BIRC5 Monoclonal Antibody

What is BIRC5/survivin and what is its significance in cancer research?

BIRC5 (Baculoviral IAP Repeat Containing 5), commonly known as survivin, is a member of the inhibitor of apoptosis (IAP) protein family that plays critical roles in cell cycle regulation and programmed cell death inhibition. In cancer research, BIRC5 has emerged as a significant biomarker due to its overexpression in various malignancies and its correlation with tumor progression and treatment resistance.

Research indicates that BIRC5 is associated with activated cell cycle programs in hepatocellular carcinoma (HCC), where it functions as a potential biomarker and inducer of myeloid-derived suppressor cells (MDSCs) infiltration in the tumor microenvironment. This relationship contributes to T-cell exclusion or dysfunction, resulting in reduced response to immune checkpoint inhibitors (ICIs) . Additionally, BIRC5 has been correlated with cell migration and immune infiltration in low-grade glioma, further highlighting its potential as a prognostic biomarker across multiple cancer types .

In which tissue types is BIRC5 expression most commonly observed?

BIRC5 expression has been documented across multiple tissue types, with particularly notable expression in various cancer tissues. According to published literature and antibody validation studies, BIRC5 expression has been confirmed in:

• Cervix carcinoma

• Lung tissue

• Mammary gland and mammary cancer

• Muscle tissue

• Myeloid leukemia cells

• Vaginal tissue

• Neuroblastoma

• Hepatocellular carcinoma

Research support for these expression patterns comes from multiple studies referenced in antibody validation reports, including publications with PubMed IDs: 18691976, 15489334, 16329164, 14702039, and 14741722 . This diverse expression pattern makes BIRC5 a valuable target for investigation across multiple cancer types.

What validation steps should be taken when using a new BIRC5 antibody for the first time?

When utilizing a BIRC5 antibody for the first time, thorough validation is essential to ensure reliable and reproducible results. The recommended validation approach includes:

• Positive control selection: Use cell lines with known BIRC5 expression such as HeLa, Jurkat, CEM, or Colo320 cell lysates, which have been documented to express BIRC5 at detectable levels .

• Western blot characterization: Perform initial validation using Western blot to confirm specific detection at the expected molecular weight (~16.5 kDa for human BIRC5).

• Cross-reactivity assessment: Test the antibody against samples from different species if cross-reactivity claims are made by the manufacturer.

• Comparative analysis: If possible, compare results with an alternative validated anti-BIRC5 antibody to confirm staining patterns.

• Negative controls: Include appropriate negative controls, such as tissues known not to express BIRC5 or samples treated with blocking peptides.

• Literature comparison: Compare your findings with published literature to verify expected expression patterns in your experimental system.

Following these validation steps ensures confidence in subsequent experimental findings and facilitates accurate interpretation of results across different experimental contexts.

What are the optimal protocols for using BIRC5 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of BIRC5 in formalin-fixed paraffin-embedded (FFPE) tissues, the following protocol has been validated in research settings:

• Sample preparation: Prepare 5mm serial sections from FFPE tissues.

• Deparaffinization and rehydration: Follow standard protocols to remove paraffin and rehydrate tissue sections.

• Antigen retrieval: Use sodium citrate buffer (0.01 M, pH 6.0) for 10 minutes at 98°C. This step is critical for unmasking epitopes that may be cross-linked during fixation.

• Endogenous peroxidase blocking: Suppress endogenous peroxidase activity with 3% hydrogen peroxide in dH₂O for 10 minutes.

• Blocking: Incubate sections with 5% normal goat serum for 30 minutes to reduce nonspecific binding.

• Primary antibody incubation: Apply polyclonal rabbit anti-BIRC5 antibody (1:100 dilution) at room temperature for 4 hours.

• Secondary antibody application: Expose sections to HRP-conjugated anti-rabbit secondary antibody for 1 hour.

• Visualization: Stain with 3,3′-diaminobenzidine and counterstain with hematoxylin.

• Analysis: Evaluate staining using ImageJ software to calculate the area percentage of positive staining, examining at least five randomly selected visual fields (×200 magnification) per section .

This protocol has been successfully used to detect BIRC5 expression in HCC tissues and correlate it with CD11b expression (a MDSC marker), demonstrating its reliability for investigating BIRC5's role in the tumor microenvironment.

How can I optimize Western blot conditions for BIRC5 detection?

Western blot optimization for BIRC5 detection requires attention to several key variables:

• Protein extraction: Extract total cell protein lysates on ice using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail to prevent protein degradation.

• Protein concentration: Determine protein concentrations using a Bio-Rad protein assay or similar method.

• Sample loading: Load 10–30 μg of total cell protein extracts per lane, with standardization across samples being critical for comparative analysis.

• Gel selection: Use 10% SDS-PAGE for optimal resolution of BIRC5 (16.5 kDa).

• Transfer conditions: Electroblot onto PVDF membranes (Millipore or equivalent) using optimized transfer conditions for low molecular weight proteins.

• Antibody incubation:

  • Primary antibody: Rabbit anti-BIRC5 polyclonal antibody (e.g., Proteintech #10508) at 4°C overnight

  • Secondary antibody: HRP-linked anti-rabbit antibody for 2 hours

• Loading controls: Include appropriate loading controls such as GAPDH (Proteintech #60004) or alpha-Tubulin (Proteintech #66031) .

• Signal detection: Use enhanced chemiluminescence for signal development, with exposure time optimized for each experimental system.

• Quantification: Perform densitometric analysis of band intensity using ImageJ or similar software for quantitative comparisons between samples.

These optimized conditions have been successfully applied in studies examining BIRC5 expression in hepatocellular carcinoma and its relationship with immune checkpoint molecules such as PD-L1.

What methods are recommended for studying BIRC5's impact on immune cell populations?

To investigate BIRC5's influence on immune cell populations, particularly MDSCs, the following methodological approaches are recommended:

• Flow cytometry analysis:

  • Isolate peripheral blood mononuclear cells (PBMCs) using Ficoll-Paque Premium

  • Block Fcγ receptors with mouse serum (10 min at 4°C)

  • Stain cells with human myeloid markers: CD11b, CD14, CD33, and HLA-DR

  • Analyze data using flow cytometry software such as FlowJo for accurate quantification

• In vitro MDSC expansion assays:

  • Generate cell lines with modulated BIRC5 expression using lentiviral vectors

  • Collect supernatant from these cells and treat PBMCs for 5 days

  • Evaluate MDSC expansion through flow cytometry analysis of markers CD11b+CD33+HLA-DR−

  • Compare MDSC expansion between BIRC5-overexpressing and control conditions

• Correlation analysis in tissue samples:

  • Perform immunohistochemistry for both BIRC5 and CD11b on serial sections

  • Quantify staining using ImageJ or similar software

  • Conduct statistical analysis using Pearson's correlation test (significant if p < 0.05 and |correlation coefficient (R)| > 0.3)

These methodologies provide complementary approaches to understand BIRC5's role in modulating immune cell populations, particularly in the context of tumor immune microenvironment.

How does BIRC5 expression impact tumor immune microenvironment and immunotherapy response?

BIRC5 expression significantly influences the tumor immune microenvironment through several mechanisms that ultimately affect immunotherapy efficacy:

• MDSC recruitment and expansion: BIRC5 overexpression promotes the expansion of immunosuppressive CD11b+CD33+HLA-DR− myeloid-derived suppressor cells (MDSCs) from peripheral blood mononuclear cells. This has been demonstrated in in vitro experimental systems where hepatocellular BIRC5 overexpression directly promoted MDSC expansion .

• T-cell exclusion and dysfunction: High BIRC5 expression correlates with reduced T-cell infiltration and function within tumors. Mechanistically, the BIRC5-induced MDSCs create an immunosuppressive environment that impairs effective T-cell responses against tumor cells .

• Immunotherapy resistance: Tumors with activated cell cycle programs and high BIRC5 expression show reduced sensitivity to immune checkpoint inhibitors (ICIs). Clinical analyses have revealed that patients with high BIRC5 expression tend to have worse clinical outcomes with immunotherapy .

• Genetic evidence: Studies using genetically modified animal models have shown that BIRC5 depletion upregulates genes related to lymphocyte-mediated immunity, natural killer cell-mediated immunity, interferon-gamma production, T-cell activation, and T-cell-mediated cytotoxicity .

These findings collectively suggest that BIRC5 functions as an immunosuppressive factor in the tumor microenvironment, creating conditions that favor tumor immune evasion and resistance to immunotherapy. Targeting BIRC5 may therefore represent a strategy to enhance immunotherapy response by reversing these immunosuppressive effects.

What is the relationship between BIRC5 and clinical prognosis in cancer patients?

BIRC5 expression has been consistently associated with clinical outcomes across multiple cancer types, with several studies highlighting its prognostic significance:

These findings establish BIRC5 as not only a biological mediator of cancer progression but also as a clinically relevant prognostic marker that could potentially guide treatment decisions, particularly regarding immunotherapy approaches.

What mechanistic pathways connect BIRC5 to immune cell regulation in cancer?

The mechanistic connections between BIRC5 and immune regulation in cancer involve complex cellular pathways that researchers are still elucidating:

• MDSC expansion pathway: BIRC5 overexpression appears to trigger signaling events that promote MDSC development from myeloid precursors. While the exact molecular mediators remain under investigation, in vitro studies have demonstrated that soluble factors from BIRC5-overexpressing cells can induce MDSC expansion, suggesting paracrine signaling mechanisms .

• Immune checkpoint regulation: There is evidence connecting BIRC5 expression with immune checkpoint molecules such as PD-L1. Experimental models have examined this relationship, suggesting potential coordination between cell survival pathways and immune evasion mechanisms .

• Cell cycle program activation: BIRC5 is integral to the activated cell cycle program in cancer cells, which has been linked to immunosuppression. This connection suggests that proliferative cancer signaling directly interfaces with immune regulation .

• T-cell exclusion pathways: Studies using genetic BIRC5 depletion models have revealed upregulation of genes related to:

  • Lymphocyte-mediated immunity

  • Natural killer cell-mediated immunity

  • Interferon-gamma production

  • T-cell activation

  • T-cell-mediated cytotoxicity

This indicates that BIRC5 normally suppresses these pathways, contributing to T-cell exclusion from the tumor microenvironment.

Further research is needed to fully map the signaling networks connecting BIRC5 to immune regulation, particularly to identify which specific cytokines, chemokines, and cell-cell interactions mediate these effects in different cancer contexts.

What are common causes of inconsistent BIRC5 staining patterns in immunohistochemistry?

Researchers often encounter variable BIRC5 staining patterns in immunohistochemistry. The following factors commonly contribute to these inconsistencies:

• Fixation variables: Overfixation or underfixation of tissue samples can significantly impact antibody accessibility to BIRC5 epitopes. Standard fixation in 10% neutral buffered formalin for 24-48 hours typically produces optimal results.

• Antigen retrieval effectiveness: Insufficient antigen retrieval is a major cause of weak or absent staining. The sodium citrate buffer (0.01 M, pH 6.0) protocol for 10 minutes at 98°C has been validated for BIRC5 detection, but optimization may be required for specific tissue types .

• Antibody specificity and sensitivity: Different anti-BIRC5 antibodies may recognize distinct epitopes with varying accessibility in fixed tissues. Validation with positive controls such as HeLa or Jurkat cell lysates is recommended before application to experimental samples .

• Endogenous peroxidase activity: Inadequate blocking of endogenous peroxidase can result in false-positive staining. The recommended 3% hydrogen peroxide treatment for 10 minutes may need adjustment based on tissue type .

• Biological heterogeneity: BIRC5 expression can vary significantly within tumors due to cellular heterogeneity. Analysis of multiple fields (minimum five randomly selected visual fields at ×200 magnification) is recommended for reliable quantification .

• Technical variability: Batch effects in staining procedures can introduce artificial differences between samples processed on different days. Including consistent positive controls across batches helps identify such technical artifacts.

Addressing these factors through careful methodology standardization and appropriate controls can significantly improve consistency in BIRC5 immunohistochemical analysis.

How should researchers interpret discrepancies between BIRC5 protein and mRNA expression data?

Discrepancies between BIRC5 protein and mRNA expression levels are not uncommon and require careful interpretation:

• Post-transcriptional regulation: BIRC5 expression is subject to extensive post-transcriptional regulation, including microRNA targeting and RNA-binding protein interactions. These mechanisms can result in protein levels that don't directly correlate with mRNA abundance.

• Protein stability differences: BIRC5 protein stability may vary across different cellular contexts or in response to treatment conditions, leading to apparent discrepancies with mRNA levels. The protein has a relatively short half-life that can be affected by various cellular conditions.

• Technical considerations:

  • Different detection sensitivities between RNA sequencing/qPCR and protein detection methods

  • Sample processing differences that may affect RNA or protein integrity differently

  • Antibody specificity issues that may detect specific isoforms or post-translationally modified forms of BIRC5

• Biological interpretations: When consistent discrepancies are observed:

  • Consider potential alternative splicing generating different BIRC5 isoforms

  • Evaluate whether post-translational modifications affect antibody recognition

  • Investigate possible differences in subcellular localization that might impact detection

  • Examine potential RNA sequestration mechanisms that prevent translation

• Validation approaches: To address discrepancies:

  • Use multiple antibodies targeting different BIRC5 epitopes

  • Employ orthogonal protein detection methods (Western blot, IHC, flow cytometry)

  • Consider protein degradation inhibitor experiments to evaluate turnover rates

  • Perform polysome profiling to assess translation efficiency

Understanding these potential sources of discrepancy is essential for accurate data interpretation and can sometimes reveal important biological mechanisms regulating BIRC5 expression.

What are recommended positive and negative controls for BIRC5 antibody validation?

Proper experimental controls are essential for rigorous BIRC5 antibody validation:

Recommended positive controls:

• Cell lines:

  • HeLa cell lysate (human cervical cancer cells)

  • Jurkat cell lysate (human T lymphocyte cells)

  • CEM cell lysate (human T lymphoblastoid cells)

  • Colo320 cell lysate (human colorectal adenocarcinoma cells)

• Tissue samples:

  • Cervix carcinoma sections

  • Mammary cancer sections

  • Myeloid leukemia samples

  • Hepatocellular carcinoma with confirmed BIRC5 expression

Recommended negative controls:

• Technical controls:

  • Primary antibody omission

  • Isotype control antibody substitution

  • Blocking peptide competition (using BIRC5-specific peptides)

• Biological controls:

  • BIRC5 knockdown/knockout cell lines (generated using siRNA, shRNA, or CRISPR-Cas9)

  • Normal tissues with minimal BIRC5 expression (carefully selected based on literature)

  • Cell lines treated with BIRC5 transcription inhibitors

• Validation specificity tests:

  • Western blot analysis showing a single band at the expected molecular weight (~16.5 kDa)

  • Immunoprecipitation followed by mass spectrometry identification

  • Comparative analysis with alternative validated anti-BIRC5 antibodies

Implementing these controls provides confidence in the specificity and sensitivity of BIRC5 detection, ensuring that experimental observations reflect genuine biological phenomena rather than technical artifacts.

What statistical approaches are appropriate for analyzing correlations between BIRC5 expression and immune cell infiltration?

When analyzing correlations between BIRC5 expression and immune cell infiltration, the following statistical approaches are recommended based on published methodologies:

• Correlation analysis:

  • Pearson's correlation test for normally distributed variables

  • Significant correlation is typically defined as p < 0.05 and |correlation coefficient (R)| > 0.3

  • This approach has been successfully applied to correlate BIRC5 expression with CD11b+ MDSC infiltration in HCC

• Survival analysis:

  • Kaplan-Meier method with log-rank test to compare survival between groups with different BIRC5 and immune cell marker expression levels

  • Cox proportional hazards regression models to adjust for confounding variables

  • These methods have demonstrated that concordant high expression of BIRC5 and intratumor infiltration of MDSCs correlates with worse prognosis

• Multivariate models:

  • Multivariable-adjusted Cox regression analysis to determine if BIRC5-based classifications are independent prognostic factors

  • Inclusion of relevant clinical variables such as tumor stage, grade, and patient demographics

• Bioinformatic approaches:

  • Gene Set Variation Analysis (GSVA) to assess enrichment of immune-related gene signatures

  • Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to predict immunotherapy response

  • TIMER2.0 database analysis for comprehensive immune infiltration assessment

• Visualization methods:

  • Scatter plots with regression lines to illustrate correlations

  • Heat maps to display patterns across multiple immune cell types and BIRC5 expression levels

  • Forest plots for hazard ratios in survival analyses

These statistical approaches provide rigorous frameworks for analyzing the relationship between BIRC5 expression and immune cell infiltration, facilitating reliable interpretation of biological significance and clinical relevance.

What are the most promising therapeutic approaches targeting BIRC5 in cancer?

Several therapeutic strategies targeting BIRC5 show promise for cancer treatment, particularly in combination with immunotherapy:

• Survivin-partner protein interaction inhibitors: These compounds disrupt essential protein-protein interactions required for BIRC5 function, potentially inhibiting its anti-apoptotic and pro-proliferative effects.

• Survivin homodimerization inhibitors: By preventing BIRC5 dimerization, these agents aim to interfere with its structural stability and functional activity.

• Survivin gene transcription inhibitors: Compounds that suppress BIRC5 gene expression at the transcriptional level could potentially reduce its abundance in cancer cells.

• Survivin mRNA inhibitors: RNA interference approaches and antisense oligonucleotides targeting BIRC5 mRNA have shown efficacy in preclinical models.

• Survivin immunotherapy: Vaccines and other immunotherapeutic approaches targeting BIRC5 as a tumor-associated antigen represent an emerging strategy .

The potential of these approaches extends beyond direct anti-tumor effects, as research suggests that BIRC5 inhibition may enhance immunotherapy efficacy by reducing MDSC infiltration and improving T-cell function in the tumor microenvironment. Further research is needed to determine which specific BIRC5-targeting strategy might most effectively complement existing immunotherapeutic approaches in different cancer contexts.

What knowledge gaps remain in understanding BIRC5's role in immune regulation?

Despite significant advances, several important knowledge gaps remain in understanding BIRC5's role in immune regulation:

• MDSC subtype specificity: It remains unclear which MDSC subtypes (polymorphonuclear-MDSCs or monocytic-MDSCs) are primarily affected by BIRC5 expression. Different studies have shown contradictory results regarding the dominant MDSC population in liver fibrosis and HCC .

• Signaling mechanisms: The precise signaling pathways, cytokines, and chemokines through which BIRC5 impacts MDSC amplification and infiltration in tumor tissue remain incompletely characterized .

• Tissue-specific effects: Whether BIRC5's immunomodulatory functions differ across cancer types and tissue contexts requires further investigation.

• Therapeutic translation: The clinical significance of targeting BIRC5 in immunotherapy of different cancer types needs additional validation, particularly through in vivo experiments to validate biomarker-directed therapy .

• Resistance mechanisms: Potential compensatory mechanisms that might emerge following BIRC5 inhibition and limit therapeutic efficacy remain poorly understood.