CXCL16 Human

What is the structure of human CXCL16 and how does it differ from other chemokines?

Human CXCL16 is a type I membrane protein containing a non-ELR motif-containing CXC chemokine domain in its extracellular region. Unlike most chemokines that are secreted, CXCL16 is one of only two transmembrane chemokines in the superfamily, along with Fractalkine (CX3CL1) . The human CXCL16 gene encodes a 273 amino acid residue precursor protein with several distinct domains: a putative signal peptide, a CXC chemokine domain, a mucin-like spacer region, a transmembrane domain, and a cytoplasmic domain with a potential tyrosine phosphorylation and SH2 protein-binding site .

This unique structure allows CXCL16 to exist in both membrane-bound and soluble forms. The functional soluble form is shed from the cell surface as an approximately 35 kDa protein . The membrane-bound form functions as both a chemokine and a scavenger receptor, while the soluble form primarily acts as a traditional chemokine that can induce migration and promote proliferation of cells expressing its receptor, CXCR6.

CXCL16 has also been independently identified as SRPSOX (scavenger receptor that binds phosphatidylserine and oxidized lipoprotein), functioning as a specific receptor for OxLDL but not LDL or acetyl-LDL .

What cell types express CXCL16 and its receptor CXCR6?

CXCL16 has a broad expression pattern in human tissues and cells. Northern blot analysis has detected CXCL16 expression in various human organs except for brain, bone marrow, skeletal muscle, and colon . At the cellular level, flow cytometry has shown CXCL16 on the surface of:

• CD19+ B cells

• CD14+ monocytes/macrophages

• CD11c+ splenic and lymph node dendritic cells

Its receptor, CXCR6 (also known as Bonzo, STRL33, or TYMSTR), is expressed primarily on T cells, particularly within the effector/memory subset . In pathological conditions such as cancer, the expression pattern extends further, with both CXCL16 and CXCR6 detected on:

• Prostate cancer cells

• Adjacent T cells in prostate cancer specimens

• Multiple human cancers associated with inflammation

Immunohistochemistry, immunofluorescence, and confocal imaging of 121 human prostate specimens have confirmed the co-expression of CXCL16 and CXCR6 on both cancer cells and adjacent T cells . This co-expression pattern suggests potential autocrine and paracrine signaling mechanisms in the tumor microenvironment.

What are the functional differences between membrane-bound and soluble CXCL16?

The two forms of CXCL16 exhibit distinct functional properties:

Membrane-bound CXCL16:

• Functions as a scavenger receptor (SRPSOX) that binds phosphatidylserine and oxidized lipoprotein

• Acts as a specific receptor for OxLDL but not LDL or acetyl-LDL

• May have inhibitory effects on cell proliferation in some contexts, as observed in colon carcinoma cell lines

• Potentially mediates cell adhesion functions

Soluble CXCL16:

• Acts as a chemokine that induces migration of CXCR6-expressing cells

• Enhances the growth of CXCR6-expressing cancer cells

• Promotes the proliferation of primary CD4 T cells

• Has demonstrated ED50 values of 2.5-12 ng/mL for certain biological effects in one recombinant formulation or 10-50 ng/mL in another

The balance between these two forms likely has important biological consequences. The conversion from membrane-bound to soluble form through proteolytic cleavage represents a key regulatory mechanism that can shift CXCL16 function from adhesion/scavenging to chemotaxis/proliferation enhancement.

How does human CXCL16 compare to its murine counterpart?

Mouse and human CXCL16 share moderate sequence homology:

This level of conservation suggests that while the core chemokine function may be similar between species, there could be significant species-specific differences in regulation, expression patterns, or interaction with other molecules. Researchers should exercise caution when extrapolating findings from mouse models to human biology, as the moderate sequence divergence might translate to functional differences.

The specific regulatory elements controlling expression, the exact shedding mechanisms, and the downstream signaling pathways might differ between species, highlighting the importance of validating murine findings in human systems whenever possible.

What signaling pathways are activated by CXCL16/CXCR6 interaction in different cell types?

CXCL16 binding to CXCR6 activates various signaling pathways depending on the cell type:

In T cells:

• CXCR6 signaling is Gi/o-protein dependent, as demonstrated in migration assays with Jurkat E6.1 T cells transfected with CXCR6-YFP

• CXCL16/CXCR6 interaction enhances proliferation rather than affecting cell survival

• The proliferative effect is specific to dividing cells, suggesting cell cycle-specific signaling mechanisms

In cancer cells:

• CXCL16/CXCR6 signaling promotes cell growth and may contribute to tumor progression

• Expression levels correlate with post-inflammatory changes in the cancer stroma, indicating potential involvement in stromal remodeling pathways

How does CXCL16 contribute to inflammation-associated cancer development?

CXCL16 appears to promote inflammation-associated cancer development through multiple mechanisms:

• Direct effects on cancer cell growth: CXCL16 enhances the proliferation of CXCR6-expressing cancer cells .

• Recruitment and activation of inflammatory cells: CXCL16 induces migration of CXCR6-expressing cells, including T cells, which may contribute to the inflammatory tumor microenvironment .

• Enhancement of T cell proliferation: CXCL16 promotes the proliferation of primary CD4 T cells, potentially increasing cytokine production in the tumor microenvironment .

• Support of tumor angiogenesis: Depletion of CXCL16 in experimental models resulted in decreased numbers of ERG+ endothelial cells, suggesting a pro-angiogenic role .

• Promotion of macrophage infiltration: CXCL16 positively correlates with M2-macrophage infiltration, and its depletion led to decreased numbers of F4/80+ macrophages in tumors .

• Correlation with stromal changes: Expression of CXCL16 and CXCR6 correlates with post-inflammatory changes in the cancer stroma, as revealed by loss of alpha-smooth muscle actin .

These observations suggest that CXCL16 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects through both direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes .

What explains the contradictory roles of CXCL16 in different cancer types?

Interestingly, CXCL16 appears to have context-dependent roles in different cancer types:

Pro-tumorigenic in prostate cancer:

• CXCL16 and CXCR6 expression correlates with poor prognostic features (high-stage and high-grade)

• CXCL16 enhances the growth of CXCR6-expressing cancer cells

Anti-tumorigenic in colon cancer:

• Membrane-bound CXCL16 inhibited cell proliferation in colon carcinoma cell lines

• No association was found between CXCL16 expression and cancer stage

• A positive correlation was reported between CXCL16 expression and patient survival

Pro-tumorigenic in thyroid cancer:

• CXCL16 expression was associated with poor prognostic factors including higher TNM staging and the BRAF V600E mutation

• A 3-gene panel including CXCL16, AHNAK2, and THBS2 showed poor recurrence-free survival in patients with higher expression

• Depletion of CXCL16 in cell models led to delayed tumor growth with decreased angiogenesis and macrophage infiltration

These contradictions likely reflect "variable roles for inflammation in cancer depending on tumor type and stage of tumor development" . Factors that may explain these differences include:

• The predominant form of CXCL16 (membrane-bound vs. soluble) in different tumor types

• Variations in the inflammatory composition of different tumor microenvironments

• Cancer-specific genetic and epigenetic contexts that modify CXCL16 signaling

• Differences in the expression pattern of CXCR6 and other interacting molecules

What is the role of CXCL16 in T cell recruitment and function?

CXCL16 plays several important roles in T cell biology:

T cell recruitment:

• CXCR6-expressing T cells migrate toward CXCL16 in a Gi/o-protein dependent manner

• This chemotactic effect likely contributes to T cell recruitment to sites of CXCL16 expression, including tumors and inflammatory tissues

T cell proliferation:

• CXCL16 enhances the proliferation of CXCR6-expressing T cells

• This effect is dose-dependent and specific to CXCR6-expressing cells

• The proliferative effect specifically targets dividing cells rather than affecting cell survival

Autocrine production:

• Activated CD4+ T cells from the effector/memory subset, particularly those expressing CXCR6, can produce CXCL16 mRNA

• This suggests a potential autocrine loop that may amplify and sustain T cell responses

Experimental approaches:

• Primary CD4+ T cells can be stimulated with plate-bound OKT3 (10 μg/ml) with or without plate-bound CXCL16 (5 μg/ml) to study proliferation effects

• Migration assays can be performed using transwell chambers with CXCL16 in the lower chamber

These findings suggest that CXCL16 may play an important role in regulating T cell responses, particularly within the effector/memory subset, through both recruitment and expansion of CXCR6-expressing T cells.

What are the optimal protocols for studying CXCL16 in experimental systems?

Researchers have employed various methodological approaches to study CXCL16:

Detection of CXCL16 expression:

• Immunohistochemistry using goat anti-CXCL16 antibodies (biotinylated and non-biotinylated, R&D Systems)

• Immunofluorescence and confocal imaging for co-localization studies

• Flow cytometry for cell surface expression analysis

• Northern blot analysis for tissue expression patterns

Functional assays:

• Migration assays:

  • Cells are washed and resuspended at 1×10^7 cells/ml in chemotaxis medium (RPMI 1640, 0.5% bovine serum albumin, 25 mM HEPES)

  • 1×10^6 cells per 100 μl are loaded in the upper transwell chamber (5.0 μm pore size)

  • CXCL16 in 600 μl chemotaxis medium is added to the lower chambers

  • After 2 hours of incubation, migrating cells are counted using flow cytometry with counting beads

• Proliferation assays:

  • For T cells: CD4+ T cells are stimulated with plate-bound OKT3 (10 μg/ml) with or without plate-bound CXCL16 (5 μg/ml) for 3 days

  • Cell proliferation is assessed by flow cytometry with counting beads

  • Dead Cell Discriminator can be used to exclude non-viable cells

Modulating CXCL16 expression:

• Gene knockdown using shCXCL16 introduced into cell lines

• Recombinant protein application (either plate-bound or in solution)

Available reagents:

• Recombinant human CXCL16 proteins (with different amino acid spans: Asn30-Pro118 or Asn49-Thr224 )

• Antibodies for detection and blocking (goat anti-CXCL16, mouse anti-CXCR6, and rat anti-CXCL16)

How can researchers effectively model CXCL16 functions in cancer?

Several experimental models have been used to study CXCL16 in cancer:

Human cancer specimens:

• Immunohistochemistry and immunofluorescence with confocal imaging of human cancer specimens allows correlation of CXCL16/CXCR6 expression with clinical parameters

• One study examined 121 human prostate specimens and an additional 461 specimens covering 12 tumor types

Cell line screening:

• Screening for chemokine expression in cancer cell lines can identify CXCL16 as a consistently expressed chemokine

• Prostate cancer cell lines and xenografts were screened for 37 chemokines in one study

Genetic manipulation:

• shCXCL16 can be introduced into cancer cells to deplete endogenous CXCL16

• Cell lines can be transfected with CXCR6 to study receptor function

In vivo models:

• Genetically modified cancer cells (e.g., with shCXCL16) can be subcutaneously injected into athymic mice

• Tumor growth can be monitored and tumors analyzed for parameters such as:

  • Angiogenesis (ERG+ endothelial cells)

  • Macrophage infiltration (F4/80+ macrophages)

  • Gene expression changes

Gene expression analysis:

• Analysis of gene expression datasets (e.g., TCGA) allows correlation of CXCL16 expression with clinical parameters and other genes

• PTCs were divided into CXCL16 expression groups for analysis of clinical characteristics

These complementary approaches allow researchers to investigate CXCL16 function from molecular mechanisms to in vivo effects and clinical correlations.

What protocols should be followed when using recombinant CXCL16 in research?

When using recombinant human CXCL16 proteins, researchers should consider the following guidelines based on the search results:

Available formulations:

• E. coli-derived human CXCL16 protein (Asn30-Pro118)

• Histidine-tagged version (Asn49-Thr224, with a C-terminal 6-His tag)

• Carrier-free options without BSA for applications where BSA might interfere

Reconstitution and storage:

• Lyophilized protein with carrier: Reconstitute at 25 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• Carrier-free formulation: Reconstitute at 100 μg/mL in sterile PBS

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

Effective concentrations:

• For biological effects, the ED50 ranges from 2.5-12 ng/mL for one formulation or 10-50 ng/mL for another

• For T cell proliferation assays: 5 μg/ml plate-bound CXCL16

• For migration assays: Optimal concentrations should be determined empirically

Experimental applications:

• Cell proliferation: Plate-bound CXCL16 (5 μg/ml) can be used alongside stimulatory antibodies (e.g., OKT3)

• Migration assays: CXCL16 is added to the lower chamber of transwell systems

• As a control, researchers should include appropriate vehicle controls and potentially other chemokines for specificity comparisons

Following these guidelines will help ensure reproducible and reliable results when using recombinant CXCL16 in research applications.

What techniques can be used to study CXCL16-CXCR6 interactions?

Several complementary techniques can be employed to study CXCL16-CXCR6 interactions:

1. Migration assays:

• Transwell migration assays can determine if cells respond to CXCL16 through CXCR6

• Detailed protocol:

  • Prepare cells at 1×10^7 cells/ml in chemotaxis medium (RPMI 1640, 0.5% BSA, 25 mM HEPES)

  • Load 1×10^6 cells (100 μl) in upper transwell chamber (5.0 μm pore size)

  • Add CXCL16 in 600 μl chemotaxis medium to lower chambers

  • Pre-incubate chambers for 30 min at 37°C in 5% CO2

  • Allow migration for 2 hours

  • Analyze migrating cells by flow cytometry with counting beads

2. Blocking experiments:

• Use anti-CXCR6 and anti-CXCL16 antibodies to block interactions

• Specific antibodies mentioned: mouse anti-CXCR6 and rat anti-CXCL16 (R&D Systems)

3. Cell engineering:

• Transfect cells with CXCR6 expression constructs (e.g., CXCR6-YFP)

• GFP-transfected cells can serve as controls

4. Gi/o protein dependency:

• Pertussis toxin can be used to determine G protein dependency of responses

5. Proliferation assays:

• Measure cell proliferation in response to CXCL16 stimulation

• Analyze specifically for effects on dividing cell populations

6. Co-expression analysis:

• Immunohistochemistry and immunofluorescence with confocal imaging to detect co-expression of CXCL16 and CXCR6 in tissues

These approaches provide a comprehensive toolkit for studying CXCL16-CXCR6 interactions at the molecular, cellular, and tissue levels.

How is CXCL16 expression correlated with prognosis in different cancer types?

CXCL16 expression shows variable associations with prognosis depending on the cancer type:

Prostate cancer:

• Expression levels of CXCL16 and CXCR6 on cancer cells correlate with poor prognostic features including high-stage and high-grade tumors

• Expression also correlates with post-inflammatory changes in the cancer stroma, as revealed by loss of alpha-smooth muscle actin

Thyroid cancer (Papillary Thyroid Carcinoma):

• CXCL16 expression is associated with poor prognostic factors including higher TNM staging and the BRAF V600E mutation

• PTCs with higher expression of a 3-gene panel including CXCL16, AHNAK2, and THBS2 showed poor recurrence-free survival compared to the lower expression group

• Experimental depletion of CXCL16 in thyroid cancer cells led to delayed tumor growth in mouse models

Colon cancer:

• In contrast to prostate cancer, no association was found between CXCL16 expression and cancer stage in colon cancer

• A positive correlation was reported between CXCL16 expression and patient survival

• In vitro experiments showed that membrane-bound CXCL16 inhibited cell proliferation in colon carcinoma cell lines

This differential association with prognosis highlights the context-dependent role of CXCL16 in cancer biology, likely reflecting "variable roles for inflammation in cancer depending on tumor type and stage of tumor development" .

How does CXCL16 interact with the tumor microenvironment?

CXCL16 interacts with multiple components of the tumor microenvironment:

1. T cells:

• CXCL16 and CXCR6 are co-expressed on both cancer cells and adjacent T cells in prostate cancer specimens

• CXCL16 enhances proliferation of CXCR6-expressing T cells, potentially increasing cytokine production within the tumor microenvironment

• Activated effector/memory T cells can produce CXCL16, suggesting potential feedback loops

2. Macrophages:

• CXCL16 positively correlates with M2-macrophage infiltration in some cancers

• Depletion of CXCL16 in experimental models led to decreased numbers of F4/80+ macrophages in tumors

• This suggests CXCL16 may recruit and potentially influence macrophage polarization in the tumor microenvironment

3. Angiogenesis:

• Tumors with depleted CXCL16 exhibited decreased numbers of ERG+ endothelial cells

• This suggests CXCL16 may promote angiogenesis, potentially by recruiting endothelial cells or inducing pro-angiogenic factors

4. Stromal remodeling:

• CXCL16 and CXCR6 expression correlates with post-inflammatory changes in the cancer stroma, revealed by loss of alpha-smooth muscle actin

• This indicates CXCL16 may influence stromal cell phenotypes and extracellular matrix remodeling

These interactions form what researchers describe as "inter-related paracrine and autocrine positive feedback loops involving these cells, pleiotropic cytokines and chemokines" . CXCL16 could enhance tumor growth both directly through effects on cancer cells and indirectly by influencing the composition and function of the tumor microenvironment.

What evidence supports the role of CXCL16 in inflammation-associated cancers?

Multiple lines of evidence support CXCL16's role in inflammation-associated cancers:

1. Expression pattern analysis:

• Screening of 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16 as the most consistently expressed chemokine

• Analysis of 461 specimens covering 12 tumor types found CXCL16 expression in multiple human cancers associated with inflammation

2. Correlation with inflammatory markers:

• CXCL16 and CXCR6 expression correlates with post-inflammatory changes in the cancer stroma

• CXCL16 positively correlates with M2-macrophage infiltration in some cancers

3. Functional evidence:

• CXCL16 enhances the growth of both CXCR6-expressing cancer cells and primary CD4 T cells

• CXCL16 induces migration of CXCR6-expressing cells, potentially recruiting inflammatory cells to the tumor microenvironment

4. Experimental manipulation:

• Depletion of CXCL16 in experimental models led to delayed tumor growth with decreased macrophage infiltration and angiogenesis

5. Clinical correlations:

• In prostate cancer, CXCL16 and CXCR6 expression correlates with high-stage and high-grade disease

• In thyroid cancer, CXCL16 expression is associated with poor prognostic factors

These findings collectively suggest that "CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes" .

How might CXCL16 be targeted therapeutically in cancer and inflammatory diseases?

While the search results don't directly discuss therapeutic targeting of CXCL16/CXCR6, they provide insights that could inform potential approaches:

Rationale for targeting:

• In prostate and thyroid cancers, CXCL16 correlates with poor prognosis and promotes tumor growth

• Depletion of CXCL16 led to delayed tumor growth with decreased angiogenesis and macrophage infiltration

• CXCL16 enhances growth of both cancer cells and tumor-associated T cells

Potential targeting strategies:

• Blocking CXCL16-CXCR6 interaction:

  • The search results mention blocking antibodies (mouse anti-CXCR6 and rat anti-CXCL16)

  • This approach could prevent both direct effects on cancer cells and recruitment of inflammatory cells

• Targeting CXCL16 shedding:

  • As soluble CXCL16 appears to promote cancer cell growth, inhibiting the enzymes that cleave membrane-bound CXCL16 could be beneficial

  • This strategy might preserve beneficial scavenger receptor functions while blocking pro-tumorigenic chemokine effects

• Inhibiting downstream signaling:

  • Targeting Gi/o-protein signaling downstream of CXCR6 could potentially block pro-tumorigenic effects

  • This approach might allow more specific inhibition of certain CXCR6 functions

Context-dependent considerations:

• Given CXCL16's contradictory roles in different cancer types, targeting strategies would need to be carefully tailored

• In colon cancer, where CXCL16 may have tumor-suppressive effects, enhancing rather than blocking CXCL16 function might be beneficial

Any therapeutic approach would need to consider both direct effects on cancer cells and indirect effects on the tumor microenvironment, including T cells and macrophages.

What are the key knowledge gaps in CXCL16 research?

Despite significant advances in understanding CXCL16 biology, several important knowledge gaps remain that warrant further investigation:

• Mechanistic details of CXCL16 shedding: While the search results mention that functional CXCL16 can be shed from the cell surface as a soluble protein , the specific proteases involved and the regulatory mechanisms controlling this process remain unclear.

• Context-dependent functions: The contradictory roles of CXCL16 in different cancer types highlight the need for better understanding of the contextual factors that determine whether CXCL16 promotes or inhibits tumor growth.

• Downstream signaling pathways: While CXCR6 signaling is known to be Gi/o-protein dependent , the detailed molecular mechanisms and downstream effectors in different cell types require further elucidation.

• Interaction with other inflammatory mediators: The search results suggest that CXCL16 may participate in "inter-related paracrine and autocrine positive feedback loops involving cells, pleiotropic cytokines and chemokines" , but the specific interactions with other inflammatory mediators are not fully characterized.

• Therapeutic targeting strategies: The optimal approaches for targeting CXCL16/CXCR6 in different disease contexts remain to be determined, particularly considering the context-dependent roles of this axis.

Addressing these knowledge gaps will be essential for fully understanding CXCL16 biology and developing potential therapeutic approaches targeting this chemokine or its receptor.

What future directions are promising for CXCL16 research?

Based on the current understanding of CXCL16 biology, several promising research directions emerge:

• Comparative studies across cancer types: Given the contradictory roles of CXCL16 in different cancers, systematic comparative studies could help identify the factors that determine whether CXCL16 promotes or inhibits tumor progression .

• Mechanistic studies of CXCL16 in the tumor microenvironment: Further investigation of how CXCL16 influences different components of the tumor microenvironment, including T cells, macrophages, and stromal cells, would provide valuable insights into its role in cancer biology .

• Development of selective modulators: Creating tools to selectively target either membrane-bound or soluble CXCL16, or to specifically inhibit certain downstream signaling pathways, could help dissect the different functions of this chemokine.

• Clinical correlation studies: Larger clinical studies correlating CXCL16/CXCR6 expression with treatment responses and patient outcomes could help establish their value as prognostic or predictive biomarkers .

• Therapeutic targeting approaches: Evaluating different strategies for therapeutic targeting of the CXCL16/CXCR6 axis, including blocking antibodies, small molecule inhibitors, or approaches targeting CXCL16 shedding, could lead to novel therapeutic options for cancer and inflammatory diseases.