Dhodh Antibody

What is DHODH and why is it relevant to cancer research?

DHODH (dihydroorotate dehydrogenase) is a 43 kDa mitochondrial enzyme essential for de novo pyrimidine biosynthesis. It catalyzes the conversion of dihydroorotate to orotate, a rate-limiting step in pyrimidine nucleotide synthesis. DHODH has gained substantial research interest because inhibitors targeting this enzyme have demonstrated robust anticancer activity across diverse cancer types . The relevance of DHODH in cancer research stems from its metabolic functions that support rapid proliferation in cancer cells and its emerging role in immune regulation. Recent studies indicate that DHODH inhibition not only affects cancer cell metabolism but also enhances antigen presentation and immune recognition, positioning it as a potential dual-action target for both direct antitumor effects and immune modulation .

What applications can DHODH antibodies be used for in experimental research?

DHODH antibodies have been validated for multiple experimental applications in research settings:

These applications enable researchers to investigate DHODH expression, localization, and interactions across various experimental models . For optimal results, antibody titration should be performed in each specific testing system to determine optimal concentration for the particular experimental setup and sample type .

How do I determine the appropriate sample preparation method for DHODH antibody-based detection?

Sample preparation methods vary depending on the application and tissue/cell type. For formalin-fixed paraffin-embedded (FFPE) tissues used in IHC applications, antigen retrieval is crucial. DHODH antibody detection typically requires either TE buffer pH 9.0 or alternative antigen retrieval with citrate buffer pH 6.0 . For protein extraction in WB applications, standard cell lysis buffers containing protease inhibitors are generally sufficient, but optimization may be required for mitochondrial proteins like DHODH. For immunofluorescence, fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.3% Triton X-100 is typically effective for accessing the mitochondrial-localized DHODH. Storage buffer composition (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3) should be taken into account when designing experiments, particularly when considering antibody stability and potential interference with downstream applications .

How can DHODH antibodies be used to study the relationship between pyrimidine metabolism and immune responses in cancer?

DHODH antibodies serve as valuable tools for investigating the mechanistic link between pyrimidine metabolism and immune responses. Recent research has revealed that DHODH inhibition induces robust upregulation of antigen presentation pathway (APP) genes and increases tumor cell antigen presentation via MHC-I .

Experimentally, researchers can use DHODH antibodies to:

• Perform co-localization studies with mitochondrial markers to assess DHODH activity in relation to cellular metabolic state

• Quantify DHODH expression levels before and after treatment with immunomodulatory agents

• Conduct chromatin immunoprecipitation (ChIP) experiments to investigate transcriptional regulation of DHODH in response to immune stimuli

• Assess correlation between DHODH expression and MHC-I levels across cell populations using flow cytometry

These approaches enable detailed investigation of how DHODH activity influences immune recognition of cancer cells. The understanding that pyrimidine nucleotide depletion can trigger interferon-like responses and enhance antigen presentation opens new research avenues for combining metabolic targeting with immunotherapy approaches.

What methodologies can be used to assess the effects of DHODH inhibition on cell proliferation and migration?

Multiple complementary methodologies can be employed to comprehensively evaluate how DHODH inhibition affects cellular functions:

• RNAi-based knockdown: Transfect cells with siRNA targeting DHODH and validate knockdown efficiency using DHODH antibodies in Western blot. Cell counting using Neubauer chamber can quantify proliferation at different timepoints (e.g., days 3 and 5) .

• EdU proliferation assay: Incorporate EdU (5-ethynyl-2'-deoxyuridine) to measure DNA synthesis as a proliferation indicator. This provides more precise quantification of actively dividing cells compared to simple cell counting .

• Wound healing assay: Create a scratch in a confluent monolayer of cells after DHODH knockdown or inhibition, then monitor wound closure at 24h and 48h intervals. Quantify healing rate using ImageJ software .

• Transwell migration assay: Assess invasive capacity by seeding cells in upper chambers containing serum-reduced medium (2% FBS) with complete medium (10% FBS) in lower chambers. After 24h, fix, stain with crystal violet, and quantify migrated cells .

These methods should be performed in parallel to obtain a comprehensive understanding of how DHODH affects cell behavior beyond simple viability measures.

How can bioinformatics approaches be integrated with DHODH antibody-based experiments to understand cancer immunity?

Integration of bioinformatics with experimental DHODH research provides powerful insights into cancer immunity. A multi-layered approach involves:

• Transcriptomic correlation analysis: Correlate DHODH expression (validated by antibody detection) with immune gene signatures using cancer datasets like TCGA. Single-sample Gene Set Enrichment Analysis (ssGSEA) can quantify enrichment of immune cell signatures in samples grouped by DHODH expression levels .

• Immune infiltration assessment: Analyze correlation between DHODH expression and specific immune cell populations (B cells, CD8+ T cells, macrophages, neutrophils, NK cells, dendritic cells). Visualize relationships using chord diagrams to identify patterns .

• Checkpoint correlation: Investigate potential relationships between DHODH expression and immune checkpoint molecules using antibody validation in experimental models and correlation analysis in patient datasets .

• Antitumor immune response analysis: Examine DHODH's effect on the seven-step cancer-immunity cycle, from antigen release to T-cell killing, using both computational approaches and experimental validation with DHODH antibodies .

This integrated approach bridges computational predictions with experimental validation, providing a more comprehensive understanding of DHODH's role in cancer immunity.

What controls should be included in experiments using DHODH antibodies?

Rigorous control inclusion is essential for experiments employing DHODH antibodies:

Additionally, for flow cytometry experiments, include unstained cells, single-color controls, and fluorescence-minus-one (FMO) controls to establish proper gating strategies, particularly important for intracellular staining protocols required for DHODH detection .

How should DHODH antibodies be optimized for use in multi-parameter flow cytometry?

Optimization of DHODH antibodies for multi-parameter flow cytometry requires several methodological considerations:

• Titration determination: Perform systematic titration experiments starting with manufacturer's recommended dilution (0.20 μg per 10^6 cells) and adjust based on signal-to-noise ratio.

• Fixation and permeabilization protocol selection: Since DHODH is mitochondrial, standard surface staining protocols are insufficient. Test different permeabilization reagents (Triton X-100, saponin, methanol) at varying concentrations to optimize mitochondrial membrane access without compromising epitope recognition.

• Fluorophore selection: Consider spectral overlap with other markers in panel. For mitochondrial proteins, brighter fluorophores (PE, APC) may provide better separation from autofluorescence.

• Compensation controls: Prepare single-stained controls for each fluorophore in your panel using the same cell type and fixation/permeabilization methods.

• FMO controls: Include fluorescence-minus-one controls to establish proper gating strategy, particularly important for distinguishing specific DHODH staining from background.

• Sequential staining consideration: For complex panels, consider whether surface markers should be stained before or after intracellular DHODH staining to preserve epitope integrity.

For co-staining experiments examining DHODH expression alongside immune markers, careful panel design is critical to obtain interpretable results that can validate bioinformatic predictions regarding DHODH's relationship to immune cell infiltration and function .

What are the recommended approaches for using DHODH antibodies in combination with DHODH inhibitors to study mechanism of action?

Integrating DHODH antibodies with inhibitor studies provides mechanistic insights through several methodological approaches:

• Temporal dynamics assessment: Treat cells with DHODH inhibitors (e.g., brequinar) across different timepoints (4h, 12h, 24h, 48h) and use DHODH antibodies to track protein expression, localization, and potential degradation or feedback regulation .

• Dose-response relationship: Combine dose-dependent inhibitor treatment with quantitative DHODH detection via immunoblotting or flow cytometry to correlate inhibitor concentration with target engagement and downstream effects.

• Target confirmation: Use DHODH antibodies to immunoprecipitate the protein from cell lysates after inhibitor treatment, then perform thermal shift assays or limited proteolysis to assess inhibitor binding. This confirms that observed effects result from on-target activity.

• Mechanism differentiation: When studying increased MHC-I expression after DHODH inhibition , combine inhibitor treatment with DHODH immunodetection and pathway component assessment (e.g., STAT1, IRF1) to distinguish between direct inhibition effects and secondary signaling consequences.

• Rescue experiments: Supplement DHODH-inhibited cells with pyrimidines (uridine, cytidine) while monitoring DHODH levels, subcellular localization, and pathway markers to identify which effects are directly attributable to pyrimidine depletion versus potential non-canonical functions.

These approaches enable researchers to definitively link observed phenotypes to DHODH inhibition while avoiding misinterpretation of off-target effects.

What are common challenges in DHODH antibody-based experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with DHODH antibodies:

When encountering reproducibility issues, reviewing all experimental parameters systematically is essential. Storage of DHODH antibodies at -20°C with minimal freeze-thaw cycles is recommended for maintaining reactivity .

How can quantitative analysis of DHODH expression be correlated with functional outcomes in cancer research?

Robust quantitative analysis of DHODH expression requires multiple complementary approaches:

• Expression level quantification: Standardize Western blot quantification using housekeeping protein normalization with densitometry software. For flow cytometry, calculate median fluorescence intensity (MFI) or percent positive cells above isotype control threshold.

• Cross-platform validation: Validate protein expression results with mRNA quantification (qPCR) and correlate with functional protein assays measuring DHODH enzyme activity.

• Survival correlation analysis: Integrate quantified DHODH expression data with patient outcome metrics using Kaplan-Meier analysis and multivariate Cox regression to identify prognostic significance.

• Immune correlation metrics: Analyze relationships between DHODH expression and immune infiltration using Spearman correlation coefficients and visualization techniques like violin plots and chord diagrams .

• Multi-parameter analysis: Employ machine learning approaches to identify patterns between DHODH expression, metabolic parameters, immune markers, and treatment responses. This can reveal non-linear relationships that simple correlation analysis might miss.

• Single-cell analysis: When applicable, combine DHODH antibody-based detection with single-cell techniques to understand expression heterogeneity and its relationship to functional states within tumor microenvironments.

These quantitative approaches provide more rigorous insights than simple qualitative assessments and enable identification of clinically relevant thresholds of DHODH expression.

How can contradictory results between DHODH antibody detection and functional outcomes be reconciled?

Reconciling contradictory results between DHODH protein detection and functional outcomes requires systematic investigation:

• Antibody validation review: Verify antibody specificity through multiple methods (Western blot, immunoprecipitation, knockdown controls) to ensure detected signals truly represent DHODH protein .

• Post-translational modification assessment: Consider that DHODH function may be regulated by modifications not reflected in total protein levels. Investigate phosphorylation, ubiquitination, or other modifications that might affect activity without changing detection.

• Localization versus expression analysis: Distinguish between changes in DHODH localization and absolute expression using subcellular fractionation and immunofluorescence microscopy, as mitochondrial protein distribution can affect function independently of total protein levels.

• Metabolic context evaluation: Assess cellular metabolic state and pyrimidine pools, as functional outcomes may depend on metabolic context rather than DHODH expression alone. Supplementation experiments with pyrimidines can help distinguish between correlation and causation.

• Temporal dynamics consideration: Evaluate whether time-dependent changes explain discrepancies, as protein expression changes may precede or lag behind functional effects.

• Genetic compensation mechanism investigation: Consider potential compensatory pathways that might be activated in response to DHODH manipulation, such as salvage pathways for nucleotide synthesis.

This systematic approach helps distinguish genuine biological complexity from technical artifacts and provides deeper mechanistic insights.

How is DHODH antibody research contributing to our understanding of DHODH as a therapeutic target in T-cell malignancies?

DHODH antibody-based research has been instrumental in revealing the unique vulnerability of T-cell malignancies to DHODH inhibition:

• Differential sensitivity mapping: DHODH antibody-based expression analysis across cancer types has identified T-cell acute lymphoblastic leukemia (T-ALL) as particularly sensitive to DHODH inhibition. This sensitivity appears linked to fundamental differences in nucleotide metabolism in T-cells compared to other cell types .

• Mechanistic pathway elucidation: Immunodetection of DHODH alongside metabolic and signaling components has revealed that T-ALL cells exhibit increased reliance on oxidative phosphorylation when treated with DHODH inhibitors. This metabolic adaptation represents a potential vulnerability distinct from normal T-cells and other hematopoietic cells .

• Normal tissue comparison: DHODH antibody-based studies have enabled comparison between malignant and normal T-cells, demonstrating that DHODH inhibition does not permanently damage the developing thymus in young mice. This finding suggests a potential therapeutic window for targeting T-ALL while sparing normal T-cell development .

• Clinical translation acceleration: The identification of T-ALL sensitivity to DHODH inhibition through rigorous antibody-validated research has accelerated the translation of DHODH inhibitors into clinical trials, building on existing safety data from autoimmune disease applications like rheumatoid arthritis and multiple sclerosis where DHODH inhibitors have already shown clinical benefit .

These advances position DHODH as a promising metabolic target for T-ALL treatment with potential for rapid clinical translation.

What is the relationship between DHODH inhibition and antigen presentation pathways in cancer immunotherapy?

Recent research has uncovered a previously unrecognized connection between DHODH inhibition and enhanced antigen presentation with significant implications for cancer immunotherapy:

• MHC-I upregulation mechanism: DHODH inhibition leads to robust upregulation of antigen presentation pathway (APP) genes and increases tumor cell presentation via MHC-I. This effect appears to be a direct consequence of pyrimidine nucleotide depletion rather than an off-target effect of DHODH inhibitors .

• Synergistic potential with immunotherapy: The enhanced MHC-I expression following DHODH inhibition creates a mechanistic rationale for combination with immune checkpoint inhibitors. In vivo evidence demonstrates additive effects between DHODH inhibitors and checkpoint blockade, suggesting a potential strategy to overcome immunotherapy resistance mechanisms related to antigen presentation defects .

• Interferon-like response: DHODH inhibition triggers expression of innate immunity-related genes and induces an interferon-like response, which likely contributes to the enhanced antigen presentation. This represents a novel metabolic pathway for modulating immune recognition of cancer cells .

• Comprehensive functional validation: Rigorous in vitro mechanistic studies across multiple cell lines have validated this phenomenon, though some researchers note that in vivo assessment of functional relevance requires additional investigation to fully substantiate therapeutic implications .

This emerging research direction offers a promising approach to enhance cancer immunotherapy efficacy through metabolic modulation of antigen presentation.

What novel applications of DHODH antibodies are being developed for cancer research and diagnostics?

Innovative applications of DHODH antibodies are expanding beyond traditional research tools:

• Biomarker development: DHODH expression assessment using validated antibodies is being explored as a potential predictive biomarker for response to both DHODH inhibitors and immunotherapies. Standardized immunohistochemistry protocols are being optimized for potential clinical implementation .

• Patient stratification approaches: Integration of DHODH expression data with immune infiltration analysis is enabling the development of multi-parameter predictive models that may help identify patients most likely to benefit from DHODH-targeted therapies or combination approaches .

• Theranostic applications: Development of DHODH antibody conjugates with imaging agents or therapeutic payloads offers potential for both diagnostic and therapeutic applications, particularly in malignancies with elevated DHODH expression.

• Real-time monitoring technologies: Adaptation of DHODH antibodies for real-time monitoring of metabolic responses to therapy using techniques like proximity ligation assays or antibody-based biosensors could provide dynamic assessment of treatment effects.

• Spatial analysis integration: Combination of DHODH antibody detection with spatial transcriptomics or multiplexed immunofluorescence enables detailed mapping of DHODH expression in relation to immune cell populations within the tumor microenvironment, providing insights into local metabolic-immune interactions.

These emerging applications demonstrate how DHODH antibodies are evolving from basic research tools to potential components of precision medicine approaches for cancer.