LMO4 Antibody

What is the biological significance of LMO4 and why is it an important research target?

LMO4 belongs to the LIM-only subfamily of LIM domain-containing transcription factors that mediate protein-protein interactions within multi-protein complexes. It plays critical roles in regulating cell proliferation and mammalian development. LMO4 is highly expressed within specific cell types in diverse epithelial-derived tissues, including the mammary gland, tongue, skin, small intestine, lung, and brain . The protein predominantly localizes to the nucleus where it controls the expression of genes involved in various biological functions .

Research interest in LMO4 stems from its involvement in several cancers—notably breast cancer, where it is overexpressed in approximately 56-62% of primary invasive carcinomas as confirmed by both in situ hybridization and immunohistochemistry techniques . More recently, LMO4 has emerged as a significant factor in enhancing CD8+ T-cell stemness and antitumor immunity, suggesting its potential role in immunotherapy applications .

What are the recommended experimental methods for detecting LMO4 expression in tissue samples?

For accurate detection of LMO4 in tissue samples, a multi-modal approach is recommended:

• In situ hybridization: This technique allows visualization of LMO4 mRNA expression patterns directly in tissue sections. Studies have successfully employed riboprobes specific to human LMO4 with appropriate sense probe controls to eliminate background signal . This method provides valuable spatial information about LMO4 expression within different cell types.

• Immunohistochemistry: For protein detection, immunohistochemistry using well-validated anti-LMO4 monoclonal antibodies has proven effective. As demonstrated in studies of breast cancer tissues, immunohistochemical staining can help categorize samples based on expression intensity (low/negative, moderate, or high) . When performing this technique, include appropriate anti-Ig antibody controls to account for non-specific binding.

• Correlation analysis: To strengthen your findings, correlate RNA and protein expression data. Research has shown strong correlation between RNA and protein overexpression in approximately 71% of breast cancer cases, though discordance may occur due to post-transcriptional regulatory mechanisms .

How should researchers optimize LMO4 antibody-based immunohistochemistry protocols for different tissue types?

Optimizing LMO4 antibody-based immunohistochemistry requires careful consideration of tissue-specific factors:

• Fixation protocol selection: Different tissues require specific fixation approaches. For breast tissue samples, standard formalin fixation has been successfully employed in studies detecting LMO4 overexpression . For neural tissues, where LMO4 plays roles in development, more gentle fixation protocols may be necessary to preserve epitope accessibility.

• Antigen retrieval optimization: Test multiple antigen retrieval methods (heat-induced vs. enzymatic) with your specific LMO4 antibody. The optimal method should maximize signal while maintaining tissue morphology.

• Antibody validation: Confirm antibody specificity through western blotting of tissue lysates and using positive controls (e.g., breast cancer cell lines known to overexpress LMO4, such as those identified in previous studies where 5 of 10 human breast cancer cell lines showed LMO4 overexpression) .

• Signal amplification considerations: For tissues with lower expression levels, consider using signal amplification systems. Research has shown that normal breast tissue and benign fibroadenomas typically display low LMO4 levels (only 35% show detectable expression), which may require more sensitive detection methods .

What controls should be included when designing experiments using LMO4 antibodies?

Rigorous experimental design for LMO4 antibody applications should include:

• Positive tissue controls: Include tissues known to express high levels of LMO4, such as proliferating epithelial cells in the small intestine crypts or the basal cell layer of skin and tongue, which have been documented to express high levels of LMO4 .

• Negative controls: Include both technical negative controls (secondary antibody only) and biological negative controls (tissues with confirmed low/absent LMO4 expression).

• Isotype controls: Use matched isotype control antibodies at the same concentration as your primary LMO4 antibody to identify non-specific binding. Studies have demonstrated that anti-Ig antibody controls show negligible staining in tumor samples .

• RNA-protein correlation controls: When possible, parallel assessment of LMO4 mRNA (by in situ hybridization or RT-PCR) and protein levels can provide important validation, especially given the documented discordance between RNA and protein levels in some tumors .

How can researchers use LMO4 antibodies to investigate its role in regulating cancer cell invasion and migration?

To investigate LMO4's role in cancer invasion and migration, consider these methodological approaches:

• Gain and loss of function studies: Use stable cell lines with LMO4 overexpression or knockdown, validated by western blotting with LMO4 antibodies. This approach has been successfully employed in lung cancer cell lines to demonstrate LMO4's involvement in invasion and migration through the AKT/PI3K pathway .

• Immunofluorescence co-localization: Use dual-staining techniques with LMO4 antibodies and markers of epithelial-mesenchymal transition to visualize potential co-regulation. LMO4 has been found to interact with signaling pathways involved in epithelial-mesenchymal signaling, leading to increased stromal cell invasion and migration .

• Invasion/migration assays with immunoblotting: Perform transwell migration or wound healing assays, followed by western blot analysis using LMO4 antibodies to correlate invasion capacity with LMO4 expression levels. These techniques have helped establish LMO4's role in cancer progression in multiple cancer types.

• Pathway analysis: Since LMO4 affects lung cancer cell invasion and migration through the AKT/PI3K pathway , use phospho-specific antibodies for AKT/PI3K pathway components alongside LMO4 antibodies to map signaling dynamics.

How can LMO4 antibodies be applied in studying the relationship between LMO4 and CD8+ T-cell function in cancer immunotherapy?

To investigate LMO4's role in CD8+ T-cell function for immunotherapy applications:

• Flow cytometry with LMO4 antibodies: Develop multi-parameter flow cytometry panels that include LMO4 alongside T-cell stemness markers (TCF1, SALL1) and markers of T-cell differentiation stages. Recent research has shown that LMO4 is downregulated upon CD8+ T-cell activation but maintained under conditions facilitating stem-like T-cell formation .

• ChIP-seq analysis: Use chromatin immunoprecipitation with LMO4 antibodies followed by sequencing to identify LMO4 binding sites in T-cell genomes. This can reveal direct transcriptional targets and regulatory mechanisms that influence T-cell stemness.

• Co-immunoprecipitation studies: LMO4 has been shown to bind to JAK1 and potentiate STAT3 signaling in response to IL-21 . Use LMO4 antibodies for pull-down experiments followed by mass spectrometry or western blotting to identify novel interacting partners in different T-cell subsets.

• Functional validation in tumor models: When testing engineered T cells with LMO4 overexpression, employ LMO4 antibodies to monitor expression levels and correlate with functional outcomes. Research has demonstrated that LMO4 overexpression boosted CD8+ T-cell antitumor immunity, resulting in enhanced tumor regression in both syngeneic and xenograft tumor models .

Why might researchers observe discrepancies between LMO4 mRNA and protein levels, and how should these be addressed?

Discrepancies between LMO4 mRNA and protein levels have been documented in approximately 29% of breast tumors . These discrepancies present both challenges and biological insights:

• Post-transcriptional regulation: LMO4 protein levels may be regulated by mechanisms affecting mRNA stability, translation efficiency, or protein turnover. Similar to observations with Cyclin D1 in breast cancer, where protein degradation pathways are deregulated , LMO4 protein levels might be influenced by aberrant post-translational modifications or protein stability mechanisms.

• Technical considerations:

  • Ensure RNA preservation in your samples by checking RNA integrity

  • Validate antibody specificity through western blotting prior to immunohistochemistry

  • Consider the heterogeneity within tumor samples that might lead to sampling bias

• Methodological approach: When discrepancies occur, employ multiple techniques:

  • Quantitative PCR alongside western blotting

  • Polysome profiling to assess translation efficiency

  • Proteasome inhibitor studies to evaluate protein degradation rates

• Interpretation strategy: Rather than viewing discrepancies as technical failures, consider them potential indicators of post-transcriptional dysregulation. This approach has revealed important cancer biology insights, such as the deregulation of protein degradation pathways in breast cancer .

What are the key considerations when using LMO4 antibodies for multiplexed immunofluorescence applications?

When implementing multiplexed immunofluorescence with LMO4 antibodies:

• Antibody panel design:

  • Select antibody clones raised in different host species to avoid cross-reactivity

  • For studying mammary tissues, consider including markers for cell proliferation, as LMO4 is expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy

  • When investigating T-cell biology, include markers like TCF7, SOCS3, JUNB, and ZFP36, which are target genes induced by LMO4-mediated STAT3 signaling

• Epitope blocking and stripping protocols:

  • Validate complete stripping between rounds of antibody staining

  • Test for potential epitope masking effects when antibodies targeting physically proximal proteins are used

• Signal calibration:

  • Establish single-stain controls for spectral unmixing

  • Use tissues with known variable expression of LMO4 (e.g., different grades of breast cancer) for threshold determination

• Data analysis approach:

  • Develop quantitative image analysis workflows that account for subcellular localization of LMO4, which predominantly localizes to the nucleus

  • Consider spatial relationships between LMO4-expressing cells and other cell types in the tumor microenvironment

How can LMO4 antibodies be employed to investigate its potential as a therapeutic target in cancer?

To explore LMO4's therapeutic potential through antibody-based techniques:

• Patient stratification studies: Use validated LMO4 antibodies for immunohistochemical analysis of patient tumor samples to correlate expression levels with treatment outcomes. The observation that 56-62% of breast cancers overexpress LMO4 suggests potential utility as a stratification biomarker.

• Functional screening approaches:

  • Develop cell-based assays with readouts dependent on LMO4 function

  • Use LMO4 antibodies to validate target engagement by potential inhibitors

  • Employ proximity ligation assays to visualize disruption of LMO4 protein-protein interactions

• In vivo imaging: Develop fluorescently-labeled LMO4 antibody fragments for non-invasive imaging of LMO4 expression in tumor xenograft models, which could aid in monitoring therapy response.

• Antibody-drug conjugates: Explore the potential of using LMO4 antibodies for targeted delivery of cytotoxic agents to cancer cells, particularly in breast and lung cancers where LMO4 overexpression has been documented .

What methodological approaches should researchers use when studying the role of LMO4 in normal development versus cancer contexts?

To differentiate LMO4's roles in development versus cancer:

• Temporal expression profiling:

  • Use LMO4 antibodies for immunohistochemistry across developmental timepoints

  • Compare with expression in corresponding adult tissues and cancer samples

  • Research has established that Lmo4 is expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy and in proliferative cap cell layers of terminal end buds in peripubertal mammary glands

• Conditional genetic models:

  • Generate tissue-specific and temporally controlled Lmo4 knockout or overexpression models

  • Use LMO4 antibodies to validate model fidelity and study phenotypic consequences

  • Consider developmental aspects, as targeted deletion of Lmo4 in mice leads to complex phenotypic abnormalities including neural tube defects and perinatal lethality

• Single-cell analysis approaches:

  • Employ LMO4 antibodies in mass cytometry (CyTOF) or for immunofluorescence in spatial transcriptomics

  • This can reveal cell type-specific expression patterns in complex tissues

  • Studies have shown that high levels of Lmo4 are frequently observed in proliferating cells, such as crypt cells of the small intestine and basal cells of the skin and tongue

• Differentiation models:

  • Use in vitro differentiation systems (e.g., mammary epithelial cell models) coupled with LMO4 antibody-based detection methods

  • This approach has revealed that forced expression of LMO4 inhibits differentiation of mammary epithelial cells , suggesting a role in maintaining proliferation