Plxdc1 Antibody

What is PLXDC1 and what is its role in cancer research?

PLXDC1 (plexin domain containing 1) is a protein that has emerged as an important biomarker in cancer research, particularly in gastric cancer. The gene ID for PLXDC1 is 57125 according to NCBI, with the corresponding GenBank Accession Number NM_020405 . Research has revealed that PLXDC1 expression is closely associated with angiogenesis (the formation of new blood vessels) .

In cancer research, PLXDC1 has garnered significant attention because it is frequently overexpressed in gastric cancer tissues compared to normal tissues . This overexpression has been linked to poor prognosis in gastric cancer patients, making it a valuable prognostic biomarker . Additionally, multivariate Cox analysis has demonstrated that PLXDC1 could serve as an independent biomarker for assessing gastric cancer risk . Beyond its prognostic value, PLXDC1 appears to play roles in immune modulation within the tumor microenvironment, with high expression correlating with immune evasion phenotypes in gastric cancer .

How is PLXDC1 antibody used in experimental procedures?

PLXDC1 antibody is utilized in various experimental procedures to detect and quantify PLXDC1 protein expression. According to the search results, PLXDC1 antibody (such as 20051-1-AP) can be used in ELISA (Enzyme-Linked Immunosorbent Assay) applications with demonstrated reactivity against human samples .

For immunohistochemical staining, the protocol typically involves:

• Fixing samples in 4% paraformaldehyde

• Embedding in paraffin

• Sectioning to 4 μm thickness

• Adhering to slides

After de-affinity treatment with xylene and rehydration, antigen retrieval is performed using citric acid buffer (pH 7.8, 0.1 M) at approximately 82°C for 24 minutes . Endogenous peroxidase activity is blocked by incubating sections with peroxidase blocking solution for 15 minutes at room temperature . The sections are then incubated with PLXDC1 primary antibody overnight at 4°C, followed by washing with phosphate-buffered saline . Subsequent incubation with biotin-conjugated secondary antibody for 10 minutes and streptavidin peroxidase for 5 minutes completes the procedure before hematoxylin counterstaining .

What tissue types show reactivity with PLXDC1 antibody?

Based on the available search results, PLXDC1 antibody has demonstrated reactivity specifically with human samples . In cancer research, PLXDC1 antibody has been effectively used to detect PLXDC1 protein expression in gastric cancer tissues .

The research indicates that PLXDC1 is overexpressed in gastric cancer tissues compared to normal gastric tissues, which can be detected using immunohistochemical staining with PLXDC1 antibody . The differential expression between cancerous and normal tissues makes PLXDC1 antibody particularly valuable for studying gastric cancer progression.

While the search results primarily focus on gastric cancer tissues, the correlation analysis between PLXDC1 expression and immune cell infiltration was conducted across multiple cancer types (pan-cancer), suggesting that PLXDC1 antibody may be applicable for studying various cancer tissues .

What experimental techniques can detect PLXDC1 expression in tissue samples?

Several experimental techniques can be employed to detect PLXDC1 expression in tissue samples:

• Immunohistochemical (IHC) Staining: The search results describe a detailed protocol for detecting PLXDC1 in paraffin-embedded tissue sections using rabbit anti-human PLXDC1 antibody, followed by HRP-coupled anti-rabbit secondary antibody . This technique allows for visualization of PLXDC1 expression patterns within tissue architecture and assessment of staining intensity.

• ELISA (Enzyme-Linked Immunosorbent Assay): PLXDC1 antibody (20051-1-AP) has been validated for ELISA applications with human samples , enabling quantitative measurement of PLXDC1 protein levels in tissue lysates or other biological samples.

• Database Analysis: The research utilized bioinformatics approaches with data from the Oncomine database and TCGA (The Cancer Genome Atlas) to analyze PLXDC1 expression across different tissues and cancer types .

• Paired and Unpaired Differential Analysis: Using the "limma" R package, researchers conducted statistical analysis to compare PLXDC1 expression between gastric cancer tissues and normal tissues, providing quantitative assessment of expression differences .

What is the calculated molecular weight of PLXDC1 protein?

According to the product information provided in the search results, the calculated molecular weight of PLXDC1 protein is 56 kDa (kilodaltons) . This molecular weight information is essential for researchers conducting protein detection experiments such as Western blotting, where the appearance of bands at the expected molecular weight helps confirm antibody specificity.

The calculated molecular weight represents the theoretical mass based on the amino acid sequence of the protein and may differ slightly from the apparent molecular weight observed in experimental procedures like SDS-PAGE due to post-translational modifications such as glycosylation, phosphorylation, or proteolytic processing.

When working with PLXDC1 antibodies, researchers should verify that detected bands align with this expected molecular weight, while also considering potential variations due to these post-translational modifications or alternative splicing that might result in different isoforms of the protein.

How does PLXDC1 expression correlate with immune cell infiltration in the tumor microenvironment?

PLXDC1 expression demonstrates significant correlations with immune cell infiltration in the tumor microenvironment, particularly in gastric cancer. Analysis revealed that PLXDC1 expression positively correlates with the infiltration of multiple immune cell types across various cancers (pan-cancer), as demonstrated in heatmap analyses .

In gastric cancer specifically, PLXDC1 expression shows significant positive correlations with numerous immune cell populations, including:

Notably, despite the increased immune cell infiltration associated with high PLXDC1 expression, patients with elevated PLXDC1 levels demonstrated poorer prognosis, suggesting functional impairment of these immune cells .

Further analysis revealed that PLXDC1 expression was significantly associated with different gastric cancer subtypes (C1/2/3/4/6), with higher expression observed in the stromal subtype (C6) compared to immune subtypes (C2/3/4) . This pattern supports the hypothesis that increased stromal cell content in high PLXDC1 expression environments may inhibit immune cell function, leading to an immune evasion phenotype in gastric cancer .

What are the best practices for immunohistochemical staining of PLXDC1 in gastric cancer samples?

Based on the methodologies described in the search results, the best practices for immunohistochemical staining of PLXDC1 in gastric cancer samples include:

Tissue Preparation:

• Fix tissue samples in 4% paraformaldehyde

• Embed samples in paraffin

• Section tissues to 4 μm thickness

• Adhere sections to slides

Deparaffinization and Antigen Retrieval:

• Remove paraffin using xylene gradient treatment

• Rehydrate the sections

• Perform antigen retrieval using citric acid buffer (pH 7.8, 0.1 M) at approximately 82°C for 24 minutes

Blocking and Antibody Incubation:

• Block endogenous peroxidase activity by covering sections with blocking solution for 15 minutes at room temperature

• Incubate with PLXDC1 primary antibody (such as rabbit anti-human PLXDC1 antibody) overnight at 4°C

• Wash gently with phosphate-buffered saline

• Incubate with biotin-conjugated secondary antibody for 10 minutes at room temperature

• Incubate with streptavidin peroxidase for 5 minutes

Staining and Visualization:

• Stain all sections with hematoxylin for counterstaining

• Clean and dry the sections

• Have a pathologist assess staining intensity under a microscope

For comprehensive analysis of the tumor microenvironment, researchers may also perform additional staining on serial sections for immune cell markers (e.g., CD8+, CD3+, CD4+) to correlate PLXDC1 expression with immune cell distribution .

How can researchers validate the specificity of PLXDC1 antibody in their experimental systems?

Researchers can implement several approaches to validate the specificity of PLXDC1 antibody in their experimental systems:

Western Blot Validation:

• Run protein extracts from tissues or cell lines known to express PLXDC1

• Confirm that the antibody detects a band at the expected molecular weight of 56 kDa

• Include both positive and negative control samples

• Consider using reducing and non-reducing conditions to evaluate antibody performance

Peptide Competition Assay:

• Pre-incubate the PLXDC1 antibody with its immunizing peptide before application

• Run parallel experiments with blocked and unblocked antibody

• Specific staining should be eliminated or significantly reduced in the peptide-blocked condition

Knockdown/Knockout Controls:

• Generate or obtain cells with PLXDC1 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9)

• Compare staining between wild-type and knockdown/knockout samples

• Specific antibodies should show reduced or absent signal in knockdown/knockout samples

Cross-Validation with Multiple Antibodies:

• Use antibodies from different manufacturers or those targeting different epitopes of PLXDC1

• Compare staining patterns to ensure consistency

• Converging results from multiple antibodies increase confidence in specificity

Correlation with mRNA Expression:

• Compare PLXDC1 protein detection with mRNA expression data (e.g., from RT-PCR or RNA-seq)

• Samples with higher mRNA levels should generally show higher protein expression

Purified antibodies, such as the antigen affinity-purified PLXDC1 antibody mentioned in the search results , generally offer higher specificity than unpurified antibodies, but validation remains essential for reliable research outcomes.

What signaling pathways are associated with PLXDC1 expression in the tumor microenvironment?

PLXDC1 expression in the tumor microenvironment is associated with several important signaling pathways, particularly those related to immune response and stromal activation. According to the search results, Hallmark pathway gene set analysis revealed that high PLXDC1 expression in gastric cancer is associated with:

Immune Activation Pathways:

• Interferon α/β response

• Allograft rejection signaling

• IL6-JAK-STAT3 signaling

• IL2-STAT5 signaling

• Inflammatory response

• TNFα-NFκB signaling

Stromal Activation Pathways:

• Epithelial-mesenchymal transition (EMT) signaling

• TGF-β signaling

• Angiogenesis

The dual activation of both immune and stromal signaling pathways creates a complex tumor microenvironment. Despite the apparent immune activation associated with high PLXDC1 expression, the simultaneous activation of stromal pathways appears to contribute to an immune evasion phenotype . This is characterized by the accumulation of immune cells in the stroma surrounding the tumor, with limited ability of these cells to penetrate the tumor parenchyma .

The positive correlation between PLXDC1 expression and multiple immune cell types, combined with its association with poor prognosis, suggests that PLXDC1 may contribute to the development of an immunosuppressive microenvironment through these signaling cascades .

What approaches can be used to study the functional relationship between PLXDC1 expression and immune evasion?

To study the functional relationship between PLXDC1 expression and immune evasion, researchers can employ several complementary approaches:

Computational Analysis:

• Utilize the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to assess immune evasion potential in PLXDC1-high vs. PLXDC1-low samples

• Perform GSVA (Gene Set Variation Analysis) and ssGSEA (single-sample Gene Set Enrichment Analysis) to identify enriched pathways and immune cell distributions

• Apply the ESTIMATE algorithm to evaluate immune cell scores, stromal scores, and tumor purity in relation to PLXDC1 expression

Immunohistochemical Analysis:

• Conduct multiplex immunohistochemistry for PLXDC1 alongside immune cell markers (CD8+, CD3+, CD4+) to evaluate spatial distribution

• Assess the pattern of immune cell localization (tumor center vs. invasive margin vs. surrounding stroma) in relation to PLXDC1 expression

• Quantify immune cell density and proximity to tumor cells in regions with varying PLXDC1 expression

In Vitro Studies:

• Establish cell lines with modulated PLXDC1 expression (overexpression, knockdown, knockout)

• Conduct co-culture experiments with various immune cell populations to assess functional interactions

• Measure cytokine/chemokine production profiles in PLXDC1-high vs. PLXDC1-low conditions

Translational Research:

• Analyze patient samples before and after immunotherapy to correlate PLXDC1 expression with treatment response

• Develop Cox risk models incorporating PLXDC1-associated immunomodulators to predict immunotherapy outcomes

• Test PLXDC1-targeting strategies in combination with immune checkpoint inhibitors

These multifaceted approaches would provide comprehensive insights into how PLXDC1 contributes to immune evasion mechanisms and potentially identify strategies to overcome this resistance.

How can conflicting data regarding PLXDC1 expression in different tumor types be reconciled?

Reconciling conflicting data regarding PLXDC1 expression across different tumor types requires a systematic approach that addresses various sources of variability:

Methodological Standardization:

• Ensure consistent antibody validation across studies using the techniques outlined earlier

• Standardize tissue processing protocols, as variations in fixation and antigen retrieval can affect detection sensitivity

• Adopt quantitative scoring systems rather than subjective assessments to reduce observer bias

Contextualization of Results:

• Recognize that PLXDC1 expression may vary by cancer type, subtype, and stage

• Consider the molecular classification of tumors (e.g., immune, stromal, proliferative subtypes) when comparing expression data

• Analyze PLXDC1 in the context of the broader tumor microenvironment rather than in isolation

Integration of Multi-Omics Data:

• Correlate protein expression with mRNA levels to identify post-transcriptional regulation

• Incorporate genomic data to identify mutations or copy number alterations affecting PLXDC1

• Analyze epigenetic modifications that might influence PLXDC1 expression variability

Meta-Analysis Approaches:

• Conduct systematic reviews and meta-analyses of published data with clear inclusion criteria

• Apply random-effects models to account for between-study heterogeneity

• Perform subgroup analyses based on tumor type, detection method, or other relevant factors

By systematically addressing these factors, researchers can develop a more nuanced understanding of PLXDC1 expression patterns across cancer types and reconcile apparently conflicting data within a coherent biological framework.

What technical considerations exist for developing PLXDC1-targeted cancer therapeutics?

Developing PLXDC1-targeted cancer therapeutics involves several important technical considerations:

Target Validation:

• Confirm the causal role of PLXDC1 in cancer progression beyond correlation studies

• Validate PLXDC1 as a driver rather than a passenger in tumor development

• Determine whether PLXDC1 inhibition affects cancer cell survival, proliferation, or immune evasion

Expression Profiling:

• Establish comprehensive expression profiles across normal and malignant tissues

• Identify cancer types with highest PLXDC1 overexpression for prioritized targeting

• Determine potential patient stratification biomarkers for PLXDC1-targeted therapy

Therapeutic Modality Selection:

• Evaluate various therapeutic approaches:

  • Monoclonal antibodies targeting PLXDC1

  • Small molecule inhibitors of PLXDC1 signaling

  • Antibody-drug conjugates for targeted delivery

  • CAR-T cells or other cellular therapies directed against PLXDC1

  • RNA interference or antisense oligonucleotides to downregulate PLXDC1

Combination Strategy Development:

• Given PLXDC1's role in immune evasion, investigate combinations with immunotherapeutics

• Explore synergies with anti-angiogenic agents given PLXDC1's association with angiogenesis

• Consider combinations with conventional chemotherapy or targeted therapies

Potential Resistance Mechanisms:

• Anticipate compensatory pathway activation upon PLXDC1 inhibition

• Monitor for potential upregulation of alternative angiogenic factors

• Develop strategies to address tumor escape mechanisms observed with other anti-angiogenic therapies

The development of PLXDC1-targeted therapeutics represents a promising but challenging opportunity, particularly given its dual role in angiogenesis and immune modulation .