ECI2 Antibody

What is the optimal protocol for ECI2 antibody validation in colorectal cancer tissues?

Rigorous validation of ECI2 antibodies for colorectal cancer research requires a multi-technique approach. Based on published protocols, researchers should:

• Perform Western blot analysis using both positive controls (normal colorectal epithelial cells like FHC that express high levels of ECI2) and negative controls (CRC cell lines with known low ECI2 expression such as HCT116 and RKO) .

• Conduct immunofluorescence experiments to confirm subcellular localization in both mitochondria and peroxisomes using co-localization markers .

• Validate with immunohistochemistry on paired normal and CRC tissues, comparing staining intensity and patterns .

• Implement siRNA knockdown experiments followed by antibody testing to confirm specificity .

Researchers should observe a distinct band at the expected molecular weight (~43 kDa) in Western blots, with stronger signals in normal tissues compared to CRC tissues. Immunofluorescence should reveal both mitochondrial and peroxisomal localization patterns.

How can researchers accurately quantify ECI2 expression levels in clinical specimens?

For quantitative assessment of ECI2 expression in clinical specimens, researchers should employ:

• RT-qPCR with carefully validated primers specific to ECI2 mRNA transcripts .

• Western blot analysis with densitometric quantification normalized to appropriate housekeeping proteins .

• Immunohistochemistry scoring based on both staining intensity and percentage of positive cells .

The established workflow based on recent studies includes:

• Tissue processing: Fresh tissue preservation in RNAlater for RNA extraction or flash-freezing for protein analysis

• RNA analysis: TRIzol extraction followed by cDNA synthesis and qPCR with ECI2-specific primers

• Protein analysis: RIPA buffer extraction followed by SDS-PAGE and immunoblotting

• Normalization: GAPDH or β-actin for loading control

When analyzing patient cohorts, researchers should stratify cases as "ECI2-high" and "ECI2-low" based on median expression values from the cohort to enable accurate survival analysis and clinicopathological correlations .

What controls are essential when using ECI2 antibodies in immunofluorescence experiments?

Essential controls for ECI2 immunofluorescence experiments include:

• Positive cellular controls: Normal colorectal epithelial cells (FHC) known to express high levels of ECI2 .

• Negative cellular controls: Validated ECI2-knockdown cell lines or cell lines with naturally low ECI2 expression .

• Peptide competition controls: Pre-incubation of the antibody with purified ECI2 peptide to verify binding specificity.

• Subcellular localization controls: Co-staining with mitochondrial markers (e.g., MitoTracker) and peroxisomal markers (e.g., PMP70) .

• Secondary antibody-only controls: To detect non-specific binding.

When conducting dual-staining experiments for ECI2 localization, researchers should observe distinct co-localization patterns in both mitochondria and peroxisomes. In SW620 cells, for example, immunofluorescence experiments have successfully demonstrated this dual subcellular distribution pattern .

How can ECI2 antibodies be used to investigate the relationship between ether lipid metabolism and NETosis in colorectal cancer?

Investigating the ECI2-ether lipid-NETosis axis requires a sophisticated experimental approach:

• Co-culture systems: Establish neutrophil-CRC cell co-cultures using dHL-60 cells (differentiated with 1.25% DMSO) and CRC cell lines with manipulated ECI2 expression .

• Neutrophil migration assays: Use conditioned media from ECI2-overexpressing or ECI2-silenced CRC cells to assess neutrophil chemotaxis .

• NETosis detection: Employ SYTOX Green immunofluorescence staining and MPO-DNA complex quantification to measure NET formation in response to different ECI2 expression levels .

• Ether lipid quantification: Use LC-MS/MS to measure ether lipid species (particularly plasmalogens) in ECI2-manipulated cells .

• Intervention experiments: Apply recombinant IL-8 (100 ng/ml optimal concentration) or anti-IL-8 antibodies to rescue or reverse phenotypic effects .

This experimental framework allows researchers to establish the mechanistic link between ECI2 expression, ether lipid metabolism, and subsequent neutrophil recruitment/NETosis. When ECI2 is silenced in CRC cells, increased ether lipid production leads to enhanced IL-8 expression, promoting neutrophil chemotaxis and NETosis, which can be reversed using anti-IL-8 interventions .

What are the methodological considerations for studying ECI2 interactions with alkylglycerone phosphate synthase (AGPS) in peroxisomes?

Investigating ECI2-AGPS interactions in peroxisomes requires specialized techniques:

• Peroxisomal fraction isolation: Use differential centrifugation with sucrose gradient to isolate intact peroxisomes from CRC cells with different ECI2 expression levels.

• Co-immunoprecipitation: Employ ECI2 antibodies to pull down protein complexes and probe for AGPS, or vice versa, to detect physical interactions .

• Proximity ligation assays: Visualize and quantify ECI2-AGPS interactions in situ within peroxisomes.

• FRET analysis: Use fluorescently tagged ECI2 and AGPS to measure interaction dynamics in living cells.

• Peroxisomal localization quantification: Develop a quantitative immunofluorescence approach to measure AGPS localization to peroxisomes in the presence or absence of ECI2 .

Research has shown that ECI2 inhibits ether lipid production by preventing the peroxisomal localization of AGPS, which is the rate-limiting enzyme for ether lipid synthesis in CRC cells . The inhibitory effect of ECI2 on AGPS localization represents a novel mechanism by which ECI2 suppresses ether lipid production, ultimately affecting IL-8 expression and neutrophil recruitment.

How can contradictions in ECI2 expression data between in vitro and in vivo models be reconciled?

Researchers have noted significant differences in ECI2 effects between in vitro and in vivo experiments. To reconcile these contradictions:

• Implement tumor microenvironment modeling: Use 3D organoid cultures or co-culture systems that incorporate immune components .

• Analyze immune infiltration: Compare neutrophil infiltration patterns in tumors with different ECI2 expression levels using immunohistochemistry or flow cytometry .

• Conduct matched in vitro and in vivo experiments: Use the same cell lines and genetic manipulations across experimental platforms, analyzing cells before and after in vivo growth .

• Apply transcriptomic analysis: Compare RNA-seq profiles from in vitro cultures versus in vivo tumors to identify differentially activated pathways .

Current research indicates that while ECI2 had minimal effects on CRC cell proliferation and invasion in vitro, it significantly inhibited tumor growth in vivo, with ECI2-silenced tumors showing increased growth . This discrepancy appears to be mediated through tumor microenvironment interactions, particularly neutrophil recruitment and NETosis, rather than direct effects on cancer cells .

How does ECI2 regulate the signaling pathways leading to IL-8 expression in colorectal cancer?

ECI2 regulation of IL-8 expression involves several interconnected signaling pathways:

• Ether lipid metabolism pathway: ECI2 inhibits peroxisomal localization of AGPS, reducing ether lipid production .

• Transcriptional regulation: RNA-seq analysis has shown that ECI2 affects cytokine-cytokine receptor interaction pathways, with IL-8 being specifically downregulated by ECI2 expression .

• Signal transduction cascade: ECI2 expression levels affect IL-8 production, which can be measured by ELISA in conditioned media from CRC cells .

Research methods to investigate this regulatory axis include:

• RNA-seq and pathway analysis to identify the cytokine-cytokine receptor interaction pathway as being significantly affected by ECI2 expression .

• RT-qPCR validation of differentially expressed genes, particularly IL-8 .

• ELISA assays to quantify secreted IL-8 protein levels in cell culture supernatants .

• Chromatin immunoprecipitation (ChIP) to assess transcription factor binding to the IL-8 promoter under different ECI2 conditions.

These interconnected pathways demonstrate how metabolic enzymes like ECI2 can influence inflammatory signaling in the tumor microenvironment, providing a mechanistic link between metabolism and immune regulation in cancer progression .

What is the prognostic significance of ECI2 expression in colorectal cancer patients?

ECI2 expression has emerged as a significant prognostic marker in colorectal cancer:

To properly assess ECI2 as a prognostic marker, researchers should:

• Use standardized immunohistochemistry scoring systems

• Apply multivariate Cox regression analysis to control for confounding factors

• Consider ECI2 in combination with established markers like TNM staging

• Validate findings across independent patient cohorts

The table below summarizes the relationship between ECI2 expression and clinicopathological parameters:

What molecular mechanisms explain ECI2's dual localization in mitochondria and peroxisomes?

The dual localization of ECI2 in both mitochondria and peroxisomes represents an intriguing aspect of its biology:

• Protein structure analysis: ECI2 contains targeting sequences for both mitochondria and peroxisomes, enabling its dual localization .

• Functional compartmentalization: In mitochondria, ECI2 primarily participates in the β-oxidation cycle, while in peroxisomes, it regulates ether lipid metabolism .

• Differential processing: Alternative splicing or post-translational modifications may direct ECI2 to different organelles.

Research approaches to investigate this dual localization include:

• Immunofluorescence co-localization studies with organelle-specific markers .

• Subcellular fractionation followed by Western blotting to quantify relative distribution.

• Mutation analysis of targeting sequences to determine their relative importance.

• Live-cell imaging with fluorescently tagged ECI2 to track dynamic distribution patterns.

Immunofluorescence experiments in SW620 CRC cells have confirmed this dual localization pattern, with ECI2 clearly detected in both mitochondria and peroxisomes . This dual localization may explain how ECI2 can influence both fatty acid metabolism (primarily in mitochondria) and ether lipid metabolism (primarily in peroxisomes), providing a molecular basis for its multifaceted roles in cellular metabolism and cancer progression .

What are the optimal experimental models for studying ECI2 function in neutrophil-cancer cell interactions?

Selecting appropriate experimental models is crucial for investigating ECI2-mediated neutrophil-cancer cell interactions:

• In vitro co-culture systems:

  • CRC cell lines (HCT116, RKO, DLD1, SW620) with manipulated ECI2 expression co-cultured with:

    • dHL-60 cells (differentiated with 1.25% DMSO)

    • Primary human neutrophils isolated from healthy donors

  • Validation of neutrophil differentiation should be performed using Giemsa staining and flow cytometry for neutrophil markers (CD11b, CD66b) .

• Ex vivo tissue explant cultures:

  • Fresh CRC tissue specimens cultured with autologous neutrophils

  • Preservation of tumor microenvironment architecture

• In vivo mouse models:

  • Intrasplenic injection of MC38 cells (mouse CRC cells) with manipulated ECI2 expression

  • Important consideration: IL-8 is not expressed in mice, but CXCL1/KC serves as a functional homolog

  • Neutrophil depletion using anti-Ly-6G antibodies to confirm neutrophil-dependent effects

• Analytical approaches:

  • Neutrophil chemotaxis assays using conditioned media from CRC cells

  • NETosis detection via SYTOX Green staining and MPO-DNA complex quantification

  • CRC cell proliferation and invasion assays in co-culture conditions

These models have successfully demonstrated that ECI2 expression in CRC cells affects neutrophil recruitment and NETosis, with downstream effects on cancer cell behavior that are only apparent in the presence of neutrophils or in vivo conditions .

How should researchers design experiments to investigate the relationship between ECI2, ether lipids, and IL-8 expression?

A comprehensive experimental design to investigate the ECI2-ether lipid-IL-8 axis should include:

• Genetic manipulation approaches:

  • Stable ECI2 overexpression in low-expressing CRC cell lines (HCT116, RKO)

  • Stable ECI2 silencing in high-expressing CRC cell lines (DLD1, SW620)

  • CRISPR/Cas9 knockout of ECI2 for complete loss-of-function studies

  • Rescue experiments with wild-type and mutant ECI2 constructs

• Ether lipid pathway analysis:

  • Measurement of ether lipid species by LC-MS/MS in cells with different ECI2 expression

  • Manipulation of AGPS expression or activity to assess interactions with ECI2 function

  • Peroxisomal localization studies of AGPS in relation to ECI2 expression

• IL-8 expression analysis:

  • RT-qPCR for IL-8 mRNA levels in response to ECI2 manipulation

  • ELISA for secreted IL-8 protein in conditioned media

  • Promoter-reporter assays to assess IL-8 transcriptional regulation

  • Chromatin immunoprecipitation to identify transcription factors involved

• Intervention experiments:

  • Addition of recombinant IL-8 (100 ng/ml optimal concentration) to ECI2-overexpressing cells

  • Application of anti-IL-8 antibodies to ECI2-silenced cells

  • Manipulation of ether lipid levels through AGPS inhibition or exogenous ether lipid addition

This experimental framework has successfully demonstrated that ECI2 inhibits ether lipid production by preventing AGPS peroxisomal localization, which in turn reduces IL-8 expression, neutrophil recruitment, and NETosis in CRC .

What technical challenges exist in quantifying neutrophil extracellular traps (NETs) in ECI2-related cancer research?

Accurate quantification of NETs in ECI2-related cancer research presents several technical challenges:

• Sample preparation challenges:

  • NETs are fragile structures easily disrupted during processing

  • Fixation methods can affect NET morphology and detection sensitivity

  • Tumor tissue processing may disrupt in situ NETs

• Detection method considerations:

  • SYTOX Green immunofluorescence staining for extracellular DNA visualization

  • MPO-DNA complex ELISA for quantitative assessment of NETs

  • Immunohistochemistry for citrullinated histone H3 (cit-H3) in tissue sections

  • Confocal microscopy for detailed structural analysis

• Quantification approach standardization:

  • Automated image analysis algorithms for consistent NET quantification

  • Standardized thresholding parameters to distinguish NETs from other DNA sources

  • Normalization strategies to account for neutrophil density differences

• Experimental validation strategies:

  • Use of NET inhibitors (e.g., DNase I) as negative controls

  • NET induction with PMA as positive control

  • Inclusion of neutrophils from healthy donors as reference standards

Current research has successfully employed SYTOX Green staining and MPO-DNA complex assays to demonstrate that conditioned media from ECI2-silenced CRC cells promotes NETosis, while media from ECI2-overexpressing cells inhibits NETosis . In vivo, citrullinated histone H3 (cit-H3) content in mouse serum and liver metastatic tissues provides a reliable measure of systemic and local NETosis activity .

How should researchers interpret discrepancies in ECI2 expression across different colorectal cancer datasets?

When confronted with discrepancies in ECI2 expression data across different datasets, researchers should consider:

• Dataset technical variations:

  • Platform differences (microarray vs. RNA-seq vs. proteomics)

  • Sample collection and processing protocols

  • Normalization methods and reference genes used

• Cohort demographic and clinical differences:

  • Patient ethnicity and geographical variations

  • Tumor stage and grade distributions within cohorts

  • Treatment history of patients included in datasets

  • Anatomical location of tumors (right vs. left colon vs. rectum)

• Analytical approaches:

  • Meta-analysis techniques to integrate multiple datasets

  • Standardization procedures to align different measurement scales

  • Stratification by clinical or molecular subtypes

  • Multivariate analysis to account for confounding variables

• Biological interpretation frameworks:

  • Consider ECI2 in relation to broader metabolic pathway alterations

  • Analyze correlations with immune infiltration patterns

  • Assess relationships with established CRC molecular subtypes

What are the best approaches for correlating ECI2 expression with neutrophil infiltration in tumor tissues?

Optimal approaches for correlating ECI2 expression with neutrophil infiltration include:

• Multiplex immunohistochemistry/immunofluorescence:

  • Simultaneous detection of ECI2 and neutrophil markers (CD66b, MPO) on the same tissue section

  • Spatial relationship analysis between ECI2-expressing tumor cells and infiltrating neutrophils

  • Digital pathology quantification using automated image analysis platforms

• Sequential tissue section analysis:

  • ECI2 staining on one section with neutrophil marker staining on adjacent sections

  • Registration of sequential images for correlation analysis

  • Quantification of neutrophil density in relation to ECI2 expression levels

• Flow cytometry of dissociated tumors:

  • Single-cell suspensions analyzed for ECI2 expression in tumor cells

  • Simultaneous quantification of neutrophil populations (CD45+CD11b+CD66b+)

  • Correlation of ECI2 expression levels with neutrophil percentages

• Transcriptomic analysis approaches:

  • RNA-seq data deconvolution to estimate immune cell proportions

  • Correlation of ECI2 expression with neutrophil gene signatures

  • Integration with spatial transcriptomics for localization information

Research has demonstrated a significant negative correlation between ECI2 expression and neutrophil infiltration in CRC tissues . Immunohistochemistry studies have shown that neutrophil infiltration is most pronounced in CRC tissues with low ECI2 expression, and the number of infiltrating neutrophils is negatively correlated with ECI2 expression levels .

How can researchers differentiate between direct and indirect effects of ECI2 on neutrophil function in the tumor microenvironment?

Differentiating between direct and indirect effects of ECI2 on neutrophil function requires sophisticated experimental designs:

• Conditioned media experiments:

  • Collect conditioned media from CRC cells with different ECI2 expression levels

  • Apply conditioned media to neutrophils and assess functional responses

  • Fractionate conditioned media to identify active components (e.g., IL-8)

• Transwell co-culture systems:

  • Physical separation of CRC cells and neutrophils while allowing soluble factor exchange

  • Comparison with direct co-culture to assess contact-dependent effects

  • Selective inhibition of candidate mediators (e.g., anti-IL-8)

• Neutrophil-specific receptor blockade:

  • Inhibition of IL-8 receptors (CXCR1/2) on neutrophils

  • Assessment of neutrophil migration and NETosis in response to CRC-derived factors

  • Comparison between wild-type and receptor-blocked neutrophils

• Direct ECI2 manipulation in neutrophils:

  • Overexpression or silencing of ECI2 in neutrophils themselves

  • Assessment of intrinsic neutrophil functions independent of tumor-derived signals

  • Comparison with effects observed in tumor-neutrophil co-culture systems

Current research indicates that ECI2 primarily exerts indirect effects on neutrophils through regulating IL-8 expression in CRC cells . Neutrophil chemotaxis assays have shown that conditioned medium from CRC cells overexpressing ECI2 significantly inhibits neutrophil migration, which can be reversed by adding recombinant IL-8 . Similarly, the enhanced neutrophil migration induced by conditioned medium from ECI2-silenced CRC cells can be reversed by anti-IL-8 antibodies .