EGFL6 Mouse

What is EGFL6 and what are its primary functions in mouse models?

EGFL6 is a secreted protein belonging to the EGF-like protein family with demonstrated roles in angiogenesis and cellular proliferation. In mouse models, EGFL6 shows tissue-specific expression patterns with particularly high expression in tumor endothelial cells compared to wound or normal endothelial cells . Functionally, EGFL6 promotes angiogenesis in tumor microenvironments through multiple signaling pathways, including FGF-2/PDGFB/VEGFA mechanisms . Unlike some angiogenic factors that function broadly, EGFL6 appears to have context-dependent activity, with significant effects on tumor angiogenesis but minimal impact on normal wound healing processes, making it a potentially attractive therapeutic target . Mouse knockout models have validated EGFL6's role in promoting tumor growth specifically, as EGFL6-deficient mice show reduced tumor burden without compromised wound healing capabilities or observable developmental abnormalities .

How does EGFL6 expression vary between normal and cancerous tissues in mouse models?

EGFL6 expression demonstrates remarkable tissue specificity in mouse models. Significant upregulation occurs in tumor endothelial cells compared to wound endothelial cells or normal tissues . This differential expression pattern suggests tumor-specific regulatory mechanisms controlling EGFL6 transcription. Immunohistochemical analyses reveal strong EGFL6 expression in tumor vasculature of wild-type mice bearing ovarian or breast cancer, while expression is absent in EGFL6 knockout mice . Additionally, tumor tissues from mouse models with EGFL6 knockout show reduced expression of angiogenic factors including FGF2, PDGFB, and VEGFA, indicating EGFL6's position in angiogenic signaling hierarchies . The difference in expression between tumor and normal tissues provides an explanation for why EGFL6 blockade affects tumor growth while sparing normal physiological processes like wound healing .

What are effective methods for generating EGFL6 knockout or modified mice?

Multiple successful approaches have been documented for generating EGFL6-modified mouse models. The classical approach involves crossing floxed EGFL6 mice with Tie2-Cre transgenic mice to achieve endothelial-specific knockout . This strategy produces mice lacking EGFL6 expression specifically in endothelial cells while maintaining expression in other tissues, allowing researchers to evaluate the endothelial-specific functions of EGFL6. For constitutive expression models, researchers have successfully inserted EGFL6 expression cassettes at the Rosa26 locus and crossed these mice with CMV-CRE mice to drive broad expression . This approach enables the study of systemic EGFL6 overexpression effects, particularly on immune cell populations.
For cellular models, CRISPR/Cas9 technology has proven effective, with documented success using sgRNAs targeting exon 2 of human EGFL6 . The technique involves designing guide RNAs with online tools (e.g., http://crispr.mit.edu/), cloning them into lentiCRISPR V2 vectors, and packaging the construct into lentiviruses for cell transduction . Single cell clones must be isolated using limited dilution methods and validated through Western blotting and sequencing to confirm successful EGFL6 knockout . These complementary approaches provide researchers with options for studying EGFL6 in various contexts, from specific cell types to whole organisms.

What tumor models are most appropriate for studying EGFL6 functions in mice?

Several mouse tumor models have demonstrated utility for investigating EGFL6 functions. Orthotopic models using murine ID8 ovarian cancer cells or E0771 breast cancer cells injected into mice allow assessment of EGFL6's effects on tumor growth, angiogenesis, and metastasis . These models are particularly valuable for studying EGFL6's role in tumor progression within the appropriate tissue microenvironment. In the ovarian cancer context, both subcutaneous and intraperitoneal injection models have been employed successfully . Intraperitoneal injection of ID8 cells mimics peritoneal metastasis, with disease progression monitored through body weight (reflecting ascites accumulation) and counting of metastatic nodules on the peritoneal wall .
The 2F8c model has proven valuable for immunotherapy studies, as tumor expression of EGFL6 in this model induces resistance to anti-PD-L1 therapy . For subcutaneous xenograft models, protocols typically involve injecting 6×10^6 cancer cells with or without EGFL6 modification into the right shoulder of BALB/c nude mice . Tumor measurements should be taken every 3 days, with volume calculated using the formula V=0.52ab² (where a=tumor length, b=tumor width) . Additionally, the ID8 p53−/− Brca2−/− model has been used effectively to study EGFL6 neutralizing antibody therapy in combination with immune checkpoint inhibitors . These diverse models enable comprehensive investigation of EGFL6's roles in different cancer contexts and therapeutic scenarios.

How should EGFL6 expression and function be assessed in mouse tissues?

A multi-modal approach is recommended for comprehensive assessment of EGFL6 expression and function in mouse tissues. For expression analysis, immunohistochemistry using rabbit anti-human EGFL6 antibodies (such as Abcam, 1:100, Cat: ab140079) effectively visualizes EGFL6 protein distribution within tissues . This should be complemented with qRT-PCR to quantify EGFL6 mRNA levels, especially when comparing expression across different conditions or tissues. Western blotting provides further validation of protein expression and enables quantitative comparisons.
For functional assessment, multiple readouts should be employed. Tumor microvessel density quantification using CD31 staining serves as a primary indicator of EGFL6's angiogenic effects . Lymphatic vessel development can be assessed using LYVE1 antibody staining . Downstream signaling pathway analysis should include evaluation of FGF-2, PDGFB, and VEGFA expression, as these have been identified as mediators of EGFL6's effects . For immune-related functions, flow cytometry analysis of tumor-infiltrating immune cells is essential, with particular attention to CD11b+ myeloid cells, granulocytic cells (CD11b+Ly6G^Hi Ly6C^Lo), and monocytic cells . NanoString nCounter panels (such as the Mouse Myeloid Innate Immune Panel) can provide comprehensive transcriptional profiling of myeloid cells in EGFL6-modified models . Functional assays should include cell proliferation assays, migration assays (scratch wound or transwell), and colony formation assays to assess cellular responses to EGFL6 modification .

How does EGFL6 differentially regulate tumor angiogenesis versus wound healing?

EGFL6 exhibits a remarkable context-dependent function, promoting tumor angiogenesis while having minimal impact on wound healing processes. This differential effect appears to stem from distinct expression patterns and tissue-specific mechanisms. In tumor microenvironments, EGFL6 is significantly upregulated in endothelial cells, promoting a cascade of angiogenic signaling through FGF-2, PDGFB, and VEGFA pathways . Knockout studies demonstrate that EGFL6 deficiency results in reduced tumor growth and decreased microvessel density in cancer models .
In contrast, wound healing processes proceed normally in EGFL6 knockout mice, with no observable defects in recovery from ischemic injury . When hind limb ischemia was induced in wild-type and EGFL6 knockout mice through femoral artery ligation, blood flow recovery was comparable between both groups by 96 hours post-ligation . This differential effect likely stems from the absence of EGFL6 upregulation in wound endothelial cells, suggesting that wound healing relies on alternative angiogenic pathways independent of EGFL6. This selective requirement for EGFL6 in pathological but not physiological angiogenesis makes it an attractive therapeutic target, potentially allowing inhibition of tumor angiogenesis without compromising normal wound healing processes that plague current anti-angiogenic therapies .

What is the role of EGFL6 in modulating immune cell populations in the tumor microenvironment?

EGFL6 significantly influences myeloid cell populations in the tumor microenvironment, promoting an immunosuppressive phenotype. In mice with broad EGFL6 expression, flow cytometry analysis reveals increased CD11b+ cells in both bone marrow and spleen compared to wild-type controls . More detailed analysis shows specific expansion of granulocytic cells (CD11b+Ly6G^Hi Ly6C^Lo) in both compartments . Transcriptional profiling of bone marrow myeloid cells from EGFL6-expressing mice demonstrates upregulation of genes associated with granulocyte differentiation and function (Csfr3, Ncf2, Clec5a, Ceacam1) and monocyte markers (CD14) .
Direct stimulation of bone marrow CD11b+ cells with recombinant EGFL6 protein increases the number of CD11b+Ly6G^Hi Ly6C^Lo cells, confirming EGFL6's direct effect on myeloid differentiation . Furthermore, EGFL6 stimulation of myeloid-derived suppressor cells (MDSCs) increases expression of immunosuppressive factors including IL-10, enhancing their inhibitory capacity . In tumor models, EGFL6 expression increases intratumoral accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAMs) . These alterations in the tumor immune microenvironment likely contribute to EGFL6's tumor-promoting effects and resistance to immunotherapy, as demonstrated in the 2F8c mouse model where tumor EGFL6 expression induces resistance to anti-PD-L1 therapy .

What signaling pathways are regulated by EGFL6 in tumor microenvironments?

EGFL6 activates multiple signaling cascades in the tumor microenvironment, with primary effects on angiogenic and immunomodulatory pathways. In tumor endothelial cells, EGFL6 regulates the FGF-2/PDGFB/VEGFA signaling axis . Immunohistochemical analysis of tumors from EGFL6 knockout mice shows significantly reduced expression of these angiogenic factors compared to tumors from wild-type mice . This suggests EGFL6 functions as an upstream regulator of these critical angiogenic pathways.
In myeloid lineage cells, EGFL6 activates differentiation pathways that promote granulocytic phenotypes. Transcriptional analysis reveals EGFL6-induced upregulation of colony stimulator factor 3 receptor (Csfr3), neutrophil cytosolic factor 2 (Ncf2), C-type lectin domain containing 5A (Clec5a), and carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1) . These molecular changes promote the development of immunosuppressive myeloid cells that facilitate tumor progression.
EGFL6 also appears to influence pathways governing cell proliferation and migration, as knockout of EGFL6 in ovarian cancer cells reduces their proliferative and migratory capacity in vitro . The cumulative impact of these signaling effects creates a tumor microenvironment that supports cancer growth through enhanced angiogenesis and immune suppression, while simultaneously promoting tumor cell proliferation and migration capabilities.

How should researchers evaluate the specificity of EGFL6 knockout phenotypes?

Rigorous assessment of EGFL6 knockout specificity requires multiple complementary approaches to distinguish direct effects from potential compensatory mechanisms or off-target impacts. First, researchers should validate knockout efficiency through both protein and mRNA measurements, using Western blotting, immunohistochemistry, and qRT-PCR to confirm complete elimination of EGFL6 expression . Sequencing of the targeted locus is essential to confirm the exact genetic modification and rule out potential in-frame mutations that might produce truncated but partially functional proteins .
Appropriate controls are crucial for phenotypic analysis. When using Cre-loxP systems for tissue-specific knockout, Cre-only controls should be included to account for potential Cre toxicity effects . For CRISPR-based knockouts, multiple independent clones (such as the E10 and G11 clones described) should be analyzed to ensure observed phenotypes are consistent across different knockout lines rather than clone-specific artifacts . Rescue experiments, where EGFL6 expression is restored through exogenous introduction, provide powerful evidence that observed phenotypes are directly attributable to EGFL6 loss rather than off-target effects .
Researchers should also evaluate potential compensatory mechanisms by examining expression of related EGF family members or alternative angiogenic factors that might be upregulated in response to EGFL6 deletion. Finally, phenotypic assessment should span multiple biological contexts (in vitro, in vivo tumor models, wound healing models) to comprehensively understand EGFL6's context-dependent functions .

What controls are essential for EGFL6 functional studies in mouse models?

Robust controls are critical for accurate interpretation of EGFL6 functional studies. For genetic models, littermate controls should be used whenever possible to minimize background genetic variation . When using conditional knockout systems (e.g., Tie2-Cre;EGFL6^fl/fl), appropriate controls include both wild-type mice and Cre-positive mice without the floxed EGFL6 allele to account for potential Cre recombinase effects . For constitutive EGFL6 overexpression models using the Rosa26 locus, comparison with standard C57BL/6J (WT) mice is appropriate, but researchers should also consider using empty vector insertions at the same locus as additional controls .
In tumor models, multiple cell lines should be tested when possible to ensure observations are not cell line-specific. Both the 2F8c and ID8 models have demonstrated utility for studying EGFL6 functions . When performing transplantation experiments, age and sex-matched mice should be used, with consistent cell numbers and injection techniques across experimental groups . For immunotherapy studies, appropriate isotype control antibodies should be included alongside therapeutic antibodies .
When using recombinant EGFL6 protein for in vitro studies, heat-inactivated protein controls can help distinguish specific biological activities from non-specific effects . Dose-response experiments are essential to establish physiologically relevant concentrations. Finally, for angiogenesis studies, comparative analysis across multiple contexts (tumor angiogenesis versus wound healing or developmental angiogenesis) provides important insights into EGFL6's context-dependent functions .

How can researchers integrate multiple data types to comprehensively analyze EGFL6 function?

Comprehensive analysis of EGFL6 function requires integration of diverse data types across multiple experimental systems. Researchers should combine genetic, molecular, cellular, and in vivo approaches to build a complete picture of EGFL6 biology. For genetic analysis, CRISPR knockout models, conditional tissue-specific models, and overexpression systems provide complementary insights into EGFL6's functions in different contexts . These should be analyzed at both genomic levels (sequencing to confirm modifications) and transcriptomic levels (RNA-Seq or targeted panels like NanoString to assess downstream effects) .
Protein-level analyses through Western blotting, immunohistochemistry, and ELISA provide essential information about EGFL6 expression patterns and quantities . Cellular phenotyping through proliferation, migration, and colony formation assays reveal cell-autonomous effects of EGFL6, while co-culture systems can illuminate intercellular communication mechanisms . Flow cytometry analysis of immune cell populations offers critical insights into EGFL6's immunomodulatory functions .
In vivo models bridge the cellular observations to organismal biology, with tumor growth, metastasis, and survival endpoints providing functional readouts . Advanced imaging techniques like immunohistochemical staining for CD31 (blood vessels) and LYVE1 (lymphatic vessels) allow visualization of EGFL6's effects on tumor vasculature . Integration of these multi-modal data requires sophisticated bioinformatic approaches, potentially including pathway analysis, gene set enrichment analysis, and network modeling to identify key nodes in EGFL6-regulated processes.

What are the most promising therapeutic applications of EGFL6 research in mouse models?

EGFL6 research in mouse models reveals several promising therapeutic directions. The most compelling application involves EGFL6 neutralizing antibody therapy, which has shown efficacy in enhancing immune checkpoint inhibitor treatment in multiple mouse models . The selective role of EGFL6 in tumor angiogenesis versus normal wound healing presents a significant advantage over existing anti-angiogenic therapies, potentially reducing side effects while maintaining anti-tumor efficacy . Development of humanized anti-EGFL6 antibodies could translate these findings toward clinical applications.
RNA interference approaches using nanoparticle delivery systems represent another promising avenue. Existing research demonstrates that targeting EGFL6 in tumor endothelial cells reduces tumor angiogenesis and growth, suggesting that endothelial-targeted delivery of EGFL6-specific siRNAs could provide therapeutic benefit . The parallel development of small molecule inhibitors targeting EGFL6 or its downstream signaling pathways might offer additional therapeutic options with potentially favorable pharmacokinetic properties.
The immunomodulatory effects of EGFL6, particularly its ability to increase myeloid-derived suppressor cells, suggest that EGFL6 blockade might enhance responses to immunotherapy . Combined approaches targeting both EGFL6 and immune checkpoint pathways showed enhanced efficacy in mouse models, pointing toward rational combination strategies for clinical development . Future therapeutic applications should focus on patient selection biomarkers, optimal combination strategies, and mechanisms to overcome potential resistance mechanisms to EGFL6-targeted therapies.

What are key unresolved questions about EGFL6 regulation and function in mice?

Despite significant advances, several critical questions about EGFL6 biology remain unanswered. The precise molecular mechanisms regulating EGFL6 expression in tumor endothelial cells versus normal endothelial cells remain poorly defined. Understanding these regulatory pathways could identify upstream targets for modulating EGFL6 expression selectively in tumors. The receptor(s) through which EGFL6 signals in different cell types (endothelial cells, myeloid cells, tumor cells) has not been fully characterized, representing a significant knowledge gap .
The relationship between EGFL6 and X-chromosome inactivation requires further investigation, particularly since EGFL6 is located in a region containing a TAD spanning from EGFL6 to GEMIN8, which may coordinate regulation . How this genomic organization influences EGFL6 expression across different tissues and species remains unclear. Additionally, the potential cross-talk between EGFL6 and other angiogenic factors deserves deeper exploration, as redundancy or complementarity in these pathways could influence therapeutic targeting strategies.
The long-term effects of EGFL6 deletion or inhibition require investigation, particularly regarding potential compensatory mechanisms that might emerge during extended treatment periods. Finally, the role of EGFL6 in metastasis formation and progression beyond primary tumor growth represents an important area for future research, as metastasis remains the primary cause of cancer-related mortality.

How might EGFL6 research in mouse models translate to human disease?

Translation of EGFL6 research from mouse models to human applications requires careful consideration of several factors. Cross-species conservation analysis indicates that while EGFL6 is present across mammals, its regulation may differ between species . In particular, X-chromosome inactivation patterns for EGFL6 may vary, potentially affecting expression levels and tissue distribution in humans versus mice . Researchers should validate key findings in human tissue samples and cell lines before advancing therapeutic development.
Encouragingly, studies utilizing human ovarian cancer cell lines like SKOV3 with EGFL6 knockout demonstrate similar effects to those observed in mouse models, suggesting functional conservation . Expression analysis of EGFL6 in human tumor samples compared to normal tissues will be essential to confirm the differential expression pattern observed in mice. Humanized mouse models, where human immune cells are introduced into immunodeficient mice, could provide valuable insights into how EGFL6-targeted therapies might interact with human immune components. For therapeutic development, human-compatible antibodies against EGFL6 need validation in appropriate preclinical models before clinical testing. Biomarker development to identify patients likely to benefit from EGFL6-targeted therapies represents another crucial translational step. Potential biomarkers might include EGFL6 expression levels in tumor samples, patterns of tumor vascularization, or immune cell profiles. Finally, careful assessment of potential toxicities, particularly effects on wound healing and normal angiogenesis in humans, will be essential before clinical implementation of EGFL6-targeting strategies.