KRT8 Antibody

What is KRT8 and which tissues typically express this protein?

KRT8 belongs to the type II (basic) subfamily of high molecular weight cytokeratins and typically exists in combination with cytokeratin 18 (CK18). It is predominantly expressed in simple epithelia including glandular epithelium of the thyroid, female breast, gastrointestinal tract, respiratory tract, and urogenital tract including transitional epithelium . Normal epidermis contains very little keratin 18, which makes KRT8/18 antibodies particularly useful for identifying specific cell types like Paget cells within epidermal tissue . Understanding the normal tissue distribution of KRT8 is essential when designing experiments to investigate its altered expression in pathological conditions.

How does KRT8 expression differ between normal and cancerous tissues?

KRT8 exhibits significant differential expression patterns between normal and malignant tissues. Adenocarcinomas and most squamous carcinomas typically show positive KRT8 staining, while keratinizing squamous carcinomas are usually negative . In lung adenocarcinoma (LUAD), KRT8 protein levels are significantly upregulated compared to normal lung tissue, as confirmed by immunohistochemistry data from The Human Protein Atlas . Similarly, in pancreatic cancer, elevated KRT8 mRNA expression has been observed, with patients showing above-median expression levels demonstrating an increased risk of mortality (hazard ratio = 1.73, p = 0.01) . These distinctive expression patterns make KRT8 a valuable marker for distinguishing malignant tissues from normal counterparts in diagnostic pathology.

What are the optimal applications for KRT8 antibodies in cancer research?

KRT8 antibodies demonstrate utility across multiple experimental applications including immunohistochemistry (IHC), flow cytometry, Western blotting, and mass cytometry (CyTOF) . For tissue-based studies, immunohistochemistry using formalin-fixed paraffin-embedded (FFPE) samples is particularly effective for evaluating KRT8 expression patterns and localization. In liquid biopsy research, enzyme-linked immunosorbent assays (ELISA) have successfully detected KRT8 in serum samples, with studies showing that serum KRT8 can discriminate between pancreatic cancer patients and healthy controls with an area under the curve (AUC) of 0.94 . For cellular studies examining KRT8's functional role, combining RNA interference techniques with proliferation and invasion assays has proven effective, as demonstrated in studies with A549 and PC9 lung cancer cell lines .

What methodological considerations are important when using KRT8 antibodies for immunostaining?

When performing immunostaining with KRT8 antibodies, several methodological factors require consideration:

• Antibody selection: Cocktail antibodies targeting both KRT8 and KRT18 (such as clones KRT8/803 + KRT18/835) provide comprehensive detection of simple epithelial tissues and their derived tumors .

• Sample preparation: Proper fixation is critical; overfixation can mask epitopes while inadequate fixation may compromise tissue morphology.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer is typically effective for cytokeratin detection in FFPE tissues.

• Signal detection: For fluorescence applications, it's important to note that conjugates with blue fluorescent dyes (CF®405S, CF®405M) are not recommended for low-abundance targets due to lower fluorescence and potentially higher non-specific background compared to other dye colors .

• Controls: Include both positive controls (tissues known to express KRT8) and negative controls (tissues lacking KRT8 expression) to validate staining specificity.

What evidence supports KRT8 as a prognostic biomarker in different cancer types?

Multiple lines of evidence support KRT8's prognostic value across different cancer types:

• Lung adenocarcinoma: In a multivariable Cox regression model adjusted for sex and age, KRT8 staining intensity significantly predicted mortality in lung adenocarcinoma patients (Hazard Ratio = 1.49, 95% CI = 1.06 – 2.10, p=0.02) . Notably, this association was not observed in squamous cell carcinoma (Hazard Ratio = 1.19, 95% CI = 0.57 – 2.52, p=0.65) .

• Pancreatic cancer: High KRT8 mRNA expression (above median level of 363.5 FPKM) was associated with increased mortality risk (Cox proportional hazard ratio = 1.73, p = 0.01) . Additionally, serum KRT8 levels were significantly higher in pancreatic cancer patients compared to healthy controls (p = 7.7e-4) .

• Multi-cancer analysis: Pan-cancer methylation analysis across 2,019 samples in 10 cancers and validated in 7,836 samples across 21 cancers has identified KRT8 as a robust biomarker that is consistently hypomethylated in various cancer types .

The prognostic value of KRT8 has been validated using diverse methodological approaches, including tissue microarrays, RNA sequencing, methylation profiling, and serum protein detection, strengthening confidence in its utility as a biomarker.

What experimental approaches are most effective for investigating KRT8's functional role in cancer?

Investigating KRT8's functional role in cancer can be approached through several complementary experimental strategies:

• Gene knockdown/knockout studies: RNA interference (siRNA, shRNA) or CRISPR-Cas9 technologies targeting KRT8 have demonstrated that depleting KRT8 in lung adenocarcinoma cell lines (A549 and PC9) suppresses tumor cell proliferation and invasion capabilities . These approaches allow direct assessment of KRT8's contribution to malignant phenotypes.

• Expression correlation analyses: Integrated analyses that correlate KRT8 expression with other molecular markers (such as p53-regulated genes) at both single-cell and bulk sample levels can reveal potential functional interactions and pathway involvement .

• Multi-omics integration: Combining DNA methylation data, RNA expression, and protein analysis provides a comprehensive view of KRT8 regulation across different molecular modalities. This approach has successfully identified KRT8 as a pan-cancer biomarker that is differentially expressed at multiple molecular levels .

• Phosphorylation studies: Evaluating phosphorylated KRT8 (p-KRT8) levels in response to cellular stressors provides insight into post-translational regulation. For example, oxidative stress induced by paraquat in retinal pigment epithelial cells (ARPE-19) enhances both KRT8 expression and its phosphorylation in a concentration and time-dependent manner .

How can researchers effectively measure KRT8 levels in liquid biopsies for minimally invasive diagnostics?

Developing KRT8 as a liquid biopsy marker requires careful methodological considerations:

• Assay selection: Enzyme-linked immunosorbent assays (ELISA) have successfully detected KRT8 in serum samples from pancreatic cancer patients, demonstrating discriminatory power between cancer and healthy controls .

• Threshold determination: Establishing appropriate cutoff values is critical for clinical utility. In pancreatic cancer studies, samples were considered KRT8-positive if they had a measured KRT8 value above the detectability limit of the ELISA (0.06 RLU) .

• Performance evaluation: Comprehensive assessment using multiple metrics is recommended. For serum KRT8 in pancreatic cancer detection, both standard ROC analysis (AUC = 0.94) and precision-recall curve analysis (AUPRC = 0.99) demonstrated excellent discriminatory performance .

• Sample processing standardization: Pre-analytical variables must be standardized to ensure reproducibility, including blood collection tubes, processing time, centrifugation protocols, and storage conditions.

What are the advantages of integrating single-cell and bulk transcriptome data when studying KRT8?

Integrating single-cell and bulk transcriptome data offers several significant advantages for KRT8 research:

• Cellular heterogeneity resolution: Single-cell RNA sequencing reveals the heterogeneity of KRT8 expression across different cell populations within the tumor microenvironment, which would be masked in bulk analyses . This cellular resolution is particularly valuable when studying early-stage lung adenocarcinoma, where four distinct subpopulations of tumor cells with varying KRT8 expression patterns have been identified .

• Transition state identification: Combined analysis enables the identification of dynamic transitions from normal epithelial cells to tumor cells, providing insight into the evolution of malignant phenotypes associated with altered KRT8 expression .

• Robust biomarker validation: When findings from single-cell analyses align with bulk tissue data, confidence in the biological significance of KRT8 alterations is substantially strengthened. This multi-scale validation approach has been successfully applied to confirm KRT8's role in lung adenocarcinoma progression .

• Pathway interaction discovery: Integrative analysis facilitates the identification of co-expressed genes and pathways that interact with KRT8, such as the discovery that KRT8 may promote metastasis and trigger epithelial-mesenchymal transition through NF-κB signaling in lung adenocarcinoma .

What technical challenges should researchers anticipate when working with KRT8 antibodies in multiplexed assays?

Researchers working with KRT8 antibodies in multiplexed assays should anticipate and address several technical challenges:

• Antibody cross-reactivity: When combining KRT8 antibodies with other antibodies in multiplexed immunofluorescence or mass cytometry (CyTOF) assays, cross-reactivity must be rigorously assessed, particularly when using cocktail antibodies targeting both KRT8 and KRT18 .

• Spectral overlap: For fluorescence-based multiplexing, careful panel design is essential to minimize spectral overlap. Notably, conjugates of blue fluorescent dyes (CF®405S, CF®405M) are not recommended for detecting low-abundance targets due to lower fluorescence and potentially higher non-specific background .

• Panel design considerations: When incorporating KRT8 antibodies into multiplexed panels, consideration of epitope accessibility and antibody compatibility is essential. The cytoplasmic localization of KRT8 must inform panel design, particularly when combining with antibodies targeting nuclear or membrane proteins.

• Signal amplification requirements: Low expression levels in some contexts may necessitate signal amplification methods, though these must be compatible with multiplexed approaches.

• Data normalization: When quantifying KRT8 across multiple samples or experiments, appropriate normalization strategies must be employed to account for technical variations in staining intensity or detection sensitivity.