Use 30 μg of protein lysate from cells or tissues of interest

  • Separate proteins using 8-15% SDS-PAGE system

  • Transfer onto nitrocellulose membrane

• Blocking and antibody incubation:

  • Block membrane using 5% bovine serum albumin and 0.1% Tween 20 in Tris-buffered saline (TBST)

  • For monoclonal HK3 antibody (67803-1-Ig): Use dilution ratio of 1:5000-1:50000

  • For polyclonal HK3 antibody (HPA056743): Use concentration of 0.04-0.4 μg/mL

• Detection and visualization:

  • Use appropriate HRP-conjugated secondary antibodies

  • Visualize with standard chemiluminescence detection systems

• Expected results:

  • HK3 typically appears as a band at approximately 99 kDa

  • Validated positive controls include Raji cells, Ramos cells, RAW 264.7 cells, human saliva, and rat spleen tissue

Note that optimization may be required depending on specific experimental conditions and sample types.

What protocols are recommended for immunohistochemistry (IHC) with HK3 antibodies?

Based on the search results, the following protocol is recommended for IHC applications with HK3 antibodies:

• Tissue preparation:

  • Fix tissues in formalin and embed in paraffin blocks

  • Section tissues into 4 μm thickness slices

• Antigen retrieval:

  • Preferred method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Antibody dilutions:

  • For monoclonal antibody (67803-1-Ig): Use 1:500-1:2000 dilution

  • For polyclonal antibody (13333-1-AP): Use 1:20-1:200 dilution

  • For polyclonal antibody (HPA056743): Use 1:1000-1:2500 dilution

• Detection system:

  • Visualize using horseradish peroxidase diaminobenzidine (HRP-DAB) immunostaining

  • For fluorescence-based detection, use appropriate fluorescence-labeled secondary antibodies followed by DAPI nuclear staining

• Validated positive controls:

  • Human lung cancer tissue has been validated for positive HK3 detection

Researchers should note that optimal dilutions may vary depending on tissue type and specific experimental conditions, so preliminary titration experiments are recommended.

How can researchers validate the specificity of HK3 antibodies in their experimental systems?

To ensure the specificity and reliability of HK3 antibody detection, researchers should implement the following validation strategies:

• Positive and negative control samples:

  • Use validated positive controls such as Raji cells, Ramos cells, RAW 264.7 cells, human lung cancer tissue, human saliva, or rat spleen tissue

  • Include negative controls (tissues known not to express HK3 or where HK3 has been knocked down)

• Knockdown/knockout validation:

  • Perform siRNA or CRISPR-mediated HK3 knockdown/knockout experiments

  • Compare antibody signal between wild-type and HK3-depleted samples

• Orthogonal validation:

  • Verify HK3 expression using orthogonal methods such as qRT-PCR or RNA-seq

  • Some antibodies, like HPA056743, have been validated using orthogonal RNAseq approaches

• Multiple antibody validation:

  • Confirm results using different HK3 antibodies targeting distinct epitopes

  • Compare monoclonal (e.g., 67803-1-Ig) and polyclonal (e.g., 13333-1-AP) antibodies to exclude epitope-specific artifacts

• Recombinant protein controls:

  • Use purified recombinant HK3 as a positive control

  • Consider using HK3 fusion proteins such as those used for immunogen generation (e.g., Ag30798, Ag4148)

These validation approaches help ensure that experimental findings are not compromised by antibody cross-reactivity or non-specific binding.

How does HK3-mediated O-GlcNAcylation of EP300 contribute to tumor immune evasion?

Recent research has uncovered a novel mechanism connecting glycolysis and immune evasion in clear cell renal cell carcinoma (ccRCC) through HK3-mediated O-GlcNAcylation of EP300 .

Key findings include:

• Mechanism of action:

  • HK3 maintains EP300 protein stability by regulating O-GlcNAcylation levels in ccRCC cells

  • O-GlcNAcylation of EP300 at Ser900 enhances its stability

  • Stabilized EP300 works with TFAP2A as a co-transcription factor to promote PD-L1 transcription

  • EP300 also functions as an acetyltransferase to stabilize PD-L1 protein

• Immunological consequences:

  • Increased PD-L1 expression contributes to tumor immune evasion by inhibiting T-cell cytotoxicity

  • Inhibition of HK3 leads to reduced PD-L1 expression, which restores T-cell cytotoxicity both in vitro and in immunocompetent mice

• Microenvironment interactions:

  • ccRCC exhibits interactive dynamics with tumor-associated macrophages (TAMs)

  • UDP-GlcNAc serves as a critical substrate for O-GlcNAcylation and facilitates TAMs polarization

  • HK3 expression in ccRCC cells is influenced by IL-10 secreted by M2 TAMs

This newly identified mechanism suggests targeting HK3 could be a promising strategy for overcoming immune evasion in cancer, potentially enhancing immunotherapy efficacy by restoring T-cell functions through downregulation of PD-L1.

What is the relationship between HK3 expression and macrophage polarization in the tumor microenvironment?

The relationship between HK3 expression and macrophage polarization represents a complex bidirectional interaction within the tumor microenvironment, as evidenced by recent research .

Key aspects of this relationship include:

• Influence of macrophages on HK3 expression:

  • M2 tumor-associated macrophages (TAMs) secrete IL-10 that influences HK3 expression in tumor cells

  • This suggests a feedback loop where TAMs can modulate metabolic programming in tumor cells

• HK3's role in macrophage polarization:

  • HK3 is involved in producing UDP-GlcNAc, a critical substrate for O-GlcNAcylation processes

  • UDP-GlcNAc has been shown to facilitate TAM polarization toward the immunosuppressive M2 phenotype

• Markers of macrophage polarization:

  • M2 phenotype is characterized by CD206 and CD163 expression

  • M1 phenotype is characterized by NOS2 and CD86 expression

• Clinical relevance:

  • High expression of HK3 correlates with immune infiltration patterns in tumors

  • This correlation suggests HK3 may play a role in shaping the immune landscape of tumors

• Potential therapeutic implications:

  • Targeting HK3 could potentially repolarize macrophages from M2 to M1 phenotype

  • This repolarization could enhance anti-tumor immune responses and potentially improve immunotherapy outcomes

This relationship highlights how metabolic enzymes like HK3 extend beyond their classical roles in energy metabolism to influence immune cell function and phenotype in the tumor microenvironment.

How does HK3 expression correlate with response to PD-1 inhibitor therapy?

Analysis of clinical data has revealed significant correlations between HK3 expression and response to PD-1 inhibitor therapy, particularly with pembrolizumab (Keytruda) :

• Predictive value:

  • HK3 expression shows a remarkable trend in predicting the efficacy of immunotherapy for patients receiving PD-1 monoclonal antibody treatment

  • This correlation suggests HK3 could potentially serve as a biomarker for patient selection in immunotherapy

• Molecular mechanisms:

  • HK3-mediated O-GlcNAcylation of EP300 promotes PD-L1 expression at both transcriptional and protein levels

  • This mechanism provides a direct link between HK3 activity and the PD-1/PD-L1 immune checkpoint pathway

  • Inhibition of HK3 leads to reduced PD-L1 expression, potentially enhancing T-cell cytotoxicity

• Clinical implications:

  • Cases with low HK3 expression were associated with more malignant entities and frequent genomic aberrations of driver oncogenes

  • This suggests that while low HK3 may indicate more aggressive disease, it might also predict different patterns of response to immunotherapy

• Future research directions:

  • Validation in larger clinical cohorts is needed

  • Investigation of combination strategies targeting both HK3 and immune checkpoints

  • Exploration of HK3 as a therapeutic target to enhance immunotherapy efficacy

These findings highlight the potential of HK3 as both a predictive biomarker and therapeutic target in the context of cancer immunotherapy.

What are common challenges when using HK3 antibodies in IHC and how can they be resolved?

Researchers may encounter several challenges when using HK3 antibodies for immunohistochemistry. Here are common issues and their solutions:

• High background staining:

  • Problem: Non-specific binding leading to high background

  • Solutions:

    • Optimize blocking conditions (increase BSA concentration to 5%)

    • Titrate antibody concentration (start with recommended dilutions: 1:500-1:2000 for monoclonal, 1:20-1:200 for polyclonal)

    • Include additional washing steps with TBST

    • Consider using animal-free blockers if animal serum causes background

• Weak or no signal:

  • Problem: Insufficient antigen detection

  • Solutions:

    • Optimize antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

    • Increase antibody concentration or incubation time

    • Use signal amplification systems (e.g., tyramide signal amplification)

    • Ensure tissue fixation is not excessive (overfixation can mask epitopes)

• Variable staining across tissue sections:

  • Problem: Inconsistent results between samples

  • Solutions:

    • Standardize tissue processing protocols

    • Use automated staining platforms if available

    • Include positive controls (e.g., human lung cancer tissue)

    • Prepare larger volumes of working antibody solutions to minimize batch effects

• Edge artifacts:

  • Problem: Stronger staining at tissue edges

  • Solutions:

    • Ensure adequate fixation of tissues

    • Apply hydrophobic barrier around tissue sections

    • Increase washing volume and ensure entire slide is submerged

• Cross-reactivity with other hexokinase isoforms:

  • Problem: Potential false positive signal from HK1, HK2, or HK4

  • Solutions:

    • Use monoclonal antibodies with validated specificity for HK3

    • Perform parallel staining with antibodies against other hexokinase isoforms

    • Include HK3 knockout/knockdown controls when possible

When troubleshooting, methodically change one variable at a time and document all modifications to identify optimal conditions for specific experimental systems.

How can researchers optimize Western blot protocols for detecting low-abundance HK3 in different tissue types?

Detecting low-abundance HK3 in Western blot applications requires careful optimization. Based on the search results and best practices, here are strategies for enhancing HK3 detection:

• Sample enrichment strategies:

  • Increase total protein loading (up to 50-60 μg per lane)

  • Use tissues with known higher HK3 expression (e.g., Raji cells, Ramos cells, RAW 264.7 cells) as positive controls

  • Consider subcellular fractionation to concentrate HK3 (primarily cytoplasmic)

  • Use immunoprecipitation to enrich HK3 before Western blot

• Protein extraction optimization:

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • Include phosphatase inhibitors to prevent dephosphorylation

  • Add N-acetylglucosamine (GlcNAc) and O-GlcNAcase inhibitors if studying O-GlcNAcylated forms

  • Consider sonication to improve extraction efficiency

• Transfer and detection optimization:

  • Use wet transfer methods for high molecular weight proteins like HK3 (99 kDa)

  • Extend transfer time or reduce voltage for more complete transfer

  • Use PVDF membranes instead of nitrocellulose for higher protein binding capacity

  • Apply enhanced chemiluminescence (ECL) substrate with extended exposure times

  • Consider using highly sensitive detection systems (e.g., femto-level ECL substrates)

• Antibody optimization:

  • Use highly sensitive monoclonal antibody (67803-1-Ig) at optimized dilutions (1:5000 initially, adjust as needed)

  • Extend primary antibody incubation to overnight at 4°C

  • Use concentration-matched secondary antibodies

  • Consider using signal amplification systems

• Tissue-specific considerations:

  • For lymphoid tissues: Use gentler lysis buffers to preserve protein integrity

  • For highly glycolytic tissues: Include additional wash steps to reduce background

  • For muscle or fibrous tissues: Extend homogenization time for complete lysis

These optimization strategies should be implemented systematically, changing one variable at a time to identify the most effective protocol for each specific tissue type and experimental context.

What controls should be included when studying HK3-mediated O-GlcNAcylation in experimental systems?

When investigating HK3-mediated O-GlcNAcylation, researchers should include several critical controls to ensure experimental validity and interpretability:

• Positive and negative controls for HK3 expression:

  • Positive control: Cell lines with confirmed high HK3 expression (e.g., Raji cells, Ramos cells)

  • Negative control: HK3 knockdown or knockout cells generated using siRNA or CRISPR-Cas9

  • Expression control: Cells transfected with HK3 expression vectors for overexpression studies

• O-GlcNAcylation detection controls:

  • Use anti-O-GlcNAc antibodies (e.g., Cell Signaling Technology #9875) as used in published studies

  • Include samples treated with O-GlcNAcase inhibitors (e.g., Thiamet G) as positive controls

  • Include samples treated with OGT inhibitors (e.g., OSMI-1) as negative controls

  • Use tissues/cells known to have high O-GlcNAcylation levels as reference standards

• Substrate-specific controls for EP300 O-GlcNAcylation:

  • Wild-type EP300 protein expression

  • EP300 Ser900 mutant (e.g., S900A) to confirm site-specificity

  • Co-immunoprecipitation controls to verify HK3-EP300 interaction

  • Functional readouts of EP300 activity (e.g., acetyltransferase activity assays)

• Metabolic controls:

  • Glucose deprivation to reduce UDP-GlcNAc availability

  • Glucosamine supplementation to increase UDP-GlcNAc pool

  • Alternative hexokinase inhibition/expression to determine HK3-specificity

  • Assessment of UDP-GlcNAc levels using metabolomics approaches

• Downstream pathway controls:

  • PD-L1 expression measurements (the downstream target of EP300)

  • T-cell cytotoxicity assays with and without HK3 manipulation

  • Macrophage polarization markers (CD206, CD163, NOS2, CD86)

  • IL-10 stimulation/blockade to assess the feedback loop with TAMs

Including these comprehensive controls will ensure that observed effects can be specifically attributed to HK3-mediated O-GlcNAcylation rather than to other confounding factors or non-specific effects.

How can HK3 antibodies be used to study the metabolic reprogramming of immune cells in the tumor microenvironment?

HK3 antibodies offer valuable tools for investigating the metabolic reprogramming of immune cells in the tumor microenvironment through several advanced experimental approaches:

• Multiplex immunofluorescence (mIF) assays:

  • Combine HK3 antibodies with immune cell markers (e.g., CD8 for T cells, CD68 for macrophages)

  • Use anti-O-GlcNAc antibodies in conjunction with HK3 to assess glycosylation patterns

  • Include metabolic markers (e.g., GLUT1, PKM2) to build comprehensive metabolic profiles

  • Analyze spatial relationships between HK3-expressing cells and immune cells in the tumor microenvironment

• Flow cytometry applications:

  • Perform intracellular staining for HK3 in isolated tumor-infiltrating immune cells

  • Combine with surface markers for immune cell identification and activation status

  • Use fluorescent glucose analogs (e.g., 2-NBDG) to correlate HK3 expression with glucose uptake

  • Sort HK3-high versus HK3-low immune populations for functional assays

• Single-cell analysis:

  • Integrate HK3 antibody staining with single-cell RNA sequencing

  • Perform cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) with HK3 antibodies

  • Correlate HK3 protein levels with metabolic gene expression signatures at single-cell resolution

  • Map metabolic heterogeneity across immune cell subpopulations

• Ex vivo functional assays:

  • Isolate tumor-associated macrophages and measure HK3 expression in M1 versus M2 phenotypes

  • Assess how HK3 inhibition affects macrophage polarization and cytokine production

  • Evaluate T cell effector functions (cytokine production, cytotoxicity) in relation to HK3 expression

  • Study how glucose availability affects HK3 expression and function in isolated immune cells

• In vivo imaging applications:

  • Develop fluorescently-labeled HK3 antibodies for intravital microscopy

  • Track HK3 expression dynamically during immune cell recruitment to tumors

  • Correlate with metabolic imaging techniques (e.g., hyperpolarized MRI with 13C-pyruvate)

These approaches leverage HK3 antibodies to provide insights into how metabolic reprogramming influences immune cell function in the tumor microenvironment, potentially identifying new targets for immunometabolic cancer therapies.

What are the implications of HK3 expression for biomarker development in immunotherapy patient selection?

The correlations between HK3 expression and immunotherapy response suggest significant potential for biomarker development with several important implications:

• Predictive biomarker potential:

  • Studies have shown HK3 expression correlates with response to PD-1 inhibitor therapy (Keytruda)

  • This correlation suggests HK3 could complement existing biomarkers like PD-L1 expression and tumor mutational burden

  • Integration into multi-parameter predictive models could enhance patient selection accuracy

• Technical considerations for clinical implementation:

  • IHC-based detection using validated antibodies (e.g., 67803-1-Ig, 13333-1-AP) offers practical implementation in clinical pathology workflows

  • Standardized scoring systems would need to be developed (e.g., H-score, percentage positive cells)

  • Quality control measures including positive controls (e.g., human lung cancer tissue) would be essential

• Biological rationale for predictive value:

  • Mechanistic link between HK3, O-GlcNAcylation of EP300, and PD-L1 expression provides biological plausibility

  • HK3's role in macrophage polarization suggests it may indicate broader immune landscape features beyond just PD-L1 expression

  • Correlation with genomic aberrations of driver oncogenes suggests potential integration with genomic biomarkers

• Therapeutic targeting implications:

  • HK3 inhibition could potentially enhance immunotherapy efficacy by reducing PD-L1 expression

  • Combined HK3 assessment and targeting could lead to personalized combinatorial approaches

  • Monitoring HK3 expression during treatment might provide insights into acquired resistance mechanisms

• Current limitations and research needs:

  • Larger prospective clinical validation studies are needed

  • Standardization of detection methods across laboratories

  • Understanding the impact of intratumoral heterogeneity of HK3 expression

  • Determining optimal cutoff values for high versus low HK3 expression

Development of HK3 as a biomarker would require thorough analytical and clinical validation, but the existing mechanistic and correlative evidence provides a strong foundation for further investigation in this direction.