Integrating individual functional moieties of CXCL10 and CXCL11 into a novel chimeric chemokine leads to synergistic antitumor effects: a strategy for chemokine-based multi-target-directed cancer therapy

Abstract

The complexity of tumor biology necessitates a multimodality approach that targets different aspects of tumor environment in order to generate the greatest benefit. IFN-inducible T cell alpha chemoattractant (ITAC)/CXCL11 and IFN-inducible protein 10 (IP10)/CXCL10 could exert antitumor effects with functional specificity and thus emerge as attractive candidates for combinatorial strategy. Disappointedly, a synergistic antitumor effect could not be observed when CXCL10 and CXCL11 were pooled together. In this regard, we seek to improve antitumor efficacy by integrating their individual functional moieties into a chemokine chimeric molecule, designated ITIP, which was engineered by substituting the N-terminal and N-loop region of CXCL10 with those of CXCL11. The functional properties of ITIP were determined by chemotaxis and angiogenesis assays. The antitumor efficacy was tested in murine CT26 colon carcinoma, 4T1 mammary carcinoma and 3LL lung carcinoma. Here we showed that ITIP not only exhibited respective functional superiority but strikingly promoted regression of established tumors and remarkably prolonged survival of mice compared with its parent chemokines, either alone or in combination. The chemokine chimera induced an augmented anti-tumor immunity and a marked decrease in tumor vasculature. Antibody neutralization studies indicated that CXCL10 and CXCL11 moieties of ITIP were responsible for anti-angiogenesis and chemotaxis in antitumor response, respectively. These results indicated that integrating individual functional moieties of CXCL10 and CXCL11 into a chimeric chemokine could lead to a synergistic antitumor effect. Thus, this integration strategy holds promise for chemokine-based multiple targeted therapy of cancer.

Fig. 1

CXCL10 and CXCL11 in combination do not generate synergistic antitumor effects. a Growth curves of CT26 colon carcinoma after intratumorally (i.t.) administrating the indicated proteins. Five days after tumor implantation, mice were i.t. administrated with CXCL10 and CXCL11, either alone or in combination, three times per week for 2 weeks. Tumor volume was measured every other day (n = 10). b Chemotaxis index (CI) of Con A/IL-2-treated T cells in response to CXCL11, CXCL10, or CXCL10 plus CXCL11 (100 ng/ml) was assessed. c Blood vessels in the cross-sections of Matrigel plugs resected from mice treated with indicated chemokines 14 days after implantation were counted and averaged (n = 6). Data are representative of three separate experiments. Data represent mean ± SD. *P < 0.05

Fig. 2

Construction and characterization of ITIP. a Schematic representation of ITIP and its amino acid sequences. b Dimensional conformation for ITIP. The molecular modeling calculation of ITIP was carried out using the Insight II software package. ITIP formed a chemokine-like conformation, including N-terminus, N-loop, three-stranded β-sheet, and C-terminal α-helix. c ITIP was purified using a nickel-chelating column and assayed by Tricine-SDS-PAGE. d The construction of ITIP was confirmed by western blotting with Abs against N-terminal (lane 1) or C-terminal (lane 2) of CXCL11, N-terminal (lane 3) or C-terminal (lane 4) of CXCL10. ITIP polyclonal Ab was prepared and tested by western blotting against CXCL11 (lane 5), CXCL10 (lane 6) and ITIP (lane 7) proteins

Fig. 3

ITIP displays both chemotactic and anti-angiogenic activities. a Chemotaxis assay of Con A/IL-2-treated T cells in response to the indicated concentration of ITIP, CXCL11, CXCL10, or CXCL10 plus CXCL11 was performed. b Con A/IL-2-treated T cells were injected intravenously followed by the indicated proteins injected into discrete areas on the dorsum of the mouse. After 20 h, skin of the injection sites were collected, sectioned and stained with H&E (×400). Inflammatory cells were counted in ten high-power fields (HPF) and results were expressed as cell number per HPF. c CI of Con A/IL-2-treated T cells pre-incubated with CXCR3 Ab or isotype Ab in response to various chemokines (100 ng/ml) was assessed. d Dose-dependent internalization of CXCR3 on Con A/IL-2-treated T cells (left) or CHO/mCXCR3 cells (right) incubated with the indicated concentrations of stimuli for 30 min. e After the different stimuli (100 ng/ml) were added (arrow), dynamic changes in calcium on Con A/IL-2-treated T cells (top) or CHO/mCXCR3 cells (bottom) were monitored continuously by plotting the shift in the fluo3-AM fluorescence over a 150-s time course. f Effect of ITIP on bEND.3 migration into denuded zone 48 h after scraping. Cells were counted in denuded zone. Arrows indicate the wound edge (×100). g H&E-stained plugs resected from ITIP or its parent chemokines treated mice 14 days after implantation. Erythrocyte-filled blood vessels in sections were indicated by bright red staining (×400). Blood vessels in the cross-sections of Matrigel plugs were counted and averaged. Representative photomicrographs are shown. Individual experiment was conducted three times with similar results (n = 6). Data show the mean ± SD. *P < 0.05; **P < 0.01

Fig. 4

ITIP exhibits high anti-tumor efficacy. a Growth curves of CT26 colon carcinoma, 4T1 mammary carcinoma and 3LL lung carcinoma after administrating i.t. the indicated proteins. Five days after tumor implantation, mice were administrated i.t. with ITIP or its parent chemokines (0.5 μg/dose/mouse) three times per week for 2 weeks. Tumor volume was measured every other day. b Survival curves of mice bearing CT26 colon carcinoma, 4T1 mammary carcinoma and 3LL lung carcinoma after treatment. Survival was monitored for 90 days. c Growth curves of ITIP treated CT26 tumor after Ab neutralization. BALB/c mice were injected subcutaneously (s.c.) on day 0 with 1 × 10⁵ CT26 cells. From day 5 on, ITIP protein (0.5 μg) was injected i.t. three times per week for 2 weeks. One day before ITIP administration, mice were given intraperitoneal (i.p.) the indicated Abs. Tumor volume was measured every other day. d Survival curves of ITIP treated CT26 tumor bearing mice after Ab neutralization. Survival was monitored for 90 days. Similar results were obtained in three separate experiments (n = 10). Data are presented as the mean ± SD. *P < 0.05

Fig. 5

ITIP induces anti-tumor immunity and tumor associated anti-angiogenic activity. a BALB/c mice were injected s.c. on day 0 with 1 × 10⁵ CT26 cells. After 5 days, ITIP or other control protein (0.5 μg) was injected i.t. three times per week for 2 weeks. One day after the last injection, tumors were surgically removed and sectioned for H&E staining and immunofluorescence staining against CXCR3, CD3, CD4 and CD8 (×400). Immunostaining with anti-CD31 Ab was used to detect microvessels in CT26 tumor (×200). b Quantification of CXCR3⁺, CD3⁺, CD4⁺ and CD8⁺ cells (left) and CD31⁺ blood vessels (right) in CT26 tumors. MVD denotes microvessel density. c Cytotoxicity assay. Splenocytes were isolated 1 day after the last injection, and cocultured with MMC-treated CT26 cells. After 5 days of culture, the in vitro CTL activity of the restimulated splenocytes was assessed against CT26 cells using a CFSE/7-AAD assay. d In vitro proliferation assay. One day after the last injection, splenocytes were isolated and labeled with CFSE, then cocultured with MMC-treated CT26 cells for 72 h and analyzed by flow cytometry for CFSE dilution. e Cytokine assay. Splenocytes were isolated and cocultured with MMC-treated CT26 cells for 24 h. The cell culture supernatant was analyzed for IFN-γ, IL-12, IL-4 and IL-10 expression by ELISA. The photomicrographs are representative of three independent experiments. An individual experiment was conducted three times (n = 6). Data are expressed as means ± SD. *P < 0.05; **P < 0.01

Fig. 6

CXCL10 and CXCL11 moieties are responsible for the potent antitumor activity of ITIP. a BALB/c mice were injected s.c. on day 0 with 1 × 10⁵ CT26 cells. From day 5 on, ITIP protein (0.5 μg) was injected i.t. three times per week for 2 weeks. 24 h before ITIP administration, mice were given i.p. the indicated Abs. One day after the last ITIP administration, tumors were surgically removed, sectioned for H&E staining (top; ×400) and CXCR3 (middle; ×400) and CD31 (bottom; ×200) immunofluorescence staining. b CXCR3⁺ cells (left) and microvessels (right) were quantitatively analyzed as above. Three independent experiments were performed, and similar results were obtained (n = 8). Representative photomicrographs of the tumor are shown. Data are expressed as means ± SD. *P < 0.05; **P < 0.01