Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors - PubMed (original) (raw)

Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors

Paul A Beavis et al. Proc Natl Acad Sci U S A. 2013.

Abstract

CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A2A/A2B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A2A(-/-) mice were strongly protected against tumor metastasis, indicating that host A2A receptors enhanced tumor metastasis. A2A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A2A blockade was due to enhanced NK cell function. Interestingly, A2B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73(+) tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A2A or A2B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A2A/A2B receptor antagonists have already entered clinical trials in other therapeutic settings.

Keywords: cancer metastasis; immunotherapy; innate immunity; tumor immunosuppression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

CD73 expression enhances spontaneous and experimental metastasis of tumor cells. (A) A total of 5 × 105 AT-3-GFP/AT-3-CD73 cells or (B) 1 × 105 B16F10-GFP/B16F10-CD73 cells were injected s.c. into WT mice and primary tumor growth compared. Results shown are the mean ± SD of five mice of a representative experiment of n = 4. (C) At day 37 after s.c. injection of AT-3-GFP/AT-3-CD73 cells, lungs were harvested and sectioned. (D) A total of 5 × 105 AT-3-GFP/AT-3-CD73 cells or (E) 2 × 105 B16F10-GFP/B16F10-CD73 cells were injected i.v. into WT mice and lungs harvested 14 d after tumor inoculation. (C and D) Results shown are the mean ± SEM of pooled data from three individual experiments. (E) Results shown are the mean ± SD of five mice from a representative experiment of n = 5. *P < 0.05, ***P < 0.001.

Fig. 2.

Fig. 2.

Activation of either the A2A or A2B adenosine receptors increases tumor metastasis. (A_–_D) WT or (C_–_E) RAG−/−cγ−/− mice were pretreated i.p. with either PSB-1115 (1 mg/kg), SCH58261 (1 mg/kg), or vehicle control before treatment with NECA (0.05 mg/kg), CGS-21680 (2 mg/kg), BAY-60-6583 (0.1 mg/kg), or PBS. One hour posttreatment, mice were injected i.v. with 2 × 105 (WT), 2 × 104 (RAG−/−cγ−/−: C and D), or 1 × 104 (RAG−/−cγ−/−: E) B16F10 GFP tumor cells. (A_–_C) Data shown are the mean ± SD from one experiment of n = 3. (D) Data are represented as the mean (n = 16) ± SEM fold increase in metastasis induced by NECA in WT and RAG−/−cγ−/− mice relative to PBS-treated controls. (E) Results shown are the mean ± SEM pooled from two individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant.

Fig. 3.

Fig. 3.

Blockade of A2A or A2B receptors with selective antagonists significantly reduces the metastasis of CD73+ tumors. WT mice were pretreated with (A) SCH58261 (1 mg/kg), (B) PSB-1115 (1 mg/kg), or vehicle control at days 0 (−1 h), 1, and 7 and injected i.v. with 2 × 105 B16F10-GFP or B16F10-CD73 tumor cells. Results shown are the mean ± SD of a representative experiment of n = 3. (C) 4T1.2 tumor cells were injected into the fourth mammary fat pad at a dose of 5 × 104. Mice were treated with either vehicle control, SCH58261 (1 mg/kg), or PSB-1115 (1 mg/kg) three times per week with therapy initiated on day 3. After 30 d, lungs were harvested and sectioned and metastases determined. Results shown are the mean ± SEM of n = 12 pooled from two individual experiments. (D) WT or A2A−/− mice treated with vehicle or SCH58261 (1 mg/kg) at days 0 (−1 h), 1, and 7 and injected i.v. with 2 × 105 B16F10 CD73+ cells. Results shown are the mean ± SEM pooled from two individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Fig. 4.

A2A blockade enhances NK cell activity in vitro and perforin-mediated cytotoxicity in vivo. Splenic NK cells were isolated from WT (A_–_D) or A2A−/− (D) mice. (A) NK cells were cultured in IL-2 (100 U/mL) and after 5 d, washed and then cocultured with 1 × 104 51Cr-labeled B16F10 tumor cells in the presence of vehicle (control) or NECA (1 μM) ±1 μM SCH58261 at indicated effector:target ratios. After 4 h coculture, supernatants were taken and the percentage chromium release determined. Results shown are the mean ± SEM pooled from three individual experiments. NK cells (5 × 104 cells/well) were stimulated with IL-18 (50 ng/mL) and indicated doses of IL-12 in the presence or absence of NECA (1 μM), SCH58261 (1 μM), PSB-1115 (1 μM), or CGS-21680 (100 nM). After 18 h, supernatants were harvested and analyzed for their concentration of IFN-γ (B). The expression of CD69 was determined by flow cytometry (C). (D) NK cells (1 × 104) cells per well were stimulated with IL-18 (50 ng/mL) and IL-12 (100 pg/mL). (B_–_D) The results shown are the mean ± SD of a representative experiment of n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Fig. 5.

Antimetastatic activity of A2A blockade is due to enhanced NK cell function in vivo. (A) WT or perforin−/− mice were treated with SCH58261 (1 mg/kg) or vehicle control at days 0 (−1 h), 1, and 7 and injected i.v. with 2 × 105 B16F10-CD73 tumor cells. Results shown are the mean ± SD of a representative experiment of n = 3. 4T1.2 tumor cells were injected into the fourth mammary fat pad of perforin−/− (B) or WT (C_–_E) mice at a dose of 5 × 104. Mice were treated with either vehicle control or SCH58261 (1 mg/kg) three times per week with therapy initiated on day 3. (B) After 26 d, lungs were harvested and sectioned. Results shown are the mean ± SEM of 12 mice pooled from two individual experiments (C_–_E). At day 30, tumors were harvested and tumor-infiltrating lymphocytes analyzed by flow cytometry. (C) Representative FACS plot showing granzyme B expression within the CD45+CD3−DX5+ (NK cell) subset. (D) Proportion of NK cells expressing granzyme B. (E) MFI of granzyme B within NK cells. (D and E) Data shown are the average MFI. Mean ± SD of five mice of a representative experiment of n = 3. *P < 0.05, **P < 0.01.

Fig. 6.

Fig. 6.

CD73 also enhances metastasis through a nonimmune dependent mechanism. (A and B) AT-3-GFP/AT-3-CD73 tumor cells were seeded at 2 × 105 on the top well of a Boyden chamber containing either serum-free media or FCS-containing media (5%) in the lower compartment. After 4 h, the lower chamber was analyzed by DAPI staining of cell inserts. The average number of cells viewed through a 10× objective was calculated over four fields of view. (A) Mean ± SEM of pooled data from five experiments. (B) Cells were treated with APCP (40 μM) and the effect on migration determined. Mean ± SD for a representative experiment of n = 3 is shown. (C) A total of 1 × 104 AT-3-GFP/AT-3-CD73 or (D) B16F10 GFP/B16F10-CD73 tumor cells were injected i.v. into RAG−/−cγ−/− mice. Lungs were analyzed for metastases 14 d after tumor inoculation. (C and D) Data shown are the mean ± SEM of pooled data from three individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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