KIF20A Antibody

What applications are validated for KIF20A antibody detection in cancer research?

KIF20A antibodies have been extensively validated for multiple applications in cancer research, with specific considerations for each technique:

• Western Blotting (WB): Effective detection at dilutions between 1:500-1:2000, with observed molecular weights primarily at 100 kDa . Multiple commercially available antibodies show reliable detection in human cancer cell lines including HeLa and Caco-2 .

• Immunohistochemistry (IHC): Validated on paraffin sections of various cancer tissues with recommended dilutions of 1:50-1:500 . Antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) has been documented for optimal staining .

• Immunoprecipitation (IP): Successfully employed to isolate KIF20A from cell lysates using 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein .

• Immunofluorescence (IF): Enables visualization of subcellular localization, particularly useful for studying KIF20A's association with microtubules and Golgi apparatus .

Robust application of these techniques has enabled researchers to correlate KIF20A expression with tumor progression across multiple cancer types .

How should KIF20A protein expression be evaluated in clinical cancer specimens?

Evaluation of KIF20A expression in clinical specimens follows specialized protocols with particular attention to scoring systems:

• Semi-quantitative scoring system: Most studies employ a scoring scale based on staining intensity and percentage of positive cells:

  • 0 (negative): No staining

  • 1 (weak): Light staining in <50% of cells

  • 2 (moderate): Light staining in >50% or moderate staining in <50%

  • 3 (strong): Moderate staining in >50% or strong staining in <50%

  • 4-5: Higher intensity staining patterns

• Expression categorization: Patients are typically stratified into "high" and "low" expression groups based on threshold scores. In hepatocellular carcinoma studies, scores 2-5 were classified as high expression while 0-1 as low expression .

• Localization assessment: Immunohistochemical evaluation should document subcellular localization patterns, with some cancers showing nuclear-enhanced staining of KIF20A .

This standardized approach enables reliable prognostic correlation across different cancer cohorts and institutions .

What are the recommended protocols for KIF20A knockdown validation studies?

Validation of KIF20A knockdown requires a comprehensive approach incorporating multiple techniques:

siRNA-mediated knockdown protocol:

• Transfection: Utilize siRNA pools (typically 50 nmol/l final concentration) targeting multiple regions of KIF20A mRNA. Example sequences include 5′-CUGUGAAGGAGAUGGUAAATT-3′, 5′-GCAAUCCCUAUGUGAAAGATT-3′, and 5′-GUUCCUGCAUGAUUGUCAATT-3′ .

• Transfection reagent selection: GenePORTER 2 Transfection reagent or X-tremeGENE HP DNA Transfection Reagent have been successfully employed .

• Verification approaches:

  • Western blot analysis using anti-KIF20A antibodies (dilution 1:100; e.g., sc-374508)

  • qRT-PCR for mRNA expression confirmation

  • Include β-actin as loading control (dilution 1:3,000)

• Rescue experiments: To confirm specificity, include KIF20A-rescue constructs containing the entire coding sequence of KIF20A cDNA inserted into expression vectors (e.g., pCMV6-Entry vector) .

• Functional validation: Assess effects on cellular processes including proliferation (colony formation assays), migration (transwell assays), and invasion assays .

This multi-faceted validation approach ensures knockdown specificity and facilitates reliable interpretation of phenotypic changes .

How should researchers optimize immunohistochemical detection of KIF20A in different cancer tissues?

Optimization of KIF20A immunohistochemistry requires systematic adjustment of multiple parameters:

• Tissue preparation:

  • Fixation: 10% neutral-buffered formalin, 24-48 hours

  • Embedding: Paraffin embedding following standard histological procedures

  • Sectioning: 4-5 μm thickness for optimal antibody penetration

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in either:

    • TE buffer (pH 9.0) - preferred primary method

    • Citrate buffer (pH 6.0) - alternative method

  • Microwave heating for 10-20 minutes followed by cooling

• Antibody optimization:

  • Titration: Test dilution range from 1:50 to 1:500 on positive control tissues

  • Incubation: Overnight at 4°C for primary antibody yields optimal signal-to-noise ratio

  • Detection system: Horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution) with DAB visualization

• Controls:

  • Positive controls: Include known KIF20A-positive tissues (e.g., certain cancer cell lines, positive tumor samples)

  • Negative controls: Primary antibody omission and isotype controls

  • Internal controls: Normal adjacent tissue for comparison with tumor areas

Optimized protocols have enabled researchers to establish KIF20A as a prognostic biomarker across multiple cancer types including cervical, hepatocellular, ovarian, and prostate cancers .

How does KIF20A expression correlate with clinical outcomes across different cancer types?

KIF20A overexpression consistently predicts poor clinical outcomes across multiple cancer types, as evidenced by comprehensive clinical studies:

Statistical analyses consistently identify KIF20A as an independent prognostic factor after adjusting for known clinical variables . The predictive value extends across early and advanced cancer stages, with subgroup analyses confirming associations in both G1/G2 and G3/G4 tumors .

What is the relationship between KIF20A expression and treatment response in cancer?

KIF20A expression significantly influences treatment outcomes through multiple mechanisms:

• Chemoresistance correlation:

  • Ovarian cancer studies demonstrate that high KIF20A expression associates with diminished response to platinum-based chemotherapy

  • Glioma patients with positive KIF20A expression show potential chemotherapy resistance, contributing to reduced survival rates

• Immunotherapy implications:

  • KIF20A functions as a tumor-associated antigen (TAA) with immunogenic properties

  • Peptide vaccines targeting KIF20A have shown promising results in clinical trials

  • KIF20A-based immunotherapeutic approaches have demonstrated feasibility and effectiveness

• Combined therapy strategies:

  • KIF20A-based thermosensitive hydrogel vaccine (K/R Lip@Gel) significantly improves the efficacy of PD-L1 blockade in hepatocellular carcinoma models

  • This combination enhances dendritic cell maturation and increases CD8+ T cell infiltration and activation

  • KIF20A may serve as an adjunctive target alongside immune checkpoint inhibitors to overcome treatment resistance

These findings suggest that KIF20A expression assessment could guide treatment selection and that targeting KIF20A may enhance conventional therapeutic approaches .

How can researchers investigate KIF20A's role in cancer cell motility and invasion mechanisms?

Investigating KIF20A's influence on cancer cell motility and invasion requires specialized experimental approaches:

• RNA granule transport visualization:

  • Immunofluorescence colocalization of KIF20A with stress granule (SG) markers and IGF2BP3

  • Live-cell imaging using GFP-tagged KIF20A to track RNA granule movement toward membrane protrusions

  • This approach revealed KIF20A's critical role in trafficking IGF2BP3-containing stress granules, which promotes pancreatic cancer cell motility

• Molecular pathway analysis:

  • Western blotting to assess expression of motility-related proteins (MMP2, MMP9) after KIF20A knockdown

  • Examination of ARF6 and ARHGEF4 expression in cell protrusions

  • These experiments demonstrated that KIF20A knockdown decreased MMP expression and inhibited membrane protrusion formation

• Functional assays with quantitative readouts:

  • Transwell migration assays: Quantify migrated cells after 24 hours (KIF20A knockdown significantly reduced migration)

  • Invasion assays: Evaluate Matrigel penetration capacity (KIF20A suppression decreased invasiveness)

  • Rescue experiments: Re-expression of KIF20A abrogated motility/invasion defects, confirming specificity

These approaches have established mechanistic links between KIF20A expression and enhanced cancer cell motility and invasiveness, particularly in pancreatic and prostate cancers .

What methodologies are effective for studying KIF20A-based cancer immunotherapy approaches?

Research into KIF20A as an immunotherapy target employs specialized techniques:

• Identifying immunogenic epitopes:

  • Computer algorithm prediction of HLA class II-binding peptides combined with known CTL-epitope sequences

  • Testing long peptides (LPs) derived from KIF20A for their ability to induce T-helper type 1 (TH1) cells and CTLs

  • IFN-γ enzyme-linked immunospot assays to quantify T-cell responses

• Vaccine formulation development:

  • Thermosensitive hydrogel vaccine formulation (K/R Lip@Gel) for KIF20A antigen delivery

  • This system enables sustained in vivo release and optimizes antigen presentation

  • Flow cytometry evaluation of dendritic cell and T-cell activation following vaccination

• Preclinical model systems:

  • Subcutaneous and orthotopic cell-derived xenograft (CDX) models

  • Immune-humanized patient-derived xenograft (PDX) models for human relevance

  • HLA-A24 transgenic mice to evaluate CTL cross-priming in vivo

• Combination therapy assessment:

  • Testing KIF20A-targeted vaccines in combination with immune checkpoint blockade (ICB)

  • Evaluating synergistic effects on tumor growth inhibition

  • Measuring immune cell infiltration and activation markers through immunohistochemistry and flow cytometry

These methodologies have demonstrated that KIF20A-based vaccines can effectively elicit robust immune responses and improve ICB therapy outcomes in hepatocellular carcinoma models .

How should researchers address inconsistent KIF20A detection across different experimental systems?

When encountering variable KIF20A detection results, systematic troubleshooting approaches are essential:

• Antibody selection considerations:

  • Different antibodies target distinct epitopes - compare monoclonal (e.g., sc-374508, D-3) versus polyclonal antibodies (e.g., ab70791)

  • Host species influences background in certain applications - rabbit polyclonal antibodies frequently demonstrate superior specificity

  • Clone-specific variations exist - observed molecular weights range from 100-120 kDa with potential additional bands at 31, 60, 68 and 73 kDa

• Technical optimization steps:

  • Sample preparation: Ensure complete protein denaturation for Western blotting

  • Blocking optimization: Test 5% non-fat milk versus BSA to reduce background

  • Increase antibody concentration for low abundance samples (recommended WB dilution range: 1:500-1:2000)

  • Antigen retrieval methods significantly impact IHC detection - compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0)

• Cell/tissue-specific considerations:

  • Expression levels vary significantly across cell types - HeLa cells consistently show strong expression

  • Subcellular localization differences exist - nuclear-enhanced staining observed in HCC tissues versus cytoplasmic distribution in normal cells

  • KIF20A expression is highly inducible - consider cell cycle phase and culture conditions

These approaches have resolved detection inconsistencies in studies across hepatocellular, cervical, ovarian, and prostate cancer research .

What controls are essential when evaluating KIF20A expression in cancer tissues?

Rigorous control implementation is critical for reliable KIF20A expression analysis:

• Technical controls:

  • Positive controls: Include known KIF20A-expressing tissues/cells (e.g., HeLa cells) to validate detection methods

  • Negative controls: Primary antibody omission and isotype controls to assess non-specific binding

  • Concentration gradients: Test multiple antibody dilutions (1:50, 1:100, 1:500) to determine optimal signal-to-noise ratio

• Biological reference standards:

  • Adjacent normal tissue: Essential for comparative analysis within the same specimen

  • Normal tissue panels: Compare expression across multiple normal tissue types (KIF20A shows notably low expression in normal liver compared to other tissues)

  • Cancer gradient specimens: Include tissues representing different grades and stages to establish expression patterns

• Validation approaches:

  • Multi-method confirmation: Correlate IHC findings with Western blot and qRT-PCR results

  • Knockdown validation: KIF20A siRNA-treated samples serve as specificity controls

  • Bioinformatic verification: Compare protein expression with mRNA expression data from sources like TCGA

• Scoring system standardization:

  • Implement established semi-quantitative scoring methods (0-5 scale)

  • Use multiple independent evaluators to ensure scoring consistency

  • Include detailed documentation of subcellular localization patterns

These comprehensive controls have enabled researchers to establish KIF20A as a reliable prognostic biomarker across multiple cancer types despite the inherent variability of immunohistochemical techniques .

How does KIF20A function in normal cellular processes versus cancer progression?

KIF20A exhibits distinct functional profiles in normal and malignant contexts:

Normal cellular functions:

• Essential regulator of cytokinesis during mitotic cell division

• Facilitates retrograde transport of Golgi membranes and vesicles along microtubules

• Interacts with GTP-bound forms of RAB6A and RAB6B to mediate vesicular trafficking

• Contains structural domains that facilitate microtubule dynamics and vesicle transport

• Exhibits microtubule plus end-directed motility

Cancer-specific mechanisms:

• Cell division dysregulation:

  • Overexpression promotes excessive cell proliferation

  • Contributes to chromosomal instability in cancer cells

  • Knockdown causes premature cell cycle exit and enhanced neuronal differentiation in medulloblastoma

• Enhanced motility pathways:

  • Transports IGF2BP3-containing stress granules toward membrane protrusions in pancreatic cancer

  • Supports expression of ARF6 and ARHGEF4 in cell protrusions

  • Knockdown decreases MMP2 and MMP9 expression, inhibiting cancer cell invasion

• Signaling pathway interactions:

  • Interacts with transcription factors like FOXM1

  • Influences JAK/STAT3 signaling in colorectal cancer

  • Subject to regulation by tumor microenvironment factors

Understanding these differential functions has informed therapeutic approaches targeting KIF20A in multiple cancer types .

What is the current evidence supporting KIF20A as a cancer immunotherapy target?

Multiple lines of evidence establish KIF20A as a promising immunotherapeutic target:

• Tumor-associated antigen characteristics:

  • Transcriptomic mRNA sequencing confirms KIF20A as an immunogenic tumor-associated antigen (TAA)

  • Minimal expression in normal tissues reduces off-target effects

  • Contains peptide sequences recognized by both CD4+ and CD8+ T cells

• Promiscuous epitope identification:

  • Long peptides (LPs) derived from KIF20A bear naturally processed epitopes

  • These peptides can induce both tumor-specific T-helper type 1 (TH1) cells and CTLs

  • HLA-A2 and HLA-A24 presented epitopes have been characterized

• Clinical evidence of immunogenicity:

  • KIF20A-specific TH1 cell responses detected in 50% of head-and-neck malignant tumor patients

  • These responses correlate with KIF20A expression in tumor tissues

  • No significant responses detected in healthy donors, confirming tumor specificity

• Vaccine development advances:

  • Thermosensitive hydrogel vaccine formulation (K/R Lip@Gel) efficiently elicits robust immune responses

  • Enhances dendritic cell maturation and T-cell activation

  • Improves therapeutic efficacy of PD-L1 blockade in multiple preclinical models:

    • Subcutaneous and orthotopic cell-derived xenografts

    • Immune-humanized patient-derived xenografts

These findings collectively support the advancement of KIF20A-targeted immunotherapeutic approaches for clinical development across multiple cancer types .