FLT4 Antibody

Introduction to FLT4/VEGFR3

FLT4 (Fms-like tyrosine kinase 4), commonly known as VEGFR3 (Vascular Endothelial Growth Factor Receptor 3), belongs to the class III subfamily of receptor tyrosine kinases (RTKs). This receptor, along with VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1), plays a fundamental role in vascular development. FLT4 functions as a cell-surface receptor primarily for VEGFC and VEGFD growth factors, making it essential for lymphangiogenesis and the development of the vascular network during embryonic development .

FLT4 plays an essential role in promoting proliferation, survival, and migration of endothelial cells. The receptor's signaling creates a positive feedback loop by enhancing production of VEGFC and, to a lesser extent, VEGFA, thereby amplifying FLT4 signaling pathways. Additionally, FLT4 can form heterodimers with KDR (VEGFR2), which modulates KDR signaling and influences vascular development .

Molecular Structure

FLT4 possesses a complex protein structure that facilitates its function as a signaling receptor. The human FLT4 gene encodes a 1298 amino acid precursor with a 24-amino acid signal peptide. The mature form of FLT4 consists of:

• A 751-amino acid extracellular domain containing seven immunoglobulin-like repeats

• A 22-amino acid transmembrane domain

• A 482-amino acid cytoplasmic domain with tyrosine kinase activity

One distinctive feature of FLT4 is the proteolytic cleavage that occurs in its fifth immunoglobulin homology domain. This cleavage results in polypeptides that remain connected by two disulfide bonds, creating a unique structural configuration compared to other VEGF receptors .

Signaling Pathways

Activation of FLT4 through binding of its ligands (VEGFC and VEGFD) initiates several important signaling cascades. The binding triggers the activation of multiple downstream pathways, including:

• MAPK1/ERK2 and MAPK3/ERK1 signaling

• MAPK8 and JUN signaling pathways

• AKT1 signaling pathway

FLT4 also mediates phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, and promotes the phosphorylation of MAPK8 at Thr-183 and Tyr-185, as well as AKT1 at Ser-473. These pathways collectively contribute to the cellular responses associated with lymphangiogenesis and vascular development .

Types of FLT4 Antibodies

FLT4 antibodies are available in various formats to accommodate different research applications. These antibodies are typically classified based on their host species, clonality, and conjugation status:

Several commercial sources provide FLT4 antibodies with varying specifications. For instance, the FLT4 Rabbit Polyclonal Antibody (CAB5605) targets amino acids 330-553 of human VEGFR3/FLT4 and has been validated for Western blot, IHC-P, IF/ICC, and ELISA applications .

Detection Methods and Protocols

FLT4 antibodies serve as valuable tools in multiple laboratory techniques for detecting and analyzing FLT4 expression and function:

Western Blot Analysis

Western blot is commonly used to detect FLT4 protein expression in cell and tissue lysates. FLT4 is typically observed as bands with different molecular weights due to glycosylation:

• ~175 kDa precursor form

• ~195 kDa mature form

• ~140 kDa non-glycosylated backbone

• ~125 kDa partially cleaved form

For Western blot applications, antibodies are typically used at dilutions ranging from 1:100 to 1:1000, depending on the specific antibody and sample type .

Immunohistochemistry

In immunohistochemistry applications, FLT4 antibodies can be used to visualize the expression of FLT4 in tissue sections, particularly in lymphatic vessels. These antibodies are valuable for distinguishing lymphatic vessels from blood vessels in histological samples, aiding in the study of lymphangiogenesis in normal development and pathological conditions .

Immunofluorescence/Immunocytochemistry

FLT4 antibodies are also used for immunofluorescence and immunocytochemistry to detect FLT4 in cultured cells. For example, the Human VEGFR3/Flt-4 Antibody has been used to detect VEGFR3/Flt-4 in HUVEC human umbilical vein endothelial cells, showing specific staining localized to cell surfaces and cytoplasm .

Research Applications

FLT4 antibodies have been extensively used in various research areas:

Vascular Biology Research

FLT4 antibodies are essential tools for studying lymphatic vessel development and function. They help researchers distinguish between blood and lymphatic vessels, enabling detailed analysis of lymphangiogenesis during development and in pathological conditions .

Cancer Research

Dysregulation of FLT4 signaling has been implicated in various cancers, particularly in promoting lymphatic metastasis. FLT4 antibodies are used to study the role of FLT4 in cancer progression and metastasis. For instance, research has shown connections between FLT4 and papillary thyroid carcinoma lymphatic metastasis through the VEGF-C and VEGFR-3 axis .

Immunological Research

Recent studies have utilized FLT4 antibodies to investigate the role of FLT4 in immune cell function. For example, research on acute myeloid leukemia has shown that targeting FLT4 can restore dysfunctional NK cells and T cells, suggesting a potential therapeutic approach .

FLT4 in Disease Pathology

FLT4 dysregulation has been implicated in several pathological conditions:

• Lymphedema: Defects in FLT4 cause hereditary lymphedema type 1 (LYH1A), a condition characterized by chronic swelling due to lymphatic vessel dysfunction .

• Cancer Metastasis: Increased FLT4 expression in tumors correlates with lymphatic metastasis in various cancers, making it a potential target for anti-metastatic therapies .

• Juvenile Hemangioma: Defects in FLT4 are found in juvenile hemangioma, suggesting its role in vascular malformations .

FLT4-targeting Peptides and Therapies

Recent research has explored the therapeutic potential of targeting FLT4. A study developed 12 peptides targeting FLT4 for clinical application, with particular focus on peptide P4 from the intracellular domain of FLT4, which demonstrated dominant negative activity .

The research showed that similar to the FLT4 antagonist MAZ51, high IFN-γ levels were expressed in acute myeloid leukemia mononuclear cells exposed to P4. Additionally, T and NK cell levels were restored, as were high IFN-γ levels, in a leukemic environment when P4 was co-cultured with cytosine β-D-arabinofuranoside .

Interestingly, the frequency of regulatory T cells was significantly decreased by P4, suggesting that FLT4-targeting peptides play a role in modulating the tumor microenvironment. This research demonstrates the potential of functionally modulating lymphocytes using peptides targeting FLT4 and proposes the development of advanced therapeutic approaches against acute myeloid leukemia by using immune cells .

Optimal Conditions and Protocols

For optimal results with FLT4 antibodies, researchers should consider the following technical aspects:

Dilution Ratios

Different applications require specific antibody dilutions:

• Western Blot: 1:100 - 1:1000

• IHC-P: 1:50 - 1:100

• IF/ICC: 1:50 - 1:200

• ELISA: Application-specific dilutions

Cross-Reactivity and Specificity

Cross-reactivity is an important consideration when selecting FLT4 antibodies:

• Some antibodies show cross-reactivity between species. For example, Human VEGFR3/Flt-4 Antibody shows approximately 15% cross-reactivity with recombinant mouse VEGFR3 in Western blots .

• Mouse VEGFR3/Flt-4 PE-conjugated Antibody detects mouse VEGF R3/Flt-4 in direct ELISAs and Western blots, with approximately 20% cross-reactivity with recombinant human VEGF R3 .

• Specificity can vary between antibodies, with some showing non-specific binding to proteins of lower molecular weight .

When selecting an FLT4 antibody, researchers should carefully consider these factors to ensure accurate and reliable results.

Performance Evaluation

Different commercial FLT4 antibodies have been evaluated for their performance in various applications:

Performance evaluations indicate that most commercial antibodies show good specificity and sensitivity, though optimal conditions may vary. For instance, a study using an antibody from Abcam reported excellent specificity towards FLT4, although some non-specificity was detected with proteins of much lower molecular weight .

Application-specific Recommendations

Based on the comparative analysis, here are recommendations for specific applications:

For Western Blot Analysis

The VEGFR3/FLT4 antibody (20712-1-AP) from Proteintech has shown reliable results in detecting FLT4 in various samples, including mouse spleen tissue, PC-3 cells, HeLa cells, and mouse heart tissue .

For Immunohistochemistry

The Human VEGFR3/Flt-4 Antibody from R&D Systems has been successfully used for immunohistochemistry in human cervical squamous metaplasia samples, producing clear staining when used with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit .

For Flow Cytometry

The APC anti-human VEGFR-3 (FLT-4) Antibody from BioLegend is specifically validated for flow cytometric analysis, with a recommended usage of 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood .

Emerging Applications

FLT4 antibody research continues to evolve with several promising directions:

Therapeutic Antibodies

The development of therapeutic antibodies targeting FLT4 represents an emerging area with potential applications in cancer treatment, particularly for preventing lymphatic metastasis. Current research on FLT4-targeting peptides provides a foundation for developing more specific and effective therapeutic antibodies .

Single-Cell Analysis

Advances in single-cell technologies are opening new possibilities for using FLT4 antibodies to study heterogeneity within lymphatic endothelial cells and to better understand the role of FLT4 in different cell populations and microenvironments .

Integration with Advanced Technologies

The integration of FLT4 antibodies with advanced technologies is expanding their research applications:

Antibody Engineering

Engineered FLT4 antibodies with enhanced specificity, stability, and functionality are being developed to improve research outcomes and therapeutic potential. These engineered antibodies may include bispecific antibodies that simultaneously target FLT4 and other relevant molecules .

Imaging Technologies

The combination of FLT4 antibodies with advanced imaging technologies, such as intravital microscopy and whole-mount imaging, is enabling more detailed studies of lymphatic vessel development and function in real-time and in three dimensions .