TPX2 Antibody, FITC conjugated

What is TPX2 and what makes it significant in cancer research?

TPX2 is a microtubule-associated protein that plays critical roles in mitotic spindle assembly and function. Research has established TPX2 as a promising biomarker and potential therapeutic target due to its significant upregulation across multiple cancer types. Integrative analyses have confirmed that TPX2 is overexpressed at both mRNA and protein levels in various solid tumors compared to normal tissues . Its role in cellular proliferation, migration, and cell cycle regulation makes it particularly relevant for cancer research. TPX2 has been validated as having potential oncogenic properties, with evidence of gene amplification in several malignancies including pancreatic adenocarcinoma .

What cellular and molecular functions does TPX2 regulate in normal and cancer cells?

TPX2 primarily functions in mitotic spindle assembly and chromosomal segregation. In cancer contexts, TPX2 has been implicated in multiple cellular processes:

• Cell proliferation and invasion regulation

• Cell cycle progression, particularly mitosis

• DNA replication processes

• Microtubule nucleation and organization

Functional studies using TPX2 knockdown in lung cancer and other cell lines have demonstrated that TPX2 downregulation significantly reduces cancer cell proliferation and migration capabilities . GeneMANIA analysis, GSEA analysis, and single-cell functional analysis have consistently shown TPX2's involvement in these critical cellular processes.

What expression patterns does TPX2 exhibit across different cancer types?

TPX2 exhibits differential expression across cancer types:

Comprehensive analysis has confirmed TPX2 upregulation across multiple solid tumors, with particularly consistent findings in lung, breast, and pancreatic cancers .

What are the optimal dilutions and conditions for using FITC-conjugated TPX2 antibodies in immunofluorescence studies?

While specific FITC-conjugated TPX2 antibody parameters were not directly addressed in the search results, standard TPX2 antibodies are recommended at dilutions of 1:200-1:800 for immunofluorescence applications . When working with FITC-conjugated antibodies, researchers should:

• Start with manufacturer-recommended dilutions and optimize based on signal-to-noise ratio

• Protect the FITC conjugate from light during all procedures

• Use proper antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Include appropriate controls to account for FITC's spectral properties

• Consider photobleaching characteristics when designing imaging protocols

Validation has been conducted in multiple cell lines including HepG2 and HeLa cells, providing reference points for optimization .

How can researchers validate TPX2 antibody specificity for experimental applications?

Rigorous validation approaches should include:

• Western blot analysis using positive control lysates (validated in HepG2, K-562, HeLa, and SMMC-7721 cells)

• Knockdown validation using TPX2-targeting siRNA to confirm signal reduction

• Comparison of staining patterns with published TPX2 localization data

• Evaluation across multiple detection methods (IF, IHC, WB) to confirm consistent results

• Use of TPX2-null or TPX2-overexpressing systems as negative and positive controls

For FITC-conjugated antibodies specifically, researchers should additionally perform:

• Spectral analysis to confirm FITC signal characteristics

• Photobleaching assessments to determine stability during imaging procedures

What protocol modifications are necessary when using FITC-conjugated TPX2 antibodies for flow cytometry compared to immunofluorescence microscopy?

While the search results don't specifically address flow cytometry protocols for FITC-conjugated TPX2 antibodies, researchers should consider these methodological differences:

For flow cytometry:

• Cell permeabilization optimization is crucial as TPX2 is primarily intracellular

• Titration experiments (starting at 1:100-1:500 dilutions) should be performed to determine optimal antibody concentration

• More stringent blocking conditions may be required to minimize background

• Proper compensation controls must be included when multiplexing with other fluorophores

For immunofluorescence microscopy:

• Dilutions of 1:200-1:800 are recommended based on standard TPX2 antibody protocols

• Antigen retrieval with either TE buffer (pH 9.0) or citrate buffer (pH 6.0) is critical for optimal results

• Counterstaining of subcellular structures can help confirm expected TPX2 localization

How does TPX2 expression correlate with tumor immune microenvironment characteristics?

TPX2 expression demonstrates significant associations with multiple immune parameters:

• Cancer-associated fibroblasts (CAF): Negative correlations with TPX2 expression observed in breast cancer (BRCA) and thymoma (THYM) across four different computational algorithms

• Macrophage populations:

  • Positive correlation with M1 macrophage infiltration in 17 cancer types (negative in THYM)

  • Positive correlation with M0 macrophage levels in 19 cancer types (negative in THYM)

  • Negative correlation with M2 macrophage levels in 7 cancer types (positive in GBM, MESO, and PRAD)

• ESTIMATE algorithm results showed negative associations between TPX2 expression and both immune score and stromal score in 16 cancer types (positive association in KIRC and THCA)

These findings suggest TPX2 may influence the immune landscape of tumors, potentially affecting immunotherapy response.

What is the relationship between TPX2 expression and immunotherapy response biomarkers?

TPX2 expression correlates with several established immunotherapy response predictors:

• Tumor Mutational Burden (TMB): Significant positive correlations observed across multiple cancer types

• Microsatellite Instability (MSI): Positive associations in several cancer types, particularly in bladder cancer

• Neoantigen load: Positive correlations across multiple tumor types

• PD-L1 expression: Significant positive correlation with CD274 (PD-L1) expression in bladder cancer

Clinical analysis from the IMvigor 210 cohort (348 patients with metastatic urothelial bladder cancer) revealed that patients with high TPX2 expression showed improved outcomes with anti-PD-L1 therapy (atezolizumab) . This suggests TPX2 might serve as a biomarker for identifying patients likely to benefit from immune checkpoint inhibitors.

How can TPX2 knockdown experiments be designed to investigate its functional role in cancer progression?

Based on published approaches, effective TPX2 knockdown studies should incorporate:

• siRNA approach:

  • Target selection: Multiple siRNA oligonucleotides targeting different regions of TPX2 mRNA

  • Concentration optimization: Serial dilutions to determine minimal effective concentration

  • Validation: qRT-PCR and Western blot to confirm knockdown efficiency

• Functional assays:

  • Proliferation: CCK8 assay demonstrated reduced cell proliferation following TPX2 knockdown

  • Migration: Wound healing assay showed reduced cell migration after TPX2 downregulation

  • Apoptosis: Cell Death ELISA assay to quantify apoptosis induction

  • Colony formation: Soft agar assays to assess anchorage-independent growth

  • In vivo growth: Xenograft models in nude mice

• Combinatorial treatments:

  • TPX2 knockdown sensitized pancreatic cancer cells to paclitaxel treatment, suggesting potential for combination therapy approaches

For comprehensive understanding, experiments should include both short-term (48-72 hour) and longer-term (1-2 week) assessments of phenotypic consequences following TPX2 depletion.

How should researchers interpret discrepancies between expected and observed molecular weight of TPX2 in Western blot studies?

TPX2 has a calculated molecular weight of 86 kDa (based on its 747 amino acid sequence), but is typically observed at approximately 100 kDa in Western blot analyses . This discrepancy should be interpreted considering:

• Post-translational modifications: Phosphorylation sites on TPX2 can increase apparent molecular weight

• Protein structure: Extended conformations may result in altered migration patterns

• Technical factors:

  • Buffer composition can affect protein migration

  • Gel percentage selection is critical (8-10% gels are typically optimal for TPX2)

  • Incomplete denaturation may cause aberrant migration

When troubleshooting unusual band patterns:

• Compare results across different cell lines with known TPX2 expression (HepG2, K-562, HeLa, SMMC-7721 cells have been validated)

• Confirm specificity using TPX2 knockdown controls

• Consider using multiple antibodies targeting different TPX2 epitopes

What factors should be considered when analyzing TPX2 expression in relation to patient outcomes?

When correlating TPX2 expression with clinical outcomes, researchers should consider:

How can researchers resolve inconsistent staining patterns when using FITC-conjugated TPX2 antibodies?

When troubleshooting inconsistent FITC-TPX2 antibody staining, consider these methodological factors:

• Fixation and permeabilization:

  • Optimize fixation time (typically 10-15 minutes with 4% paraformaldehyde)

  • Test different permeabilization reagents (0.1-0.5% Triton X-100 vs. methanol)

  • Consider cell type-specific differences in membrane permeability

• Antigen retrieval:

  • Test both recommended methods: TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • Optimize retrieval duration and temperature

• FITC-specific considerations:

  • FITC is pH-sensitive; ensure buffers are maintained at correct pH

  • Protect from light throughout all procedures

  • Use anti-fade mounting media to prevent photobleaching

  • Consider photobleaching during imaging optimization

• Blocking optimization:

  • Test different blocking solutions (BSA vs. serum)

  • Extend blocking time to reduce background

  • Include detergents (0.1% Tween-20) in wash buffers

• Validate with alternative approaches:

  • Compare patterns with non-conjugated TPX2 antibodies

  • Confirm specificity through TPX2 knockdown

  • Compare localization patterns with published data showing expected nuclear and spindle-associated localization during different cell cycle phases

How might FITC-conjugated TPX2 antibodies be utilized in multiplex imaging approaches for tumor microenvironment characterization?

FITC-conjugated TPX2 antibodies offer valuable opportunities for multiplex imaging strategies:

• Panel design considerations:

  • TPX2 (FITC channel) can be combined with immune cell markers (CD4, CD8, CD68) in other channels

  • Include cancer-associated fibroblast markers given known correlations with TPX2

  • Incorporate proliferation markers (Ki-67) and cell cycle regulators

• Methodological approaches:

  • Sequential immunofluorescence with spectral unmixing

  • Multiplexed immunohistochemistry with tyramide signal amplification

  • Imaging mass cytometry for highest-dimensional analysis

• Analytical frameworks:

  • Spatial relationship analysis between TPX2-expressing cells and immune infiltrates

  • Correlation of TPX2 expression patterns with TMB and MSI status

  • Machine learning approaches for pattern recognition

This approach could provide insights into how TPX2 expression impacts the spatial organization of tumor immune microenvironments, potentially revealing mechanisms behind the observed correlations with immunotherapy response.

What mechanistic hypotheses might explain the correlation between TPX2 expression and immunotherapy response?

Several potential mechanisms warrant investigation:

• Genomic instability pathway:

  • TPX2 overexpression may drive chromosomal instability

  • Increased genomic instability could elevate neoantigen production

  • Higher neoantigen load correlates with better immunotherapy response

• Immune checkpoint modulation:

  • Positive correlation between TPX2 and PD-L1 expression in bladder cancer

  • TPX2 may directly or indirectly regulate immune checkpoint molecules

• Macrophage polarization effects:

  • TPX2 correlates positively with M1 macrophages in most cancers

  • M1 macrophages typically promote anti-tumor immunity

• Cell death mechanisms:

  • TPX2 inhibition increases apoptosis

  • Immunogenic cell death may be enhanced in high-TPX2 contexts

These hypotheses align with the observation that bladder cancer patients with high TPX2 expression showed improved outcomes with atezolizumab treatment despite TPX2's general association with poor prognosis in conventional treatment contexts .

How might combined targeting of TPX2 and immune checkpoints enhance cancer immunotherapy?

Based on the correlative evidence, combination strategies hold promise:

• Rational for combination:

  • TPX2 knockdown sensitizes cancer cells to paclitaxel

  • High TPX2 expression correlates with better anti-PD-L1 response

  • TPX2 targeting could enhance immune recognition while checkpoint inhibition activates T cells

• Experimental approaches:

  • Sequential therapy: TPX2 inhibition followed by checkpoint blockade

  • Concurrent inhibition with optimized dosing

  • Cell-specific targeting using antibody-drug conjugates

• Biomarker-guided patient selection:

  • Multi-parameter assessment including TPX2 expression, TMB, MSI status

  • Immune infiltrate characterization

  • PD-L1 expression levels

Preliminary evidence suggests that "patients who did not benefit from classic anticancer therapies might be responsible for the immunotherapy" , supporting investigation of TPX2-related biomarkers for immunotherapy selection.