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. 2012 Jan 1;188(1):198-205.
doi: 10.4049/jimmunol.1101845. Epub 2011 Nov 23.

Adenosine A2B receptor blockade slows growth of bladder and breast tumors

Caglar Cekic  1 , Duygu Sag, Yuesheng Li, Dan Theodorescu, Robert M Strieter, Joel Linden
Affiliations

Adenosine A2B receptor blockade slows growth of bladder and breast tumors

Caglar Cekic et al. J Immunol. .

Abstract

The accumulation of high levels of adenosine in tumors activates A(2A) and A(2B) receptors on immune cells and inhibits their ability to suppress tumor growth. Deletion of adenosine A(2A) receptors (A(2A)ARs) has been reported to activate antitumor T cells, stimulate dendritic cell (DC) function, and inhibit angiogenesis. In this study, we evaluated the effects of intermittent intratumor injection of a nonselective adenosine receptor antagonist, aminophylline (AMO; theophylline ethylenediamine) and, for the first time to our knowledge, a selective A(2B)AR antagonist, ATL801. AMO and ATL801 slowed the growth of MB49 bladder and 4T1 breast tumors in syngeneic mice and reduced by 85% metastasizes of breast cancer cells from mammary fat to lung. Based on experiments with A(2A)AR(-/-) or adenosine A(2B) receptor(-/-) mice, the effect of AMO injection was unexpectedly attributed to A(2B)AR and not to A(2A)AR blockade. AMO and ATL801 significantly increased tumor levels of IFN-γ and the IFN-inducible chemokine CXCL10, which is a ligand for CXCR3. This was associated with an increase in activated tumor-infiltrating CXCR3(+) T cells and a decrease in endothelial cell precursors within tumors. Tumor growth inhibition by AMO or ATL801 was eliminated in CXCR3(-/-) mice and RAG1(-/-) mice that lack mature T cells. In RAG1(-/-) mice, A(2B)AR deletion enhanced CD86 expression on CD11b(-) DCs. Bone marrow chimera experiments demonstrated that CXCR3 and A(2B)AR expression on bone marrow cells is required for the antitumor effects of AMO. The data suggest that blockade of A(2B)ARs enhances DC activation and CXCR3-dependent antitumor responses.

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Conflict of interest statement

Conflicts of interest: Joel Linden has a consulting relationship with Forest Labs, the supplier of ATL801.

Figures

Figure 1
Figure 1
AMO inhibits growth of MB49 bladder carcinoma. Subcutaneous inoculation of 105 tumor cells into the right flank of C57BL/6 mice was followed in 2–3 days by injection into the same site of 100 μl of 200 μM AMO or PBS, and repeat injections 3 time weekly. The effects of AMO are shown in: A) C57BL/6 mice (n=15); and B) in mice lacking both A2A and A2B receptors (n=10). Panel C) overlays the effects of AMO and deletion of A2A and A2B receptors. In panel D) AMO injection was started 9 days after tumor cell innoculation and repeated at the times indicated by arrows (n=9).
Figure 2
Figure 2
Adaptive immune responses are required for tumor growth inhibition by AMO. A) CD69, and B) Intracellular IFNγ, expression were evaluated by flow cytometry to detect the activation status of T cells from PBS or AMO treated MB49 tumors grown for three weeks in WT animals, (**P<0.01, *P< 0.05). Data are pooled from three independent experiments, each with 3–4 mice per treatment group). C) MB49 bladder carcinoma cells (105) were inoculated. s.c. in the right flank of RAG1−/− mice. Intratumor injections of 200μM AMO in 100μL PBS were performed three times weekly starting from day 2 or 3 after tumor cell injection, N=13.
Figure 3
Figure 3
AMO activates the IFN-γ-CXCR3 signaling pathway to inhibit tumor growth. A,B)Nine days after inoculation of 105 MB49 bladder cells, tumors were injected three times with PBS or 200 μM aminophylline in 100 μl PBS on days 9, 11 and 14. On day 15 tumors were harvested and assayed for mRNA expression or FACS analysis.A) Relative transcript levels of IFN-γ and interferon inducible chemokines, CXCL9, CXCL10 and CXCL11 were detected by qPCR. B) Frequencies of CXCR3+ lymphocyte populations were detected by flow cytometry, *P<0.05 and **P<0.01. C) MB49 bladder carcinoma cells (105) were inoculated. s.c. in the right flank of CXCR3−/− mice and treated with intratumor injections 200 μM aminophylline in 100 μl PBS three times weekly. N=7.
Figure 4
Figure 4
AMO inhibits bladder carcinoma growth primarily by blocking A2BARs. AMO (100 ul of 200uM) or PBS was injected three after inoculation of 105 MB49 bladder carcinoma s.c. on the right flanks of A) WT, B) A2BAR−/− and C) A2AAR−/− mice. Results are from two independent experiments, N=8–9. Two way ANOVA was performed to test statistical significance (**P<0.01, ***P<0.0001).
Figure 5
Figure 5
Aminophylline targets A2BARs and attracts CXCR3+ immune cells, to inhibit tumor growth and angiogenesis. Lethally irradiated WT mice received WT, A2BAR−/− or CXCR3−/− bone marrow transplantation. Recipient mice were inoculated with MB49 bladder carcinoma cells (105) s.c. 7 weeks after bone marrow transfer. Intratumor injection of 100 ul of 200 μM aminophylline or PBS was performed three times weekly starting 2–3 days after tumor inoculation. A) Tumor volumes as measured with calipers before each injection of AMO, ***P<0.0001 by two way anova (time vs. treatment). B) Primary tumors were harvested three weeks after tumor inoculation and tested for CD34+CD45- cell frequency by Flow cytometry as an index of neo-vascularization. **P<0.01. N=9.
Figure 6
Figure 6
Selective A2BAR blockade inhibits MB49 bladder carcinoma growth. In separate experiments, wildtype, A2AAR−/− or A2BAR−/− mice were divided into two groups and inoculated in the right flank with 105 MB49 bladder cancer cells. The site of inoculation were then injected 3 times weekly with 100 μl vehicle or 1 μM ATL801. The ratio of tumor sizes in mice receiving ATL801/vehicle control is plotted vs. time after tumor inoculation. Compared to vehicle, ATL801 reduced tumor size by about 50% between days 12–19 of treatment in wildtype and A2AAR−/− mice but ATL801 had no effect in A2BAR−/− mice. Two way ANOVA was performed to test statistical significance (***P<0.001), N=8–9.
Figure 7
Figure 7
Selective A2BAR blockade requires CXCR3 to inhibit MB49 bladder carcinoma growth. A) ATL801 injection into tumors was started 9 days after injection of 105 MB49 cells and repeated at the times indicated by arrows. B,C) On day 15 tumors were harvested and analyzed for mRNA expression or by FACS. B) Relative transcript levels of IFN-γ and interferon inducible chemokines, CXCL9, CXCL10 and CXCL11, between ATL801 vs. PBS treatment groups were detected by qPCR. C) Frequencies of CXCR3+ lymphocyte populations were detected by flow cytometry, *P<0.05 and **P<0.01. MB49 bladder carcinoma cells (105) were inoculated. s.c. in the right flank of D) RAG1−/− and E) CXCR3−/− mice and treated with intratumor injections 1 μM ATL801 in 100 μl PBS three times weekly. N=9.
Figure 8
Figure 8
AMO and ATL801 inhibit the growth and metastasis of 4T1 breast cancer cells. 14T1-12B breast cancer cells (105) expressing luciferase were injected into the mammary fat pads of Balb/c mice and treated with AMO (A; top, n=13) or ATL801 (A; bottom, n=5) 3 times weekly starting on day 0. E) Lung metastasis was quantified in kidney and lung following luciferin injection based on luminescence detected using an IVIS 200 imaging system. (Representative of n=5). ANOVA and post-hoc tests were performed to test significance differences between AMO and PBS (*P<0.05, ***P<0.001).
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