Recombinant Human C-C motif chemokine 25 protein (CCL25)

What is CCL25 and what are its primary biological functions?

Recombinant Human C-C motif chemokine 25 (CCL25) is a chemokine protein that plays significant roles in immune cell trafficking and regulation. As a chemotactic factor, CCL25 functions primarily as a potent chemoattractant for lymphocytes, particularly T cells, making it crucial for immune system development and function . The protein is expressed in specific tissues including the thymus and small intestine, suggesting tissue-specific immune regulatory functions . CCL25 demonstrates full biological activity in chemotaxis bioassays using human monocytes at an effective concentration range of 1.0-10 ng/ml . Unlike many other CC chemokines, CCL25 has less than 30% sequence identity with other members of its family and is encoded by a gene located on chromosome 19 in humans rather than clustering with other CC chemokine genes .

How is recombinant human CCL25 typically produced for research applications?

For research applications, recombinant human CCL25 is typically produced using E. coli expression systems. The protein usually covers the mature form (amino acids 24-150 or 21-151, depending on the manufacturer), which represents the biologically active region . The production process involves expressing the protein in bacterial systems followed by purification steps that typically include chromatographic techniques to achieve high purity (>97% as determined by SDS-PAGE and HPLC analysis) . Quality control measures include testing for endotoxin levels (maintained below 1.0 EU/μg using the LAL method) and biological activity verification through chemotaxis assays . The final product is commonly provided as a lyophilized powder that can be reconstituted with sterile water or appropriate buffer solutions for experimental use .

What receptors does CCL25 interact with and how do these interactions differ?

CCL25 primarily interacts with two receptors: CCR9A and CCR9B. These receptors are alternative splice variants that differ in their N-terminal regions . The functional characteristics of these interactions include:

This differential responsiveness suggests that CCR9A has a higher affinity for CCL25 than CCR9B . Flow cytometry experiments with calcium flux measurements have demonstrated that the EC50 for CCL25 is consistently lower on CCR9A-expressing cells compared to CCR9B-expressing cells, even when controlling for receptor expression levels . This indicates fundamental differences in the signaling properties of these two receptor variants that may have biological significance in different cellular contexts.

What are the recommended methods for handling and reconstituting lyophilized CCL25?

When working with lyophilized recombinant human CCL25, proper handling and reconstitution techniques are essential to maintain protein activity. The recommended protocol includes:

• Allow the lyophilized protein to reach room temperature before opening the vial

• Reconstitute in sterile water or appropriate buffer (PBS or HEPES buffer at pH 7.2-7.4) to a concentration of 0.1-1.0 mg/ml

• Gently swirl or rotate the vial until complete dissolution; avoid vigorous shaking or vortexing which can cause protein denaturation

• Allow the solution to stand for at least 15 minutes at room temperature to ensure complete reconstitution

• The reconstituted protein can be stored at 4°C for up to one week

• For longer storage, prepare small aliquots and store at -20°C or -80°C to avoid repeated freeze-thaw cycles which can compromise protein activity

When using CCL25 in chemotaxis assays, it's typically effective at concentrations ranging from 1.0-10 ng/ml, though this may vary depending on the specific cell type being studied .

How does CCL25 contribute to cancer migration and invasion mechanisms?

CCL25 has been shown to significantly enhance the migration and invasion capabilities of non-small cell lung cancer (NSCLC) cells through specific molecular pathways . The mechanism involves:

• Binding of CCL25 to CCR9 receptors on cancer cells

• Upregulation of key metastasis-associated factors, including:

  • Vascular endothelial growth factors (VEGF-C and VEGF-D)

  • Matrix metalloproteinases (MMP-1 and MMP-7)

Research using NSCLC cell lines (A549 and SK-MES-1) demonstrated that stimulation with 100 ng/ml CCL25 significantly increased both the transcriptional and protein expression levels of these factors . This upregulation was specifically mediated through CCL25/CCR9 interaction, as evidenced by the reversal of these effects when cells were pre-treated with 5.0 μg/ml anti-CCR9 antibody .

The migration and invasion assays showed that CCL25 functions as a potent chemoattractant for cancer cells, drawing them through Matrigel-coated membranes with 8-μm pores when added to the bottom chamber at 100 ng/ml . This effect was significantly inhibited in cells pre-incubated with anti-CCR9 antibody, confirming the specificity of the CCL25/CCR9 pathway in driving these metastatic behaviors .

What methodologies are effective for studying CCL25-mediated chemotaxis in different cell types?

Several methodologies have proven effective for studying CCL25-mediated chemotaxis, with specific protocols depending on the cell type under investigation:

• Transwell Migration Assay:

  • Use Matrigel invasion chambers with 8-μm pore size

  • Hydrate membrane with serum-free media for 2 hours at 37°C

  • Seed 1×10⁴ cells in the top chamber

  • Add CCL25 (100 ng/ml) to the bottom chamber as a chemoattractant

  • Incubate overnight at 37°C with 5% CO₂

  • Fix migrated cells with 100% methanol for 10 minutes

  • Stain with crystal violet for 2 minutes

  • Count migrated cells using light microscopy (200× magnification)

• Calcium Flux Assay:

  • Load cells (2×10⁷ cells/ml) with indo-1 acetoxymethylester (10 μM) and Pleuronic (300 μg/ml)

  • Incubate for 45 minutes at 30°C with occasional shaking

  • Wash twice with HBSS/FBS buffer

  • Perform measurements using flow cytometry with dual-wavelength detection (390/20 and 530/20 nm)

  • Record baseline, add sham injection at 30 seconds, then add CCL25 at various concentrations

  • Analyze data using appropriate flow cytometry software

• Receptor Antagonism Studies:

  • Pre-incubate cells with 5.0 μg/ml anti-CCR9 antibody

  • Conduct migration assays as described above

  • Compare migration in the presence of CCL25 with and without receptor blocking

These methodologies allow for comprehensive assessment of CCL25's chemotactic activity across different experimental conditions and cell types.

What is the role of CCL25 in enhancing immunotherapy against triple-negative breast cancer?

CCL25 has emerged as a promising enhancer of immunotherapy against triple-negative breast cancer (TNBC), particularly when combined with CD47-targeted approaches. Research has revealed that:

• CCL25 is not naturally expressed in human or murine TNBC tumors

• Intratumoral delivery of CCL25 can significantly enhance antitumor immunity

• CCR9⁺CD8⁺ T cells have elevated potential for activation and strong antitumor responses

A novel approach using tumor acidity-responsive nanoparticle delivery system (NP-siCD47/CCL25) has shown significant promise. This system:

• Sequentially releases CCL25 protein and CD47 small interfering RNA within the tumor

• Significantly increases tumor infiltration of CCR9⁺CD8⁺ T cells

• Down-regulates CD47 expression in the tumor microenvironment

• Inhibits tumor growth and metastasis through T cell-dependent immunity

• Shows synergistic enhancement when combined with PD-1/PD-L1 checkpoint blockade therapies

This research demonstrates that the strategic delivery of CCL25 to the tumor environment can overcome the natural absence of this chemokine in TNBC and effectively recruit antitumor T cells, representing a promising strategy to enhance cancer immunotherapy effectiveness.

How does CCL25/CCR9 signaling compare with other chemokine signaling pathways in disease contexts?

The CCL25/CCR9 signaling pathway exhibits several distinctive features compared to other chemokine pathways in disease contexts:

• Tissue Specificity: Unlike many chemokines with broad expression patterns, CCL25 expression is highly restricted, primarily to the thymus and small intestine, with limited expression in spleen after LPS challenge . This restricted expression pattern suggests specialized roles in T cell development and intestinal immunity.

• Genomic Organization: The CCL25 gene is not located within any of the known CC chemokine gene clusters, residing instead on chromosome 19 in humans, suggesting evolutionary divergence from other chemokines .

• Receptor Variants: The existence of two splice variants (CCR9A and CCR9B) with different affinities for CCL25 provides a level of signaling complexity and regulation not seen with all chemokines . The EC₅₀ values differ significantly between these variants, with CCR9A showing higher sensitivity to CCL25.

• Disease Relevance: In cancer contexts, particularly NSCLC, the CCL25/CCR9 axis shows remarkably low mutation rates compared to other signaling pathways like EGFR . This genetic stability makes it potentially advantageous as a therapeutic target that might be less prone to developing resistance mechanisms.

• Therapeutic Application: Unlike some chemokines that may promote tumor growth, engineered delivery of CCL25 in TNBC can enhance anti-tumor immunity, suggesting context-dependent effects that can be therapeutically exploited .

This unique profile positions the CCL25/CCR9 axis as a specialized signaling pathway with distinct regulatory mechanisms and therapeutic implications that differ from more broadly expressed chemokines.

What are the optimal experimental conditions for evaluating CCL25-induced signaling in primary versus transformed cells?

When evaluating CCL25-induced signaling, experimental conditions must be optimized differently for primary cells versus transformed cell lines:

For Primary Cells (e.g., primary T cells, monocytes):

• Cell Isolation: Use negative selection methods to avoid pre-activation through surface receptor binding

• Culture Conditions: Short-term culture (24-48 hours) in serum-free or low-serum (0.5-1% FBS) media to minimize background signaling

• CCL25 Concentration Range: 1-100 ng/ml, with dose-response curves recommended

• Timing: Examine early signaling events (calcium flux) within seconds to minutes and later events (gene expression) at 6-24 hours

• Controls: Include both positive controls (other established chemokines) and negative controls (heat-inactivated CCL25)

For Transformed Cells (e.g., cancer cell lines):

• Cell Preparation: Serum starvation for 12-24 hours before CCL25 stimulation to synchronize cells and reduce background signaling

• CCL25 Concentration: Higher concentrations may be required (50-200 ng/ml) due to potential desensitization in cancer cells

• Incubation Time: Extended exposure (12-48 hours) may be necessary to observe phenotypic changes like migration and invasion

• Antagonism Studies: Include receptor blocking experiments (5.0 μg/ml anti-CCR9 antibody) to confirm specificity

• Endpoint Measurements: Focus on metastasis-related gene expression (VEGF-C, VEGF-D, MMP-1, MMP-7) and functional assays (migration, invasion)

Common Technical Considerations:

• Fresh preparation of CCL25 solution is recommended for each experiment

• For calcium flux assays, maintain consistent temperature (room temperature) and timing of measurements

• When examining receptor desensitization, sequential stimulation protocols with increasing concentrations provide valuable insights

• For gene expression studies, validate findings at both mRNA and protein levels

These optimized conditions account for the biological differences between primary and transformed cells, enabling more accurate evaluation of CCL25 signaling in different experimental contexts.

What are the current challenges and contradictions in CCL25 research regarding its pro-tumor versus anti-tumor effects?

Research on CCL25 presents several challenges and apparent contradictions regarding its role in cancer, particularly concerning its pro-tumor versus anti-tumor effects:

Contradictory Effects:

Research Challenges and Limitations:

• Context-Dependent Functions: The seemingly contradictory roles of CCL25 highlight its context-dependent functions, which remain incompletely understood. The same signaling pathway can promote tumor progression when acting on cancer cells but enhance anti-tumor immunity when directing immune cell infiltration.

• Technical Limitations: Current research is limited by:

  • Inconsistent CCL25 preparations across studies (variations in N-terminal sequence)

  • Differing experimental models (in vitro versus in vivo)

  • Variable receptor expression levels affecting sensitivity to CCL25

• Mechanistic Gaps: Several unanswered questions remain:

  • How does the tumor microenvironment modulate CCL25/CCR9 signaling?

  • What determines whether CCL25 will primarily affect tumor cells versus immune cells?

  • How do the two receptor variants (CCR9A and CCR9B) differentially influence cancer progression?

• Therapeutic Implications: These contradictions create challenges for therapeutic development:

  • Simple CCL25 administration might simultaneously promote both pro-tumor and anti-tumor mechanisms

  • Targeted delivery systems may be required to direct CCL25 effects toward desired cell populations

  • Combination approaches may be necessary to enhance beneficial effects while suppressing detrimental ones

• Low Mutation Rate Advantage: Despite these challenges, the low mutation rates of CCL25 and CCR9 compared to other therapeutic targets like EGFR suggest potential advantages for long-term therapeutic efficacy with potentially lower rates of resistance development .

These challenges point to the need for more comprehensive research that simultaneously examines CCL25 effects on both tumor and immune cells within the same experimental system, particularly in in vivo models that capture the complexity of the tumor microenvironment.