LGALS2 Antibody

What is LGALS2 and what are its primary functions in normal tissues?

LGALS2 encodes galectin-2, a member of the glycan-binding protein family. It functions primarily as a soluble beta-galactoside binding lectin found as a homodimer that can bind to lymphotoxin-alpha . LGALS2 is predominantly expressed in the gastrointestinal tract and is associated with several physiological processes :

• Regulation of collateral arteriogenesis

• Cell adhesion mechanisms

• T cell apoptosis modulation

• Involvement in mucosal immune system function

Immunohistochemical studies reveal that LGALS2 tends to aggregate around CD11b+ cells (including monocytes and macrophages) but is not directly expressed on these cells . This localization pattern suggests a role in intercellular communication within tissue microenvironments.

What are the optimal methods for detecting LGALS2 expression in experimental models?

Detection of LGALS2 can be accomplished through multiple complementary techniques:

For protein detection:

• Western blot analysis: Using specific anti-LGALS2 antibodies (1:1,000 dilution) followed by appropriate secondary antibodies and ECL Detection Systems

• Immunohistochemistry (IHC): Using antibodies at 1:50-1:200 dilution depending on the application

• ELISA: Particularly effective for quantitative measurement of LGALS2 in cell supernatants or serum

For mRNA detection:

• qRT-PCR using primers:
  Forward: 5'-ATGACGGGGGAACTTGAGGT-3'
  Reverse: 5'-CAGGTTCAGCTTGTCTGTCC-3'

For scoring IHC results, a standardized scoring system can be employed as follows:

The immunoreactivity score (IRS) is calculated by multiplying the proportion score by the intensity score (range: 0-12) .

How does LGALS2 contribute to immune escape mechanisms in triple-negative breast cancer?

LGALS2 has been identified as a key mediator of immune escape in TNBC through a complex mechanism involving tumor-associated macrophages:

• Macrophage recruitment and polarization: Tumor cell-intrinsic LGALS2 induces an increased number of tumor-associated macrophages and promotes their M2-like polarization through the CSF1/CSF1R axis

• Alteration of immune cell populations: LGALS2-overexpressing tumors exhibit:

  • Lower proportion of leukocytes (CD45+ cells)

  • Increased percentage of myeloid cells (CD45+CD11b+)

  • Decreased T cells (CD45+CD3+), B cells (CD45+CD19+), and NK cells (CD45+CD3-CD49b+)

  • Higher proportion of regulatory T cells (Tregs; CD45+CD3+CD4+CD25+FOXP3+)

  • Increased M2-like macrophages (CD45+CD11b+F4/80highCD206+)

  • Higher levels of myeloid-derived suppressor cells (MDSCs) (CD45+CD11b+Ly6g+)

• Impact on cytotoxic lymphocytes: LGALS2 overexpression significantly decreases the cytotoxic function of CTLs and NK cells, while increasing the exhaustion phenotype of CTLs

• Differential effects in vitro vs. in vivo: Notably, LGALS2 overexpression accelerates tumor growth in vivo but shows no effect on proliferation in vitro, indicating that its mechanisms are dependent on the tumor microenvironment

What is the relationship between LGALS2 and oxidative stress response in colorectal cancer?

Genome-wide CRISPR screens have identified LGALS2 as a significant regulator of oxidative stress response:

• Cell survival regulation: Depletion of LGALS2 renders cells resistant to H₂O₂ treatment, suggesting its role in oxidative stress-induced cell death mechanisms

• Paradoxical effects in colitis and cancer: LGALS2 deficiency:

  • Ameliorates DSS-induced acute colitis

  • Promotes colorectal tumor growth in the AOM/DSS-induced model

• Mechanistic explanation: This apparent paradox may be explained by:

  • LGALS2 inhibits epithelial cell proliferation or promotes their death under oxidative stress in acute colitis

  • LGALS2 suppresses tumor cell proliferation under oxidative stress, thus its disruption accelerates tumor growth

• Context-dependent effects: The dose of DSS used in experimental models significantly impacts outcomes - at lower doses used for tumor models, the impact of LGALS2 disruption on epithelial cell death and colitis development is less prominent

How do researchers reconcile contradictory findings regarding LGALS2's role across different cancer types?

LGALS2 exhibits seemingly contradictory roles across different cancer types, requiring careful consideration of context:

Researchers should consider:

• Tissue-specific effects: LGALS2 function depends on the tissue microenvironment and predominant immune cell populations

• Species differences: Different mouse strains exhibit varying susceptibility to experimental models (BALB/c vs. C57BL/6J)

• Methodological differences: Studies using exogenous LGALS2 treatment versus genetic disruption approaches yield different outcomes

• Clinical correlation: N stage and T stage in papillary thyroid carcinoma correlate significantly with LGALS2 expression (p=0.0020 and p=0.0029 respectively)

What are the best approaches for validating LGALS2 knockout efficiency in CRISPR experiments?

For rigorous validation of LGALS2 knockout using CRISPR-Cas9 systems:

• Genomic verification:

  • NGS to confirm on-target editing and assess potential off-target effects

  • T7 endonuclease assay to detect indels at the target site

• Protein level confirmation:

  • Western blot to confirm absence of LGALS2 protein expression

  • Multiple antibodies targeting different epitopes should be used

• Functional validation:

  • H₂O₂ challenge assays to confirm altered oxidative stress response

  • Cell viability measured by CCK8 assay following oxidative challenge

• Rescue experiments:

  • Reintroduction of LGALS2 expression to confirm phenotype reversibility

  • Use of multiple sgRNAs targeting different regions of LGALS2 to ensure consistency

How should researchers design co-culture experiments to examine LGALS2's effects on macrophages?

Based on successful protocols from published studies:

• Transwell co-culture setup:

  • Seed macrophages (1×10⁵) in the lower chamber 24 hours before co-culture

  • Add tumor cells (1000 cells) in the upper chamber

  • Collect macrophages after 72 hours for subsequent analysis including qPCR and flow cytometry

• Macrophage proliferation assessment:

  • Label macrophages with CFSE prior to co-culture

  • Quantify proliferation using flow cytometry analysis after co-culture period

• Controls to include:

  • LGALS2-overexpressing cells

  • LGALS2-knockout cells

  • Vector control cells

  • Transwell-only controls (no tumor cells)

• Analysis parameters:

  • M1/M2 polarization markers (CD206 for M2-like macrophages)

  • Cytokine production profile

  • Gene expression changes in macrophages post-coculture

What is the optimal approach for in vivo CRISPR screens targeting LGALS2 and related pathways?

For conducting effective in vivo CRISPR screens similar to those that identified LGALS2:

• Lentiviral vector design:

  • Clone sgRNAs targeting individual genes into lentiGuide-Puro vectors

  • For overexpression studies, clone corresponding DNA fragments into pCDH vectors

• Cell preparation for transplantation:

  • Viral transduction with centrifugation at 2000 rpm for 2 hours in the presence of polybrene

  • Selection with puromycin (5 μg/ml) for 7 days

  • Wash cells twice in sterile PBS and resuspend at 1.5×10⁷ cells/ml for transplantation

• Mouse model selection:

  • Use multiple mouse models with different immune-deficiency characteristics to capture multidimensional immune responses

  • Consider both BALB/c and C57BL/6J backgrounds for comprehensive analysis

• Analytical approaches:

  • Single-cell RNA sequencing to characterize changes in the transcriptome

  • Flow cytometry to analyze infiltrating immune cells in tumors

  • Use Cell Ranger and Seurat for cell type classification

How does LGALS2 expression correlate with clinical parameters in different cancer types?

In papillary thyroid carcinoma, LGALS2 expression shows significant correlation with nodal status and tumor stage:

These correlations suggest that higher LGALS2 expression is associated with earlier nodal stage (N0) and lower tumor stage (T1/T2), indicating its potential role as a tumor suppressor in this cancer type .

In breast cancer, LGALS2 appears to be downregulated at both mRNA and protein levels, suggesting it may play a vital role in breast cancer progression, with overexpression predicting better prognosis .

What are the promising therapeutic approaches targeting LGALS2 in cancer?

Based on preclinical studies, several therapeutic approaches show promise:

• LGALS2 inhibitory antibodies:

  • Blockade of LGALS2 using inhibitory antibodies successfully arrested tumor growth and reversed immune suppression in TNBC models

  • Provides theoretical basis for LGALS2 as a potential immunotherapy target

• Combination with immune checkpoint inhibitors:

  • Given the role of LGALS2 in immune escape mechanisms, combining LGALS2 blockade with established immune checkpoint inhibitors may enhance efficacy

  • Particularly relevant for TNBC, where current immune checkpoint inhibitors show limited response rates

• Targeting the CSF1/CSF1R axis:

  • LGALS2 mediates immune escape through the CSF1/CSF1R axis

  • Inhibitors of this pathway could potentially overcome LGALS2-mediated immunosuppression

• Context-dependent approaches:

  • In cancers where LGALS2 acts as a tumor suppressor (papillary thyroid carcinoma), strategies to enhance its expression or activity may be beneficial

  • In contexts where it promotes immune escape (TNBC), inhibition would be preferred