ANTXR1 Antibody

What is ANTXR1 and what is its significance in biomedical research?

ANTXR1 (anthrax toxin receptor 1), also known as TEM8 (tumor endothelial marker 8) and ATR, is a type I transmembrane glycoprotein that functions as a tumor-specific endothelial marker. It was initially identified as a receptor for Bacillus anthracis toxin but has since gained significant attention in cancer research . ANTXR1 contains a von Willebrand factor A (vWA) domain responsible for binding to anthrax protective antigen (PA) as well as collagens I and IV . The protein plays crucial roles in cell adhesion, migration, and interaction with the extracellular matrix .

ANTXR1 is particularly significant in cancer research due to its overexpression in multiple malignancies including colorectal, gastric, breast, and pancreatic tumors, making it both a potential biomarker and therapeutic target . Additionally, pathogenic variants in ANTXR1 lead to the rare GAPO syndrome (Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy) .

How do I select the appropriate ANTXR1 antibody for my specific research application?

Selecting the optimal ANTXR1 antibody requires consideration of multiple factors to ensure experimental success:

• Application compatibility: Different antibodies are optimized for specific applications. Review validation data for your intended application (WB, IHC, IF, IP, ELISA).

• Species reactivity: Verify cross-reactivity with your experimental species. Many commercial ANTXR1 antibodies show reactivity with human, mouse, and rat samples .

• Epitope recognition: Consider which region or isoform of ANTXR1 you intend to detect. Some antibodies specifically recognize the C-terminus , while others target different regions. Note that ANTXR1 has multiple isoforms with the 15091-1-AP antibody detecting 80-85 kDa bands .

• Clonality considerations: Polyclonal antibodies (like 18717-1-AP, 15091-1-AP) recognize multiple epitopes and often provide stronger signals, while monoclonal antibodies offer higher specificity .

• Validation evidence: Review published literature and manufacturer validation data, including Western blot images, IHC staining patterns, and KO/KD validation .

Based on published data, here are application-specific antibody recommendations:

Always perform your own validation with appropriate controls to confirm specificity in your experimental system .

What are the optimal protocols for detecting ANTXR1 by Western blotting?

Optimizing Western blot protocols for ANTXR1 detection requires attention to several key parameters:

Sample preparation and protein loading:

• Extract proteins using standard lysis buffers containing protease inhibitors

• Load 20-30 μg of total protein per lane

• Use 8-10% SDS-PAGE gels (appropriate for the ~80 kDa ANTXR1 protein)

Primary antibody conditions:

• Antibody 15091-1-AP: 1:500-1:1000 dilution

• CST ANTXR1/TEM8 Antibody #23494: 1:1000 dilution

• Antibody ABIN499365: 1-2 μg/mL

• Incubate overnight at 4°C in 5% BSA or non-fat milk in TBST

Important considerations:

• Expect bands at 80-85 kDa despite calculated MW of 63 kDa due to glycosylation

• Some antibodies recognize only specific isoforms; 18717-1-AP only detects isoform 1

• Positive controls include HeLa cells, mouse testis tissue, mouse lung tissue, and PC-13 cells

Troubleshooting tips:

• If multiple bands appear, verify specificity with ANTXR1 knockdown/knockout controls

• For weak signals, extend exposure time or consider using more sensitive detection reagents

• For high background, increase washing steps and optimize blocking conditions

The observed molecular weight of ANTXR1 varies between 80-85 kDa in Western blots, which differs from its calculated molecular weight of 63 kDa due to post-translational modifications, particularly glycosylation .

How should I optimize immunohistochemistry protocols for ANTXR1 detection in tissue sections?

Successful immunohistochemical detection of ANTXR1 in tissue sections requires careful optimization:

Tissue preparation:

• Use 4-6 μm sections from formalin-fixed, paraffin-embedded tissues

• Mount on positively charged slides to prevent tissue loss

Antigen retrieval (critical step):

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

• Heat treatment: Water bath or pressure cooker for 20 minutes

Antibody dilution and detection:

• For antibody 15091-1-AP: Start with 1:50-1:100 dilution, then optimize (range: 1:20-1:200)

• For R&D Systems' AF3886: 3 μg/mL has shown good results

• Incubation time: Overnight at 4°C or 1-2 hours at room temperature

• Detection system: HRP-polymer detection systems provide optimal sensitivity

• Chromogen: DAB (3,3'-diaminobenzidine) with 2-10 minutes development time

• Counterstain: Hematoxylin (adjust timing to avoid obscuring ANTXR1 staining)

Expected staining pattern:

• ANTXR1 shows cytoplasmic and plasma membrane localization in positive cells

• Human colon cancer and liver cancer tissues serve as reliable positive controls

Control sections:

• Positive control: Colon cancer tissue, which consistently expresses ANTXR1

• Negative control: Primary antibody omission or isotype control

• Tissue-matched controls to evaluate specific versus background staining

The optimization process should include a dilution series and careful evaluation of signal-to-noise ratio across different conditions. Document all parameters for reproducibility .

What controls are essential when evaluating ANTXR1 expression in experimental systems?

Rigorous experimental design for ANTXR1 studies requires comprehensive controls to ensure data validity:

Essential positive controls:

• Cell lines with confirmed ANTXR1 expression: HeLa cells, certain cancer cell lines

• Tissue samples known to express ANTXR1: Colon cancer, liver cancer tissues

• Recombinant ANTXR1 protein (useful for antibody validation)

• ANTXR1 overexpression systems (transfected cell lines)

Critical negative controls:

• ANTXR1 knockout or knockdown samples (gold standard for specificity)

• Primary antibody omission control (tests for non-specific secondary antibody binding)

• Isotype-matched control antibody at equivalent concentration

• Peptide competition assay (pre-incubation of antibody with immunizing peptide)

Method-specific controls:

• Western blot: Loading controls (β-actin, GAPDH); molecular weight markers

• IHC/IF: Adjacent sections with H&E staining; autofluorescence controls

• qPCR: No-RT controls, reference gene validation

Biological context controls:

• Multiple experimental models (cell lines, primary cells, tissue samples)

• Multiple detection methods (protein and mRNA quantification)

• Functional validation connecting ANTXR1 expression to biological outcomes

When working with ANTXR1 antibodies, it's particularly important to validate specificity due to the presence of multiple isoforms and potential cross-reactivity. The observed molecular weight pattern (typically 80-85 kDa bands despite a calculated 63 kDa size) can serve as an internal validation checkpoint .

How does ANTXR1 expression correlate with cancer progression and patient outcomes?

ANTXR1 expression has emerged as a significant biomarker with prognostic value across multiple cancer types:

Prognostic associations:

Progression indicators:

• ANTXR1 plays roles in tumor angiogenesis, invasion, and metastasis across cancer types

• Overexpression has been documented in gastric, breast, colon, and pancreatic tumors

• High ANTXR1 expression correlates with reduced progression-free survival (PFS) in bladder cancer

Therapeutic relevance:

• Melanoma patients treated with immunotherapy show significant survival differences based on ANTXR1 expression levels

• High ANTXR1 expression tumors demonstrate reduced sensitivity to adjuvant chemotherapy

• ANTXR1 expression correlates inversely with tumor mutation burden (TMB), potentially indicating differential responsiveness to immune checkpoint inhibitors

These findings position ANTXR1 as not only a prognostic biomarker but also as a potential predictor of treatment response. The correlation between ANTXR1 expression and clinical outcomes appears consistent across different cancer types, suggesting its broad utility in oncology research .

What is the relationship between ANTXR1 and the tumor microenvironment?

ANTXR1 plays a complex role in shaping the tumor microenvironment (TME) through multiple mechanisms:

Stromal and immune cell correlations:

• ANTXR1 expression positively correlates with stromal score, immune score, and ESTIMATE score in gastric cancer patients, indicating its influence on the infiltration of stromal and immune cells

• Analysis using the TIMER algorithm revealed positive correlations between ANTXR1 expression and infiltration of CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells

Cell type-specific relationships:

• Advanced analysis using xCell algorithm identified 49 cell types significantly correlated with ANTXR1 expression (30 positively, 19 negatively)

• Stromal cells and myeloid cells show predominantly positive correlations with ANTXR1

• Lymphoid cells generally demonstrate negative correlations with ANTXR1 expression

TME remodeling and therapy implications:

• ANTXR1 contributes to TME remodeling, particularly affecting stromal elements

• This remodeling appears to influence chemotherapy resistance mechanisms

• Patients with low ANTXR1 expression may respond better to immune checkpoint inhibitors due to higher tumor mutation burden

The data suggest ANTXR1 as a key regulator of the TME composition, with potential implications for therapeutic strategies targeting the tumor-stroma interface .

What mechanisms link ANTXR1 to tumor growth and metastasis?

ANTXR1 promotes tumor progression through several interconnected mechanisms:

Angiogenesis regulation:

• ANTXR1 was initially identified as tumor endothelial marker 8 (TEM8) due to its upregulation in tumor vasculature

• It plays a critical role in the angiogenic response of endothelial cells

• This angiogenic function supports tumor growth by facilitating blood vessel formation

Cell adhesion and migration:

• ANTXR1 mediates cell adhesion to type 1 collagen and gelatin through its vWA domain

• It promotes reorganization of the actin cytoskeleton and enhances cell spreading

• These functions facilitate tumor cell migration and invasion into surrounding tissues

Extracellular matrix interactions:

• ANTXR1 functions as a sensor of extracellular mechanical cues (ECM rigidity, hydrostatic pressure)

• It connects the intracellular cytoskeleton with ECM components, similar to integrins

• These interactions may drive epithelial-to-mesenchymal transition in cancer cells

Tumor microenvironment modulation:

• ANTXR1 expression shapes the composition of tumor-infiltrating immune and stromal cells

• High ANTXR1 expression correlates with specific immune infiltration patterns that may support tumor growth

• ANTXR1-mediated TME remodeling contributes to therapy resistance mechanisms

Expression in cancer stem cells:

• ANTXR1 is selectively expressed on cancer stem cells and invasive cancer cells

• This expression pattern suggests roles in maintaining stemness and promoting invasion

• Targeting ANTXR1+ cells potentially addresses tumor-initiating cell populations

Collectively, these mechanisms position ANTXR1 as a multifunctional protein that influences cancer progression through effects on tumor cells themselves, supporting stromal cells, and the extracellular matrix .

How does ANTXR1 function as a mechanosensor in cellular systems?

ANTXR1 has emerged as an important mechanosensor that detects and responds to extracellular mechanical cues:

Structural basis for mechanosensing:

• ANTXR1 shares high structural similarity with integrins, which are canonical mechanosensors

• Like integrins, ANTXR1 contains a von Willebrand factor A (vWA) domain that mediates interactions with the extracellular matrix

• Despite these similarities, ANTXR1's functions and downstream signaling are independent from integrins

Mechanical cue detection:

• Experimental evidence indicates ANTXR1 can sense:

  • Extracellular matrix rigidity

  • Hydrostatic pressure

  • Other mechanical signals in the cellular microenvironment

Mechanotransduction mechanisms:

• ANTXR1 connects intracellular cytoskeleton to ECM molecules, forming a mechanical linkage

• This linkage enables bidirectional force transmission between the cell interior and exterior

• The mechanical coupling regulates cellular processes including adhesion, migration, and ECM homeostasis

Biological significance:

• In tumor biology, ANTXR1's mechanosensing may influence cancer cell migration and invasion

• In normal physiology, it likely contributes to tissue homeostasis and cellular responses to mechanical stimuli

• The mechanosensory function may be particularly relevant in contexts where ECM properties are altered, such as fibrosis or tumor stroma

This mechanosensing role represents a novel aspect of ANTXR1 biology beyond its functions as an anthrax toxin receptor and tumor marker, highlighting its importance in cellular adaptation to mechanical environments .

What is the relationship between ANTXR1 deficiency and GAPO syndrome?

ANTXR1 deficiency causes GAPO syndrome through mechanisms involving cellular senescence and extracellular matrix dysregulation:

GAPO syndrome clinical manifestations:

• Named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy

• Associated with complex phenotypes affecting cardiovascular, skeletal, pulmonary, and nervous systems

• Pathogenic variants in ANTXR1 have been established as the genetic cause

Cellular mechanisms in ANTXR1 deficiency:

• Recent evidence demonstrates that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts

• This senescence correlates with defects in nuclear architecture and actin dynamics

• The findings suggest GAPO syndrome should be reconsidered as a progeroid (premature aging) disorder

Extracellular matrix abnormalities:

• Aberrant accumulation of ECM components and fibrosis are hallmarks of GAPO syndrome

• These ECM abnormalities contribute significantly to disease pathogenesis and reduced life expectancy

• ANTXR1's normal role in ECM homeostasis appears critical for preventing these abnormalities

Aging-related implications:

• ANTXR1 deficiency reveals an unexpected role for this integrin-like ECM receptor in human aging

• The senescence phenotype in ANTXR1-deficient cells provides insights into both normal and pathological aging processes

• This connection to aging biology opens new research directions for understanding ANTXR1 function

This relationship between ANTXR1 and GAPO syndrome highlights the critical importance of ANTXR1 in normal development and tissue homeostasis, extending our understanding beyond its roles in cancer and anthrax toxin entry .

What are current approaches for targeting ANTXR1 in cancer therapy?

ANTXR1 has emerged as a promising target for cancer therapy with multiple targeting strategies under development:

Antibody-drug conjugates (ADCs):

• Anti-TEM8 antibody-drug conjugates have shown promise in preclinical studies across diverse cancer types

• This approach leverages ANTXR1's selective expression on tumor vasculature and cancer cells

• ADCs combine the specificity of anti-ANTXR1 antibodies with cytotoxic payloads

Chimeric antigen receptor (CAR) T-cell therapy:

• Byrd and colleagues developed anti-ANTXR1 CAR-T cells for triple-negative breast cancer

• This approach successfully induced regression of established patient-derived xenograft tumors

• CAR-T targeting leverages the selective expression of ANTXR1 on tumor cells and tumor vasculature

Biomarker-guided therapy selection:

• ANTXR1 expression levels can potentially guide therapy selection:

  • Low ANTXR1 expression correlates with higher tumor mutation burden

  • Patients with low ANTXR1 expression may benefit more from immune checkpoint inhibitors

  • Survival differences based on ANTXR1 expression have been observed in melanoma patients receiving immunotherapy

Rationale for ANTXR1 as a therapeutic target:

• Selective expression pattern: ANTXR1 is expressed on tumor cells, cancer stem cells, and tumor-associated stromal cells

• Limited normal tissue expression reduces potential toxicity

• ANTXR1 is rarely mutated in cancers, providing a stable therapeutic target

• Targeting ANTXR1 may simultaneously affect tumor cells and the supporting microenvironment

The therapeutic targeting of ANTXR1 represents a promising approach that could address multiple aspects of cancer biology simultaneously, potentially overcoming resistance mechanisms associated with other targeted therapies .

How do I resolve issues with varying molecular weights observed for ANTXR1 in Western blots?

Researchers frequently encounter molecular weight variations when detecting ANTXR1 in Western blots. Understanding the sources of these variations is essential for proper data interpretation:

Expected molecular weight patterns:

• Calculated molecular weight: 63 kDa based on amino acid sequence

• Typically observed molecular weights: 80-85 kDa in Western blots

• This discrepancy is primarily due to post-translational modifications

Sources of molecular weight variation:

• Multiple isoforms:

  • ANTXR1 has several alternatively spliced isoforms:

    • Type I transmembrane isoforms: 564 aa (80-85 kDa) and 368 aa (60 kDa)

    • Potentially secreted isoforms: 330 aa and 297 aa (45 kDa)

  • Antibody specificity for particular isoforms varies; 18717-1-AP only recognizes isoform 1

• Post-translational modifications:

  • Glycosylation significantly affects ANTXR1's apparent molecular weight

  • Other modifications (phosphorylation, ubiquitination) may further alter migration

• Tissue/cell-specific differences:

  • Expression patterns of isoforms and extent of modifications vary across cell types

  • Compare your results with positive controls (HeLa cells, mouse testis)

Resolution strategies:

When reporting ANTXR1 Western blot results, always specify the observed molecular weight, antibody used, and experimental conditions to facilitate cross-study comparisons .

How do I interpret conflicting data on ANTXR1 function across different experimental systems?

Resolving conflicting data on ANTXR1 function requires systematic analysis of biological and methodological variables:

Context-dependent function analysis:

• ANTXR1 demonstrates different functions in different cellular contexts

• Example: ANTXR1 correlates positively with myeloid cells but negatively with lymphoid cells

• Cancer type-specific effects: Different relationships with immune infiltration between cancer types

Methodological evaluation:

• Examine antibody epitopes and isoform specificity (different antibodies recognize different isoforms)

• Compare detection methods (WB, IHC, IF, functional assays) which may yield different results

• Evaluate sample preparation differences that might affect ANTXR1 detection or function

Experimental system reconciliation:

• Compare in vitro cell culture vs. in vivo animal models vs. human patient samples

• Consider 2D vs. 3D culture systems which may reveal different ANTXR1 functions

• Analyze genetic background differences that might influence ANTXR1 activity

Integrative analysis approaches:

• Combine data from multiple experimental approaches to build a comprehensive model

• Conduct meta-analysis of published studies to identify consistent patterns

• Use computational methods to integrate diverse datasets and reveal context-specific functions

Design of decisive experiments:

• Create experiments specifically designed to resolve contradictions

• Employ genetic approaches (CRISPR-Cas9, RNA interference) to directly test functional hypotheses

• Include time-course analyses to capture temporal aspects of ANTXR1 function

What factors might affect ANTXR1 detection and expression in experimental models?

Multiple factors can influence ANTXR1 detection and expression in experimental models, requiring careful consideration for accurate data interpretation:

Biological factors affecting ANTXR1 expression:

• Cell type and origin:

  • Expression varies significantly across cell types

  • Cancer cells (particularly colorectal, gastric, breast) typically show higher expression

  • Endothelial cells, especially from tumor vasculature, express significant ANTXR1

• Culture and microenvironmental conditions:

  • Cell density affects expression (ANTXR1 functions in cell-cell interactions)

  • Extracellular matrix composition influences expression (ANTXR1 binds collagens)

  • Oxygen levels may impact expression (association with tumor vasculature)

  • Mechanical forces likely modulate expression (ANTXR1 functions as a mechanosensor)

• Growth phase and cellular state:

  • Cell cycle stage may influence ANTXR1 levels

  • Cellular senescence affects expression (ANTXR1 deficiency promotes senescence)

Methodological factors affecting ANTXR1 detection:

• Antibody selection:

  • Epitope recognition varies between antibodies

  • Some antibodies recognize only specific isoforms

  • Antibody affinity and specificity affect detection sensitivity

• Detection method optimization:

  • Western blot: Sample preparation, protein extraction method, gel percentage

  • IHC/IF: Fixation method, antigen retrieval conditions, detection system

  • Flow cytometry: Cell preparation, antibody concentration, gating strategy

• Controls and validation:

  • Positive controls should be consistent across experiments

  • Genetic manipulation (overexpression, knockdown) to validate specificity

  • Multi-method validation to confirm expression patterns

For reproducible ANTXR1 research, systematically document all experimental conditions and standardize protocols when possible. When comparing results across studies, consider how these factors might contribute to observed differences in ANTXR1 expression or function .