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The CXC chemokine receptor-4 (CXCR4) plays a critical role in cancer by positively regulating cancer cell metastasis and survival. We previously showed that high concentrations of the CXCR4 ligand, wild-type CXCL12 (wtCXCL12), could inhibit colorectal cancer metastasis in vivo, and we have hypothesized that wtCXCL12 dimerizes at high concentration to become a potent antagonist of CXCR4. To address this hypothesis, we engineered a covalently locked, dimeric variant of CXCL12 (CXCL122). Herein, we show that CXCL122 can not only inhibit implantation of lung metastasis of CXCR4-B16-F10 melanoma cells more effectively than AMD3100, but that CXCL122 also blocks the growth of established pulmonary tumors. To identify a basis for the in vivo efficacy of CXCL122, we conducted Western blot analysis and ELISA analyses, which revealed that CXCL122 was stable for at least 12 hours in serum, whereas wtCXCL12 was quickly degraded. CXCL122 also maintained its antagonist properties in in vitro chemotaxis assays for up to 24 hours in serum, whereas wtCXCL12 was ineffective after 6 hours. Heat-inactivation of serum prolonged the stability and function of wtCXCL12 by more than 6 hours, suggesting enzymatic degradation as a possible mechanism for wtCXCL12 inactivation. In vitro analysis of amino-terminal cleavage by enzymes dipeptidylpeptidase IV (DPPIV/CD26) and matrix metalloproteinase-2 (MMP-2) resulted in 25-fold and 2-fold slower degradation rates, respectively, of CXCL122 compared with wtCXCL12. In summary, our results suggest CXCL122 possesses greater potential as an antimetastatic drug as compared with AMD3100 or wtCXCL12, potentially due to enhanced serum stability in the presence of N-terminal degrading enzymes. Mol Cancer Ther; 11(11); 2516–25. ©2012 AACR.

PDF file, 137K, Full length blot of Figure 3A.

PDF file, 58K, IC50 of CXCL122 for 10 nM wtCXCL12-induced THP-1 migration.

PDF file, 77K, Full length blots of Figure 3B and Figure 5A.

Supplementary Methods, Figures 1-9 from A Novel Function of Junctional Adhesion Molecule-C in Mediating Melanoma Cell Metastasis

Background Chemokines represent a superfamily of immune-modulatory small protein molecules that regulate leukocyte migration to inflammatory sites through their chemoattractant and cell signaling properties. This review focuses on the immunological functions of the CCR6 chemokine receptor and is chemokine ligand, CCL20, that contribute to it role in inflammation in human psoriasis. Methods Peer-reviewed relevant articles are searched and selected from 2000 to 2022 using the search engines including PubMed and Google Scholar. Results After selectively reviewing and evaluating over seventy articles, a comprehensive overview on the immunology of CCL20-CCR6 axis in psoriasis and psoriatic arthritis, the X-ray crystal structures of CCL20 monomers, and the potential of developing clinical therapies targeting this axis is summarized. Conclusions Over the past decade, preclinical studies carried out in animal models of psoriasis involving agents targeting CCL20-CCR6 axis have yielded promising results. Other studies that this axis may play a role in a number of other autoimmune diseases, including rheumatoid arthritis, suggesting a rationale for further investigation into this key signaling/migratory pathway.

Dendritic cells (DCs) emigrate to regional lymph nodes (LNs) during immune responses via afferent lymphatic channels. Secondary lymphoid-tissue chemokine (SLC), a CC chemokine, is expressed in secondary lymphoid organs and mediates the chemotaxis of lymphocytes and DCs via its receptor, CC chemokine receptor 7 (CCR7). By dual-label fluorescence confocal microscopy, we showed MHC class II-positive cells within SLC-staining lymphatic channels in the mouse dermis. SLC was a potent in vitro chemoattractant for cultured, migratory skin DCs, and it enhanced the emigration of MHC class II-positive DCs from mouse skin explants by an average of 2.5-fold. Mature or cytokine-activated, but not resting, Langerhans cells expressed CCR7 mRNA by RT-PCR. Anti-SLC Abs, but not control or anti-eotaxin Abs, blocked the in vivo migration of 51Cr-labeled, skin-derived DCs from footpads to draining LNs by 50% (n = 9, p < 0.005). Thus, we provide direct evidence that SLC and CCR7 participate in the emigration of DCs from peripheral tissue to LNs via lymphatics.

The chemokine receptor, CXCR4, is expressed by human melanomas, and its ligand, CXCL12, is frequently produced at sites of melanoma metastasis. Herein, we examine CXCR4-enhanced binding of B16 murine melanoma cells to endothelial cells (ECs) and recombinant adhesion molecules in vitro to determine the role of tumor- and EC-derived adhesion molecules in tumor metastasis. By flow cytometry, unstimulated primary lung ECs showed constitutive expression of vascular cellular adhesion molecule 1 (VCAM-1), whereas skin-derived ECs did not. All B16 cell lines tested showed constitutive expression of alpha(4) and beta(1) integrin chains but showed no expression of beta(2) integrins. CXCR4-B16 arrest on VCAM-1/immunoglobulin-coated plates and tumor necrosis factor alpha-stimulated ECs under physiological shear stress conditions (1.5 dynes/cm(2)) was rapid, resistant to shear stress of 10 dynes/cm(2), and showed no evidence of rolling before arrest. In vitro, CXCR4-B16 cell binding to ECs was blocked by anti-beta(1) and anti-CXCL12 monoclonal antibodies. In vivo, metastasis of CXCR4-B16 cells to murine lungs was strongly inhibited by anti-CXCL12 and two different anti-beta(1) monoclonal antibodies. Finally, CXCR4-B16 exposed to CXCL12 rapidly increased binding affinity for soluble VCAM-1/immunoglobulin as detected by a flow cytometric assay. Thus, beta(1) integrins play a critical role in CXCR4-mediated B16 tumor cell metastasis in vivo and may be a potential target for inhibition of tumor metastasis, particularly to the lung.