Blockade of A_2b Adenosine Receptor Reduces Tumor Growth and Immune Suppression Mediated by Myeloid-Derived Suppressor Cells in a Mouse Model of Melanoma^,

Abstract

The A_2b receptor (A_2bR) belongs to the adenosine receptor family. Emerging evidence suggest that A_2bR is implicated in tumor progression in some murine tumor models, but the therapeutic potential of targeting A_2bR in melanoma has not been examined. This study first shows that melanoma-bearing mice treated with Bay 60-6583, a selective A_2bR agonist, had increased melanoma growth. This effect was associated with higher levels of immune regulatory mediators interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1) and accumulation of tumor-associated CD11b positive Gr1 positive cells (CD11b⁺Gr1⁺) myeloid-derived suppressor cells (MDSCs). Depletion of CD11b⁺Gr1⁺ cells completely reversed the protumor activity of Bay 60-6583. Conversely, pharmacological blockade of A_2bR with PSB1115 reversed immune suppression in the tumor microenvironment, leading to a significant melanoma growth delay. PSB1115 treatment reduced both levels of IL-10 and MCP-1 and CD11b⁺Gr1⁺ cell number in melanoma lesions. These effects were associated with higher frequency of tumor-infiltrating CD8 positive (CD8⁺) T cells and natural killer T (NKT) cells and increased levels of T helper 1 (Th1)-like cytokines. Adoptive transfer of CD11b⁺Gr1⁺ cells abrogated the antitumor activity of PSB1115. These data suggest that the antitumor activity of PSB1115 relies on its ability to lower accumulation of tumor-infiltrating MDSCs and restore an efficient antitumor T cell response. The antitumor effect of PSB1115 was not observed in melanoma-bearing nude mice. Furthermore, PSB1115 enhanced the antitumor efficacy of dacarbazine. These data indicate that A_2bR antagonists such as PSB1115 should be investigated as adjuvants in the treatment of melanoma.

Introduction

Adenosine has been described as an important regulator of immune response in the tumor microenvironment [1,2]. The immune-suppressive effects of adenosine in tumors are dependent on the A_2a receptor subtype (A_2aR), which inhibits T cell functions, favoring tumor development [3]. In contrast, stimulation of A₃ adenosine receptor (A₃R) subtype can markedly limit tumor growth by promoting an efficient antitumor immune response in mice [4,5]. There is growing evidence that the A_2b receptor subtype (A_2bR) can also influence tumor progression in some murine tumor models. We studied the effects of PSB1115, a selective A_2bR antagonist, in a well-established mouse melanoma model. A_2bR is activated by high levels of adenosine [6], achieved in hypoxic tumor microenvironments [1]. Ryzhov and colleagues [7] provided the first genetic evidence for a pivotal role of A_2bR in tumor development. The growth of Lewis lung carcinoma was reduced in A_2bR-deficient mice compared to that in wild-type controls. This was due to an effect on adenosine-mediated release of angiogenic factors, such as vascular endothelial growth factor, from host immune cells [7]. Together with previous evidence on A_2bR-mediated up-regulation of angiogenic factors in cancer cell lines [8,9], these observations highlight the critical role of A_2bR in supporting tumor angiogenesis. More recently, it has been demonstrated that A_2bR promotes the expansion of myeloid-derived suppressor cells (MDSCs) from mouse hematopoietic progenitors in vitro [10]. MDSCs contribute to tumor immune tolerance by releasing adenosine in a CD73-dependent manner [10,11]. Furthermore, A_2bR blockade can reduce the growth of bladder and breast cancers in mice, by promoting a T cell-mediated response in a chemokine C-X-C receptor 3 (CXCR3)-dependent manner [12]. These studies suggest that A_2bR is implicated in tumor progression and that blocking A_2bR could contribute to improve immune response in the tumor environment and thus limit tumor growth. Although our knowledge of the role of A_2bR in promoting cancer development is growing, the antitumor activity of A_2bR blockade in melanoma has not been investigated.

Melanoma is the most aggressive skin tumor, with high metastatic potential. Advanced melanoma is resistant to most chemotherapeutics [13]. Immunotherapy has shown promise in preclinical and clinical studies, and currently, melanoma is one of few malignancies for which there is a Food and Drug Administration (FDA)-approved immunotherapeutic agent, ipilimumab [14–17]. However, in most cases of advanced melanoma, the prognosis remains dismal, and the current scientific challenge is to further improve the efficacy of melanoma therapy. The tumor microenvironment is critical to modulate antitumor immune responses. Immune-suppressive cells in tumor microenvironment, including MDSCs, promote tumor progression by suppressing antitumor immune responses and/or modulating angiogenesis [18–21]. MDSCs accumulate in the blood, lymphoid tissue, and tumor tissue, in human cancers and animal tumor models [18]. MDSCs, identified in mice as CD11b positive Gr1 positive (CD11b⁺Gr1⁺) cells [21], are potent suppressors of T cell-mediated responses, and strategies aimed at reducing MDSC accumulation in tumors or suppressing MDSC function improve T cell activity, resulting in tumor growth inhibition [20,21].

In this study, we show that Bay 60-6583, a selective A_2bR agonist, enhanced melanoma growth by enriching MDSCs in the tumor lesion. This effect was associated with higher levels of immune-suppressive mediators such as interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1). These mediators can further stimulate MDSC accumulation in the tumor [22–24]. Conversely, administration of PSB1115 inhibited the accumulation of tumor-infiltrating MDSCs. This enhanced T helper 1 (Th1)-like response in the tumor environment and significantly delayed melanoma growth. PSB1115 also increased the efficacy of melanoma chemotherapeutic dacarbazine in our model. These results suggest that selective A_2bR antagonists such as PSB1115 can reverse the immune suppression in the tumor environment and potentially enhance the efficacy of melanoma chemo- and immunotherapy.

Materials and Methods

Mice and Cells

Female C57Bl6j and Athymic Nude-Foxn1^nu (6–8 weeks old) mice were purchased from Harlan (Harlan Laboratories, Udine, Italy) and maintained in a pathogen-free animal facility. All the experiments were conducted according to institutional animal care guidelines, Italian DL No. 116 of 27 January 1992, and European Communities Council Directive of 24 November 1986 (86/609/ECC). B16-F10 murine melanoma cell line was purchased from American Type Culture Collection (LGC Standards Srl, Milan, Italy) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (0.1 mg/ml; Sigma-Aldrich, Milan, Italy).

In Vivo Studies

For tumor challenge, 2 x 10⁵ B16-F10 cells were subcutaneously injected on the right flank of anesthetized mice [25]. The specific adenosine A_2bR agonist Bay 60-6583 [26–28] and the selective A_2bR antagonist PSB1115 [26,29,30] were from Tocris Cookson Ltd (London, United Kingdom). Ten days after tumor cell implantation, when palpable tumors had developed, Bay 60-6583 (0.2 mg/kg) or PSB1115 (1 mg/kg) was delivered to the mice for four consecutive days by the peritumoral (p.t.) route, which is an important route of administration to evaluate directly the effect of these drugs on tumor growth. Phosphate-buffered saline alone was used as vehicle control for PSB1115. Phosphate-buffered saline containing 0.01% DMSO was used as control for Bay 60-6583. Gemcitabine (Gem, 120 mg/kg; Sigma-Aldrich) [31,32] was intraperitoneally (i.p.) injected once at day 10 after tumor cell implantation into mice receiving PSB1115 or Bay 60-6583 or vehicle. Dacarbazine (DTIC, 100 mg/kg; Sigma-Aldrich) [33] was i.p. injected once at day 10 after tumor cell implantation into mice receiving PSB1115 or vehicle as described above. Mice were killed the day after the last injection, and melanoma tissues and spleens were isolated for further analyses. Tumor growth was daily monitored and calculated as we previously reported [25]. For long-term experiments, mice were to be killed according to the animal care protocol when the tumor volume reached ∼1000 mm³.

Isolation and Adoptive Transfer of MDSCs

Spleens of melanoma-bearing mice were digested and passed through 70-µm cell strainers, and red blood cells were lysed. CD11b⁺Gr1⁺ cells were sorted by FACSAria III (BD Biosciences, Milan, Italy; purity was >92%, as assessed by flow cytometer). An amount of 1.5 x 10⁴ freshly isolated CD11b⁺Gr1⁺ cells was peritumorally injected into melanoma-bearing mice at day 10 after B16.F10 tumor cell implantation, when PSB1115 treatment started, as described above.

Flow Cytometry Analysis

Single-cell suspensions from tumors and spleens of treated mice were prepared for flow cytometry analysis as previously described [25]. Briefly, tissues were digested and passed through 70-µm cell strainers, and red blood cells were lysed. Cell samples were preincubated with anti-mouse CD16/CD32 (eBioscience, San Diego, CA) to block nonspecific Fc-mediated interactions. The following mouse-specific antibodies were used: CD11c-fluorescein isothiocyanate, CD11b-phycoerythrin cyanine 5.5 (PeCy5.5), Gr1-phycoerythrin (PE) or Gr1-allophycocyanin, CD3-PeCy5.5, CD8-allophycocyanin or CD8-PE, CD4-allophycocyanin, NK1.1-PE, F4/80-PE, major histocompatibility complex II (MHC II)-allophycocyanin, CD80-PE, CD25-PE, and forkhead box P3 (FoxP3)-PeCy5.5 (all from eBioscience). Data were acquired with FACSCalibur flow cytometer (BD Biosciences).

Intracellular Staining

For intracellular cytokine staining, splenocytes from melanoma-bearing mice treated with PSB1115 or vehicle (Ctr) were stimulated with Dynabeads Mouse T-Activator CD3/CD28 (Invitrogen, Milan, Italy), according to the manufacturer's instructions, for 20 hours. Cells were stained with CD3-PeCy5.5 and CD8-allophycocyanin or CD4-allophycocyanin. After fixation/permeabilization (eBioscience), cells were then stained with anti-mouse interferon (IFN)-γ-fluorescein isothiocyanate antibody (Ab). Data were acquired with FACSCalibur flow cytometer (BD Biosciences). Carboxyfluorescein succimidyl ester (CFSE) proliferation assay was performed by labeling splenocytes with 1 µmCFSE (eBioscience), cocultured with FACS-sorted CD11b⁺Gr1⁺ cells (1:1 ratio), and stimulated with CD3/28 monoclonal antibodies (mAbs). Flow cytometry analysis of CFSE dilution in CD3⁺ T cells was performed on day 5 of culture. Bay 60-6583 was tested at 1µm.

Enzyme-Linked Immunosorbent Assays

IFN-γ, tumor necrosis factor (TNF), granzyme B, IL-10, and MCP-1 were analyzed by ELISA kits (R&D Systems, Abingdon, United Kingdom, and eBioscience) in melanoma tissue homogenates. IFN-γ levels were also measured in the supernatant of splenocytes harvested from both control and PSB1115-treated mice. IL-10 was also measured in the supernatant of FACS-sorted CD11b⁺Gr1⁺ cells stimulated with Bay (1 µm) for 20 hours.

Statistical Analysis

Results are expressed as means ± SEM. All statistical differences were evaluated by either two-tailed Student's t test or one-way analysis of variance (ANOVA) as appropriate. P values of <.05 were considered statistically significant.

Results

Bay 60-6583 Promotes Melanoma Growth In Vivo by Inducing an Immune-Suppressive Environment in the Tumor Tissue

We first investigated the effect of Bay 60-6583, a selective A_2bR agonist [26–28], on tumor growth in B16 melanoma-bearing mice. Treatment with Bay 60-6583 (0.2 mg/kg, p.t.) significantly enhanced melanoma growth compared to control (Figure 1A).

Figure 1.

Bay 60-6583 promotes tumor accumulation of MDSCs, which favors melanoma growth. (A) Tumor volumes (mm³) in C57Bl6j mice treated with Bay 60-6583 (0.2 mg/kg, p.t.) compared to control (Ctr) are expressed as means ± SEM (n = 12 per group). (B) CD11b-PeCy5.5-positive (⁺) Gr1-PE⁺ cells were assessed by flow cytometry in the melanoma lesions from Bay 60-6583-treated and control mice and expressed as percentage of leukocytes (n = 11 per group). Representative dot plots of tumor-infiltrating CD11b⁺Gr1⁺ cells are shown. CD11b-PeCy5.5⁺ Gr1-PE⁺ cells in the melanoma lesions (C) and spleen (D) of tumor-bearing mice received a single injection of Gem (120 mg/kg, i.p.) and/or Bay 60-6583. Data as percentage are expressed as means ± SEM (n = 12 per group). (E) Tumor volumes (mm³) in treated and untreated mice (n = 6–10 per group). Data are from three independent experiments and represent means ± SEM. *P < .05, **P < .01, and ***P < .001 (one-way ANOVA analysis and Student's t test, as appropriate).

Because tumor progression can be promoted by an immune-suppressive environment, we first analyzed immune cell populations in tumors of Bay 60-6583-treated mice compared to controls. We found that the percentage of tumor-infiltrating MDSCs, identified as CD11b⁺Gr1⁺ cells [21], was increased in Bay 60-6583-treated animals compared to control (Figure 1B). There was no change in FoxP3⁺CD4⁺CD25⁺ T cells (regulatory T cells) in melanoma lesions of mice treated with Bay 60-6583 compared to controls (data not shown). To better understand the role of CD11b⁺Gr1⁺ cells in Bay 60-6583-induced tumor growth in mice, we depleted CD11b⁺Gr1⁺ cells by using Gem, which preferentially decreases CD11b⁺Gr1⁺ cell levels in tumor-bearing mice as previously reported by other groups [31,32], most likely by inducing selective death of Gr1⁺CD11b⁺ cells [31]. Gem treatment decreased CD11b⁺Gr1⁺ cell levels both in tumor tissues and in spleens of melanoma-bearing mice (Figure 1, C and D, respectively, and Figure W1, A and B). Moreover, mice treated with Gem + Bay 60-6583 had decreased numbers of CD11b⁺Gr1⁺ cells both in tumor tissues and in spleens, similar to Gem alone (Figure 1, C and D, respectively). This effect was associated with decreased tumor growth compared to control (Figure 1E). Notably, treatment of B16.F10 cells in vitro with Gem caused only ∼10% reduction of cell viability (data not shown). Depletion of CD11b⁺Gr1⁺ cells eliminated the increase in tumor volume caused by Bay 60-6358 (Figure 1E). Tumor growth in mice receiving Gem + Bay 60-6583 was not significantly different than those observed in mice treated with Gem alone (Figure 1E). We also tested whether Bay 60-6583 could affect MDSC functionality. Bay 60-6583 treatment in vitro did not affect either the ability of CD11b⁺Gr1⁺ cells to suppress T cell proliferation in a CFSE proliferation assay or the production of IL-10 (Figure W2, A and B, respectively). These results suggest that Bay 60-6583 promotes CD11b⁺Gr1⁺ cell accumulation in tumor but did not directly influence their function.

We next investigated Bay 60-6583-mediated effects on inflammatory factors in melanoma lesions. The levels of IL-10 and MCP-1 were significantly increased in the tumor tissue of mice treated with Bay 60-6583 compared with control (Figure 2, A and B, respectively). These inflammatory mediators have been reported to be crucial for MDSC accumulation in melanoma tumor lesions [21,24]. Therefore, the increased number of MDSCs in Bay 60-6583-treated mice is associated with alteration in inflammatory mediators in the tumor environment.

Figure 2.

Effects of Bay 60-6583 on immune regulatory mediators within tumor tissue. IL-10 (A) and MCP-1 (B) levels were analyzed in tumor tissue homogenates by ELISA as picogram per milligram protein (n = 7–12 per group). Data are from three independent experiments and represent means ± SEM. *P < .05 (Student's t test).

PSB1115 Arrests Melanoma Growth in Mice

The effect of Bay 60-6583 on melanoma growth was completely abolished in mice treated with PSB1115 (1 mg/kg, p.t.), a selective A_2bR antagonist [26,29,30] (Figure 3A). Importantly, melanoma growth was significantly decreased after PSB1115 administration compared to control mice (Figure 3A). In addition, we performed long-term experiments where tumor growth was monitored up to 20 days. PSB1115 significantly delayed melanoma growth compared with control (Figure W3).

Figure 3.

Effect of PSB1115 on melanoma growth and immune-suppressive mediators in tumors. (A) Tumor-bearing mice were treated with PSB1115 (1 mg/kg, p.t.), Bay 60-6583, or both, Bay 60-6583 and PSB1115. Tumor volumes (mm³) of treated and untreated mice are expressed as means ± SEM (n = 12–20 per group). (B) CD11b-PeCy5.5⁺ Gr1-PE⁺ cells measured in tumor tissue of control mice and PSB1115 treated mice are expressed as means ± SEM (n = 11 per group). Representative dot plots of CD11b⁺Gr1⁺ cells are shown. MCP-1 (C) and IL-10 (D) levels as picogram per milligram protein were determined in the tumor tissue homogenate of control mice or mice treated with PSB1115 and expressed as means ± SEM (n = 12 per group). Results are from three independent experiments. *P < .05 and ***P < .001 (one-way ANOVA analysis and Student's t test, as appropriate).

We next investigated the mechanism responsible for the antitumor activity of PSB1115. Because Bay 60-6583 increased the level of tumor-infiltrating CD11b⁺Gr1⁺ cells, we hypothesized that the antitumor activity of PSB1115 was due to inhibition of the accumulation of CD11b⁺Gr1⁺ cells within melanoma tissue. We found that the number of tumor-infiltrating CD11b⁺Gr1⁺ cells of PSB1115-treated mice was significantly reduced compared to control mice (Figure 3B). This effect appeared to be selective for CD11b⁺Gr1⁺ cells, because the numbers of CD11c⁺Gr1^- dendritic cells (DCs) or CD11b⁺Gr1^- cells in tumor tissue were not significantly altered in mice treated with PSB1115 (Figure W4, A and B, respectively). Administration of PSB1115 did not affect the maturation of DCs, because the expression of MHC II and CD80 on DCs was unchanged (Figure W4, C and D, respectively). The levels of MCP-1 and IL-10 in tumors of mice treated with PSB1115 were decreased compared to controls (Figure 3, C and D, respectively), suggesting that the antitumor activity of PSB1115 is associated with a reduced immune-suppressive tumor environment.

T Cells Contribute to the Antitumor Effect of PSB1115

Numerous studies reported that MDSCs, which are abundant in melanoma tissue, are potent suppressors of T cell-mediated immune responses in the tumor milieu [23,34]. We tested whether a reduced number of CD11b⁺Gr1⁺ cells within tumor tissue after PSB1115 administration was associated with improved antitumor immune response. Mice treated with PSB1115 showed an increased number of tumor-infiltrating CD8 positive (CD8⁺) T cells and natural killer T (NKT) cells (identified as CD3⁺NK1.1⁺ cells) in melanoma lesions compared to controls (Figure 4, A and B). This effect was associated with increased levels of TNF-α (Figure 4C), IFN-γ (Figure 4D), and granzyme B (Figure 4E) in tumor homogenates compared with control. We also performed ex vivo experiments on splenocytes of PSB1115-treated mice. IFN-γ production (Figure 4F) and the number of IFN-γ-producing CD8⁺ T cells and CD4⁺ T cells (Figure W5, A and B, respectively) were significantly increased in splenocytes from PSB1115-treated mice compared with controls.

Figure 4.

T-mediated response is restored in PSB1115-treated mice. (A) CD3-PeCy5.5⁺CD8-allophycocyanin⁺ cells and (B) CD3-PeCy5.5 ⁺NK1.1-PE⁺ cells in tumor tissue from control or treated mice (n = 8–14 per group). Representative dot plots for each population are shown. TNF-α (C), IFN-γ (D), and granzyme B (E) levels as picogram per milligram protein in the tumor tissue homogenate of treated and control mice (n = 6–10 per group). Data are expressed as means ± SEM and are from three independent experiments. (F) Splenocytes isolated from tumor-bearing mice receiving vehicle or PSB1115 were cultured and stimulated with anti-CD3/28 mAbs (black bars). IFN-γ production was measured in culture supernatants of nonstimulated and stimulated splenocytes by ELISA and expressed as mean ± SEM (n = 10 per group). (G) Tumor volumes (mm³) were calculated in melanoma-bearing nude mice treated or not treated with PSB1115 (n = 8 per group). Data are from two independent experiments and expressed as means ± SEM. *P < .05, **P < .01 and ***P < .001 (one-way ANOVA analysis or Student's t test, as appropriate).

To elucidate the potential role of nonimmunologic effects in the antitumor activity of PSB1115, we tested its effects in allograft tumors in Athymic Nude-Foxn1^nu mice. Nude mice, implanted subcutaneously (s.c.) with B16.F10 cells, were treated with PSB1115 as described above for C57Bl6j mice. No antimelanoma effect of PSB1115 was observed in nude mice (Figure 4G), indicating that the antitumor activity of PSB1115 in our model is entirely due to immune modulation and underscoring the critical role of T cells in the antitumor effect of PSB1115.

Taken together, our data indicate that PSB1115 administration had significant antitumor activity in melanoma-bearing mice. This effect was associated with reduced accumulation of CD11b⁺Gr1⁺ cells in the tumor environment and increased numbers of tumor-infiltrating CD8⁺ T cells and NKT cells, as well as Th1-like cytokine production.

PSB1115 Reduces MDSC Accumulation within Melanoma Tissue

Our results show that PSB1115 decreases CD11b⁺Gr1⁺ cell accumulation within melanoma lesions, leading to reduced tumor growth. To clarify the role of these cells in the antitumor effect of PSB1115 in melanoma-bearing mice, we administered PSB1115 in CD11b⁺Gr1⁺ cell-depleted mice. Gem was administered at day 10 after B16.F10 cell implantation as reported above. Depletion of CD11b⁺Gr1⁺ cells with Gem did not alter the antitumor activity of PSB1115 compared to mice treated with PSB1115 alone (Figure 5A). The accumulation of CD11b⁺Gr1⁺ cells in both tumors and spleens after treatment with Gem and/or PSB1115 was markedly reduced compared with controls (Figure 5, B and C, respectively). Decreased accumulation of CD11b⁺Gr1⁺ cells was also observed in the spleens of mice treated with PSB1115 alone (Figure 5C). However, this was most likely not due to a systemic effect of PSB1115, which was peritumorally administered, but a consequence of reduced tumor volume, consistent with previous studies [35,36]. Indeed, the frequency of CD11b⁺Gr1⁺ cells in the spleens of mice of both groups (PSB1115 and Ctr) with tumors of equal sizes was similar (data not shown). These data suggest that inhibition of CD11b⁺Gr1⁺ cell recruitment with PSB1115 or depletion of these cells with Gem have comparable effects on tumor growth and that these effects are not additive.

Figure 5.

The antitumor activity of PSB1115 is associated with impaired CD11b⁺Gr1⁺ cell accumulation in tumor. (A) Tumor volumes (mm³) in mice treated with vehicle (Ctr) or PSB1115 and Gem as previously described (n = 7–10 per group). CD11b-PeCy5.5⁺Gr1-PE⁺ cells in the tumor tissue (B) or spleen (C) from treated mice are expressed as means ± SEM (n = 6–8 per group). Data are from two independent experiments. (D) Tumor volumes (mm³) in melanoma-bearing mice receiving PSB1115 or vehicle, adoptively transferred or not with CD11b⁺Gr1⁺ cells, are expressed as means ± SEM (n = 4–5 per group). CD3-PeCy5.5⁺CD8-allophycocyanin⁺ cells (E) and CD3-PeCy5.5⁺NK1.1-PE⁺ cells (F) were determined in tumor tissues of treated and untreated mice. Numbers indicate the percentage of positive cells gated on lymphocyte cells.

Next, to determine whether the antitumor activity of PSB1115 was attributable to impaired accumulation of CD11b⁺Gr1⁺ cells within tumor tissue, we adoptively transferred CD11b⁺Gr1⁺ cells to melanoma-bearing mice receiving PSB1115 or vehicle. Adoptive transfer of CD11b⁺Gr1⁺ cells from tumor-bearing mice abrogated the antitumor activity of PSB1115 (Figure 5D). The percentage of tumor-infiltrating CD8⁺ T cells and NKT cells in mice adoptively transferred with CD11b⁺Gr1⁺ cells and treated with PSB1115 returned to values similar to those observed in control mice groups (Figure 5, E and F, respectively).

These results indicate that decreased numbers of tumor-associated CD11b⁺Gr1⁺ cells in PSB1115-treated mice restored a T cell-dependent antitumor response and accounted for the reduced melanoma growth.

PSB1115 Treatment Enhances the Therapeutic Efficacy of the Antimelanoma Chemotherapeutic Dacarbazine

We then explored the therapeutic potential of PSB1115 in combination with dacarbazine, a chemotherapeutic currently used for treating metastatic melanoma [37]. Dacarbazine (100 mg/kg, i.p.) [33] alone significantly reduced tumor growth compared to controls (Figure 6A). Interestingly, the combination of dacarbazine and PSB1115 resulted in enhanced antitumor activity in melanoma-bearing mice compared to single agents (Figure 6A). The percentage of CD8⁺ T cells and NKT cells in the melanoma lesions was significantly increased compared with control (Figure 6, B and C, respectively), as well as the levels of granzyme B in tumor tissue (Figure 6D). Similar results were also obtained in mice treated with oxaliplatin, a chemotherapeutic not currently used for melanoma therapy (data not shown).

Figure 6.

PSB1115 enhances the efficacy of dacarbazine against melanoma. (A) Tumor volumes (mm³) of mice receiving dacarbazine (100 mg/kg, i.p.) and/or PSB1115 are expressed as means ± SEM (n = 7–10 per group). CD3-PeCy5.5⁺CD8-allophycocyanin⁺ cells (B) and CD3-PeCy5.5⁺NK1.1-PE⁺ cells (C) were determined in the tumor tissue (n = 6–8 per group). (D) Granzyme B levels as picogram per milligram protein measured in the tumor tissue homogenate of treated mice are expressed as means ± SEM (n = 6–8 per group). Data are from two independent experiments. *P < .05, **P < .01, and ***P < .001 (one-way ANOVA analysis).

Discussion

This study shows that A_2bR promotes the accumulation of MDSCs in the melanoma tissue. Hence, the selective A_2bR antagonist PSB1115 may be an attractive therapeutic drug to reverse MDSC-mediated immune suppression and limit melanoma growth.

MDSCs are potent immune regulatory cells that accumulate in blood, in lymphoid organs, as well as in tumor lesions, in mouse tumor models, and in patients with cancer [18]. These cells originate from bone marrow (BM) myeloid progenitor cells and, under normal conditions, differentiate in peripheral organs into DCs, macrophages, or granulocytes [21,23]. In the context of cancer, soluble factors released in the tumor microenvironment can inhibit this maturation process and drive the accumulation of myeloid cells with immune-suppressive features (MDSCs) [21–24,34]. In melanoma mouse models, the production of inflammatory mediators during tumor progression is thought to attract MDSCs into tumor lesions [34,36]. These cells markedly impair the proliferation and functions of T cells, hampering their antitumor activity. Several lines of evidence indicate that therapeutic strategies that selectively reduce the number of MDSCs inhibit their function or modulate their differentiation and can inhibit tumor growth [23,24].

Although the role of many inflammatory mediators in inducing these processes is well known, recent evidence suggests that adenosine is also critical in controlling the accumulation of MDSCs in tumor-bearing mice. It is well established that adenosine accumulates in tissue under hypoxic conditions typical of tumor microenvironment. Importantly, adenosine can critically hamper the adaptive immune response [1,2,38,39]. A recent study by Ryzhov et al. demonstrated that A_2bR-deficient mice showed reduced Lewis lung carcinoma tumor development [10]. Tumor-bearing A_2bR-/- mice had lower number of MDSCs compared with wild-type mice, suggesting a critical role of this receptor in MDSC expansion [10]. Hence, A_2bR stimulation in BM hematopoietic progenitor cells could prevent their differentiation into mature myeloid cells and thereby promote MDSC accumulation. Moreover, a previous study reported that adenosine, through A_2bR, skews the differentiation of mouse hematopoietic progenitor cells from DCs toward a tolerogenic, angiogenic, and proinflammatory phenotype [40]. Expansion of immature myeloid cells in BM of tumor-bearing animals results in an accumulation of MDSC in peripheral lymphoid organs and in the tumor tissue. Our data show that PSB1115 preferentially decreased the accumulation of tumor-associated CD11b⁺Gr1⁺ cells. We cannot formally exclude the possibility that PSB1115 might inhibit the differentiation of myeloid progenitor cells into MDSCs. However, under our experimental conditions in which PSB1115 was administered peritumorally, a localized inhibitory effect of PSB1115 on MDSC accumulation in the tumor microenvironment is more likely. Notably, we did not observe any effect on MDSC accumulation in nontumoral peripheral organs, consistent with an effect on tumor recruitment rather than with a putative systemic activity of PSB1115. We cannot rule out that PSB1115 may promote the differentiation of MDSCs in macrophages. However, Corzo et al. have shown that, in tumor microenvironment, MDSCs differentiate preferentially to tumor-associated macrophages [41]. Tumor-associated macrophage promotes tumor growth rather than inhibiting it [41]. The antitumor activity we have observed in PSB1115-treated mice is not consistent with this hypothesis. Furthermore, no clear difference was found for DCs between PSB1115-treated and control mice, suggesting that PSB1115 does not influence the process of MDSC maturation. MDSC accumulation in melanoma is driven by soluble factors released both from tumor and stoma cells [24,34]. Previous reports have demonstrated that agents able to alter the levels of inflammatory mediators in tumor microenvironment reduce MDSC accumulation [36,42,43]. In this work, we found that PSB1115-treated mice showed reduced concentrations of IL-10 and MCP-1 within tumors. Melanoma B16.F10 cells stimulated with Bay 60-6583 in vitro produced greater amounts of IL-10; this effect was completely blocked by PSB1115 (data not shown). In our experimental model, the impaired accumulation of MDSCs in melanoma lesions of mice treated with PSB1115 might be, at least in part, ascribed to a modulation of the cytokine milieu in tumor microenvironment from an immune-suppressive to a nonimmune-suppressive type. To what extent these inflammatory factors are involved in PSB1115-induced effects in our model remains to be elucidated in future studies. MDSCs are potent suppressors of T cell-mediated immune responses in the tumor milieu [19,23,24]. PSB1115 treatment enhanced the frequency of tumor-infiltrating CD8⁺ T cells and NKT cells and the release of IFN-γ compared with controls. When nude mice were used to test the antitumor activity of PSB1115, we did not observe any effect on melanoma growth, proving that the activity of PSB1115 was dependent on T cells. On the basis of our results, we propose that the impaired recruitment of MDSCs to tumor lesions in PSB1115-treated mice reduces immune evasion, thereby restoring a T cell immune response. Although our data support a role for A_2bR in mediating MDSC accumulation in our melanoma model, other receptors may also contribute to the effects of adenosine. We have previously shown [4,5] that the A₃ AR subtype has an opposite effect to A_2bR. Specifically, activation of A3 by a selective agonist suppresses melanoma growth in an NK- and CD8 T cell-dependent fashion. Preliminary experiments with A_2a receptor subtype inhibitors indicate that this receptor (which is highly expressed in T cells) does not modulate MDSC accumulation but has direct effects on T cells. These results will be reported elsewhere. Finally, we showed that the combination of PSB1115 and dacarbazine was more effective in suppressing melanoma growth than either drug alone. This data is translationally relevant, as it supports a possible study of chemoimmunotherapy combination regimens including standard-of-care chemotherapy with dacarbazine and immune-stimulatory A_2bR antagonists.

The therapeutic potential of A_2bR blockade has been tested in few murine tumor models to date. Cekic et al. [12] demonstrated that administration of nonselective AR antagonist aminophylline, currently used for treating respiratory diseases [44,45], can reduce the growth of bladder and breast cancers in mice in an A_2bR-dependent manner. To our knowledge, this is the first study demonstrating that selective blockade of A_2bR by PSB1115 can be effective in delaying melanoma growth by inhibiting tumor MDSC accumulation and restoring antitumor immunity. An interesting question for future studies is whether A_2bR antagonists also increase the efficacy of other immunotherapeutic strategies in melanoma, such as ipilimumab or tumor vaccines. As small molecules that presumably have better tumor penetration than protein-based biologics, A_2bR antagonists may be very attractive candidates for clinical development.

Abbreviations

AR

adenosine receptor