A locked, dimeric CXCL12 variant effectively inhibits pulmonary metastasis of CXCR4-expressing melanoma cells due to enhanced serum stability

Abstract

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.

Figure 1. CXCL12₂ inhibits CXCR4-B16 metastasis more effectively than wtCXCL12 and AMD3100

(A) CXCR4-luc-B16-F10 cells (4 × 10⁵) were injected i.v. into the tail vein of C57BL/6 mouse with the indicated concentrations of drugs. On day 1, the animals were given the same treatment i.v. Lungs were harvested 14 days after inoculation, and luciferase activity was measured to evaluate metastatic tumor. (B) CXCL12₂ (n=4), (C) wtCXCL12 (n=4), and (D) AMD3100 data (vehicle: n=9, others: n=8, Data is a summation of 2 independent experiments) are shown. (E) Representative lung images from mice treated with each drug are shown. (F) Calcium response of CXCR4-luc-B16-F1 cells induced by 500 nM CXCL12₂ or wtCXCL12 was measured in the presence and absence of 5 μM AMD3100. Experiments were recorded in quadruplicate on two separate days. Tumor burden was assessed by measuring luciferase-dependent light production using relative light units (RLU).

Figure 2. CXCL12₂ inhibits outgrowth of established CXCR4-B16 metastatic lesions in lung

(A) CXCR4-luc-B16-F10 cells (4 × 10⁵) were injected into the tail vein of C57BL/6 mouse, allowed to establish, and then treated with 5 μM CXCL12₂ on days 5 and 6. Lungs were harvested 20 days after inoculation, and luciferase activity was measured to evaluate tumor burden. (B) Representative images of treated and untreated lungs are shown. (C) Tumor burden in the lungs were quantitatively assessed (n=12, Data is a summation of 2 independent experiments). (D) Representative images of the thoracic cavity wall after lungs were harvested. Yellow arrows indicate tumor invasion. (E) The tumor lesions (nodules) invading into the thoracic cavity wall were quantified (n=7, Representative of 2 independent experiments with similar results).

Figure 3. WtCXCL12 is quickly degraded in mouse serum, but CXCL12₂ is stable for at least 12 hr

(A) CXCL12₂ and wtCXCL12 were incubated with 90% normal mouse serum. Western blot analysis was performed using anti-hCXCL12 ELISA antibody (R&D Systems ELISA DuoSet). Representative of 2 independent experiments with similar results. (B) WtCXCL12 was also incubated with heat-inactivated normal mouse serum and monitored by western blot. Cropped bands are shown; full-length blots are presented in Fig. S3 and Fig. S4.

Figure 4. Mouse serum quickly diminishes wtCXCL12 function whereas CXCL12₂ remains active for at least 24 hours

(A) Degradation of 10 nM wtCXCL12 and CXCL12₂ were monitored by ELISA in the presence of mouse serum (n=3). Representative of 2 independent experiments with similar results. (B) WtCXCL12 (initial starting concentration, 10 nM) degradation monitored in the presence of a protease inhibitor cocktail (cOmplete, Mini, EDTA-free (Roche); n=3). (C) Degradation of CXCL12₂ (initial starting concentration, 10 nM) was monitored in the presence of protease inhibitors. Negative control (labeled “−”) contained no chemokine but contained 1% mouse serum (n=3). (D) WtCXCL12-induced chemotaxis as a function of time the chemokine is incubated with 90% mouse serum (n=3). (E) Inhibition of 10 nM wtCXCL12-induced migration by either 10 nM or 100 nM CXCL12₂ incubated with 90% mouse serum for indicated time. Chemotaxis assays were performed using THP-1 monocyte cells (n=3). Negative control (labeled “−”) contained no CXCL12₂ but contained 10 nM wtCXCL12 and 1% mouse serum. As a positive control (labeled “fresh”), the indicated chemokines were incubated only with 1% mouse serum, which did not result in degradation (data not shown).

Figure 5. CXCL12₂ is resistant to both MMP-2 and DPPIV/CD26 cleavage

(A) Histidine-tagged Smt-wtCXCL12 was incubated with mouse serum and heat-inactivated mouse serum. The western blot was performed using an anti-hCXCL12 antibody (R&D Systems ELISA DuoSet). (B) MMP-2 cleaves CXCL12 between Ser4 and Leu5 while DPPIV/CD26 cuts between Pro2 and Val3. (C) Cleavage reactions of 10 μM Smt-wtCXCL12 (left) and 5 μM Smt-CXCL12₂ (right) in the presence of 1.4 ng/μl human MMP-2 were assessed by reducing SDS-PAGE. Reactions were quenched at the indicated time points and (D) quantified using densitometry to yield half-lives for Smt-wtCXCL12 (61 ± 9 min) and Smt-CXCL12₂ (129 ± 26 min). Representative of 2 independent experiments with similar results (n=3). (E) 10 μM CXCL12 and 5 μM CXCL12₂ were incubated with 0.2 ng/μl human DPPIV/CD26 for the indicated time points and analyzed by MALDI-TOF MS. For each MS spectra, the intensity of the full-length CXCL12 variants was normalized to an internal Smt3 standard. The half-lives of wtCXCL12 and CXCL12₂ are 26.5 ± 8.7 min and 665 ± 888 min, respectively. Representative of 2 independent experiments with similar results (n=3).