Background
Head and neck squamous cell carcinoma (HNSCC), arising in the oral cavity, larynx, hypopharynx and oropharynx, is the 6th most common cancer worldwide [
1]. Despite the intense research interest and efforts in developing therapeutic approaches based on surgical intervention, chemotherapy, radiotherapy, and monoclonal antibody-based therapy, the overall survival rate of HNSCC patients has barely improved [
2]. Currently, only 50%–60% of patients can survive 5 years after diagnosis [
3]. Generally, smoking, alcohol consumption, and human papillomavirus (HPV) infections are the main risk factors of HNSCC [
3]. Notably, HNSCC has been recently identified as an immunosuppressive disease with exhausted T cells, poor antigen-presenting function and accumulation of immunosuppressive cells consisting myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) [
4,
5]. Therefore, a greater understanding of immunosuppression during the initiation and progression of HNSCC will be valuable to formulate improved therapies.
Solid tumors are often infiltrated by immune cells, predominantly T lymphocytes and myeloid cells [
6]. More T cells infiltrating the tumor microenvironment has always been considered as a sign of better prognosis in head and neck cancer [
7]. However, a subpopulation of these T cells recruited to human solid tumors are immunosuppressive CD4
+ Foxp3
+ regulatory T cells (Tregs) [
8]. As compared with the healthy controls, increased accumulation of Tregs was reported in tumor site and peripheral blood of patients with cancer, including HNSCC [
9]. Moreover, increased frequency of Tregs has been shown to be closely associated with the poor clinical outcome, presumably due to Tregs-mediated suppression of anti-tumor immunity [
10,
11]. Tregs exerts its immunosuppressive functions through distinct and often unexpected mechanism. For instance, Tregs interfere with effector T cell proliferation and cytokine production, inhibit the maturation of antigen presenting cells (APCs) and produce immune suppressive cytokines, such as IL-10 [
12,
13]. Therefore, management of Tregs in tumor microenvironment has been underlined as a potential anti-tumor strategy [
14].
Immunosuppressive adenosine 3′5’-monophosphate (cAMP)-mediated pathway, signaling through adenosine A2A receptor (A2AR), can inhibit T lymphocytes and natural killer (NK) cells in hypoxic, inflamed, and cancerous microenvironment [
15]. A2AR interferes with the trafficking and activities of T cells and NK cells due to the heterologous desensitization of chemokine receptors and reduction in the levels of pro-inflammatory cytokines [
15‐
17]. In the mice that are genetically deficient in A2AR, or in the presence of A2AR specific antagonists, an enhanced T cell- and NK cell-associated tumor rejection was observed [
15,
18‐
20]. In addition, blocking the adenosine-generating pathway CD39/CD73 also induced potent regression of breast cancer, colorectal cancer and melanoma [
21‐
24]. A recent study revealed that A2AR stimulation by agonist in vitro expanded CD4
+ Foxp3
+ cells [
25]. These researches revealed the potential relevance between adenosine pathway and Tregs. However, to date, the expression and function of A2AR in HNSCC are far from clear.
In the present study, we aimed to identify the correlation between A2AR expression and clinicopathological characteristics in HNSCC tissue microarrays. In vivo, we sought to investigate the anti-tumor effect induced by A2AR pharmacological blockade in genetically defined immunocompetent HNSCC mouse model.
Methods
Human tissue samples
Human tissue microarrays (TMAs) include 43 histologically confirmed normal oral mucosae, 48 dysplasia (Dys), 165 primary HNSCC (PH), 12 recurrent HNSCC, and 17 HNSCC with induction chemotherapy (cisplatin, docetaxel and fluorouracil, TPF). HPV infection was determined by immunohistochemistry staining of p16 and further confirmed by HPV DNA in situ hybridization as previously described [
26]. The patients received 2 rounds of TPF therapy in accordance with the protocol of Zhang’s clinical trial [
27]. Clinical stages of HNSCC patients were classified according to the guidelines of the UICC (International Union Against Cancer) 2002. Pathological grade was determined according to the scheme of WHO (World Health Organization). All the tissues were obtained from the Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology Wuhan University with the approval of Wuhan University Medical Ethics Committee. The informed consents were obtained from the patients before surgery.
Mice
The time inducible tissue-specific
Tgfbr1/
Pten double conditional knockout (2cKO) mice (
Tgfbr1
flox/flox;
Pten
flox/flox;
K14-CreER
tam+/−) were bred as previously described [
28]. The mice maintained and genotyped as previously reported and
Tgfbr1
flox/flox/
Pten
flox/flox mice (
Tgfbr1
flox/flox;
Pten
flox/flox;
K14-CreER
tam−/−) from same cage were separated as wide type (WT) control. All the mice were bred in the FVBN/CD1/129/C57 mixed background.
In vivo SCH58261 treatment
The
Tgfbr1/Pten 2cKO mice were given tamoxifen by oral gavage for 5 consequent days [
28]. And these mice randomly divided into two groups including vehicle group (DMSO diluted in PBS,
n = 6) and A2AR antagonist SCH58261 treatment group (
n = 6). A week later, SCH58261 (1 mg/kg) or vehicle was intraperitoneally injected into
Tgfbr1/Pten 2cKO mice every other day until the end point. The endpoint was determined according to a systematic evaluation by the veterinarian. Photographs of tumor-bearing mice were taken at day 19 and day 34. Body weight and the tumor volumes were measured every other day. All mice were euthanized at the end of the study
.
Flow cytometry
Single cell suspension was isolated from spleen, lymph nodes, peripheral blood and tumors according to a standardized protocol [
29]. Cells from different groups including wild type (WT) mice and 2cKO mice in vehicle group or SCH58261 treated group were re-suspended in staining buffer (PBS with 2% FBS) at 4 °C and non-specific Fc was blocked for 10 min. Fluorochrome-conjugated monoclonal antibodies were used for staining: isotype-matched IgG controls, Percp-Cy5.5-conjugated F4/80; PE-conjugated CD11b, IFN-γ; PE-Cy5-conjugated Foxp3, FITC-conjugated CD4, CD8 and Gr1 (eBioscience), Adenosine A2A-R Antibody Alexa Fluor® 647 (Santa Cruz Biotech). For IFN-γ staining, cells were processed with Cell Stimulation Cocktail (plus protein transport inhibitors, eBioscience), which contains Phorbol-12-myristate-13-acetate (PMA), ionomycin, Brefeldin A and Monensin for 12 h following the manufacture’s instruction. Dead cells were excluded by staining 7AAD (Invitrogen). Isotype control and positive control were set for each antibody and each experiment. Different gating strategy was used to identify the cell populations. Data were analyzed with Flowjo 7.6 (Tree Star).
Isolation of CD8+ T cells
CD8+ T cells were purified from freshly isolated tumor infiltrated lymphocytes of the 2cKO mice from vehicle group or SCH58261 treated group by immunomagnetic sorting using the mouse CD8+ T cell isolation kit and following the manufacturer’s instructions (Miltenyi Biotech). The purity of the isolated CD8+ T cells was measured by surface staining with anti-CD8 mAb. The overall purity of the resulting cells was 85.3% ± 1.2%. Cell viability was >90% as measured by trypan blue exclusion.
Cytokine measurement
Freshly isolated CD8+ T cells were cultured in RPMI medium at a concentration of 1 × 106 for 8 h. The supernatants were collected for IFN-γ and TNF-α measurement. The levels of IFN-γ and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) (BD Pharmingen and R&D System).
Immunofluorescence
Briefly, the human HNSCC tissue sections were hydrated and antigen retrieval. Then sections were blocked with goat serum and incubated with rabbit polyclonal antibody against A2AR (Abcam) at 4 °C overnight, followed by incubation with fluorochrome conjugated secondary antibodies (Alexa 594 anti-rabbit; Invitrogen) and DPAI (Vector Laboratories). The images were observed and taken using C2+ confocal microscope system (Nikon).
Immunohistochemistry
Paraffin sections of human HNSCC tissue microarrays or mouse HNSCC section were rehydration in graded alcohol. The antigen retrieval was performed in boiled sodium citrate. All the sections were incubated in 3% hydrogen peroxide for endogenous peroxidase blockade. Goat serum or rodent block (for mouse section) was used to block the non-specific binding at 37 °C for 1 h. Next, sections were incubated with antibody for A2AR (Abcam 1:200), HIF-1α (Abcam 1:200), CD73 (Genetex 1:200), Foxp3 (Abcam 1:100), CD8 (ZSGB-BIO 1:100, for human samples), CD8α (Novus, 1:200, for mouse samples) at 4 °C for 12 h. On the day 2, sections were incubated with secondary biotinylated immunoglobulin G antibody solution and an avidin-biotin-peroxidase reagent. Then, the section stained with DAB kit (Mxb Bio) and the sections lightly counterstained with haematoxylin (Invitrogen, USA). Negative control with primary antibody replaced by PBS, isotype control and commercial available positive control for each antibody were set in parallel.
Western blot
The mouse tumor tissues were carefully dissected (n = 6, respectively) and stored at −80 °C. All samples were lysed with RIPA Lysis Buffer (Beyotime), which contains protease inhibitors and phosphatase inhibitors. Lysates were denatured in loading buffer (Beyotime) at 95 °C for 5 min. Protein samples were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). Rabbit A2AR antibody (Abcam, 1:1000), Rabbit CD73 antibody (Genetex, 1:1000) and Rabbit HIF-1α antibody (Abcam 1:1000) were used as primary antibody, GAPDH (Abcam, 1:3000) was used as loading control. Western blot staining was performed by using enhanced chemiluminescence detection kit (Advansta). All Western blots were repeated at least 3 times.
Scoring system
The scanning of all slices was performed by Aperio ScanScope CS scanner (Leica, USA) with background subtraction. The histoscore of each slice was quantified by Aperio Quantification System (Version 9.1). The histoscore of pixel quantification was calculated as the total intensity/total cell member.
Statistical analysis
Statistical analysis was performed GraphPad Prism 5.0 (Graph Pad Software Inc). One-way ANOVO followed Tukey test was applied to analyze the difference in A2AR expression in normal oral mucosa, oral epithelial dysplasia and HNSCC, pathological grades, the population change of CD8+ T cells, MDSCs and TAMs in spleen, lymph nodes and peripheral blood of different groups and the spleen index. Unpaired t test was used to analyze the difference of A2AR expression in tumor size (T1 + T2 vs T3 + T4), lymph node metastasis (N0 vs N1 + N2), HPV infection status (HPV+ vs HPV-), primary HNSCC and recurrence HNSCC (primary vs recurrence), primary HNSCC and TPF chemotherapy specimen (primary vs post TPF), population change of CD4+ Foxp3+ Tregs and CD4+ Foxp3+ A2AR+ cells from each group, the immunohistochemical staining of Foxp3+ and CD8+ cells from each group and the increased body weight. The data are presented as the Mean ± SEM, and statistical significance was determined as P < 0.05. The correlation of A2AR and HIF-1α, CD73, Foxp3, and CD8 was analyzed by two-tailed Pearson’s statistics. The Kaplan-Meier method and the log-rank test were applied to analyze the overall survival rate (OS) between A2AR high group and A2AR low group. The median of A2AR histoscore was chosen as cut-off. For the hierarchical cluster, the histoscore were converted into −3 to 3 using Microsoft excel. Then, the results were imported and hierarchical analysis was performed with Cluster 3.0. The heatmap was visualized with Java TreeView 1.0.5.
Discussion
Adenosine plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, benefiting the progression of cancer [
41]. Tissue hypoxia seems to be essential to the increase of intratumoral adenosine levels [
24]. On one hand, hypoxia elevates the expression of adenosine generation pathway CD39 and CD73 [
33,
42]. On the other hand, hypoxia decreases adenosine kinase and inhibits the conversion of adenosine [
43]. As a consequence, specific adenosine receptors such as A2AR are activated by increased levels of extracellular adenosine in tumor microenvironment [
43]. In the present study, elevated expression of A2AR, which was positively correlated with HIF-1α and CD73, was detected in HNSCC tissue microarrays, indicating the activation of hypoxia-CD73-A2AR pathway. Hypoxia appears quite frequently in a variety of solid tumors when tumor growth exceeds the angiogenic growth [
44,
45]. An earlier study demonstrated that nuclear overexpression of HIF-1α was detected in 69.64% of analyzed oral squamous cell carcinoma (OSCC), being positively correlated with the rate of tumor progression (tumor size, lymph node metastasis and histological differentiation) [
46]. In the current study, overexpression of A2AR was linked to larger tumor size, lymph node metastasis and pathological grade. Considering the positive correlation between A2AR and HIF-1α in HNSCC tissues, we suggested that A2AR interfere the tumor progression rate partially depend on hypoxia status. To date, there is no convincing research indicating that A2AR overexpression was correlated with poor clinical outcome in HNSCC. In the present study, Kaplan-Meier analysis data implicated high expression of A2AR was associated with unfavorable clinical prognosis.
Under the influence of adenosine pathway, CD8
+ T cells become less cytotoxic with decreased TCR signaling and production of proinflammatory cytokines, such as IFN-γ [
47]. Considering the immunosuppressive role of A2AR in cancerous tissues, we subsequently assessed the correlation between A2AR and CD8 and found that A2AR expression was negatively correlated with the CD8 expression in human HNSCC tissues. To some extent, these results suggested A2AR might play an immunosuppressive role through influencing CD8
+ T cells population in HNSCC. Notably, it has been earlier reported that hypoxia-A2AR pathway was not only an immunosuppressive signaling that inhibits the TCR signaling, but also facilitated the development of immunosuppressive Tregs [
48]. Moreover, a recent study demonstrated that in vitro A2AR stimulation by agonist numerically and functionally enhanced the Tregs sorted from the human peripheral blood [
25]. These studies emphasized the regulatory role on Tregs expansion and function by A2AR signaling. Indeed, in this present study, the positive correlation between the expression of A2AR and Foxp3 indicated the potential relevance between A2AR signaling and Tregs in HNSCC.
Alterations of PTEN/PI3K/AKT/mTOR pathway and TGF-β1 are the most frequent molecular events during HNSCC tumorigenesis and progression [
49]. In the previous study, by combining knockout of
Tgfbr1 and
Pten (2cKO), we constructed a spontaneous immune competent HNSCC mouse model, which was suggested as an appropriate pre-clinical animal model for HNSCC research. The emerging tumors were not only pathologically indistinguishable from the human HNSCC, but also presented major molecular alternations in human HNSCC [
35]. In the present study, deletion of
Tgfbr1 and
Pten in mouse head and neck epithelia activated hypoxia-CD73 pathway and consequently induced the elevation of A2AR on the immune cells, including CD4
+ Foxp3
+ Tregs and CD8
+ T cells. Additionally, deletion of
Tgfbr1 in murine head and neck epithelia resulted in enhanced paracrine effect of TGF-β1 in tumor stroma, which facilitated the immunosuppressive status and promoted the tumor progression [
37]. Given that the development of Tregs is under the influence of various inductive signals, most importantly TGF-β1 [
50], we found a significantly increased population of CD4
+ Foxp3
+ Tregs in the tumor of 2cKO mice. It has been reported that A2AR stimulation enhanced the proliferation of Tregs [
25]. In the present study, elevated A2AR was detected on the surface of CD4
+ Foxp3
+ Tregs in 2cKO tumor bearing mice, emphasizing the potential role of A2AR signaling in regulating the expansion or functions of Tregs in HNSCC. These findings also provided us with the rationale for decreasing Tregs by A2AR antagonist. Indeed, pharmacological blockade of A2AR by antagonist repressed the tumor growth of 2cKO mice and reduced the population of CD4
+ Foxp3
+ Tregs. Meanwhile, an enhanced anti-tumor response of CD8
+ T cells was observed in 2cKO tumor bearing mice treated with A2AR antagonist SCH58261, indicating the improvement of the immunosuppressive status. This result was partially in accordance with the study that A2AR protected tumor cells from anti-tumor CD8
+ T cells [
15]. It has been reported that selective deletion of A2AR on myeloid cells caused potent tumor rejection which was associated with significant increases of MHC II and IL-12 expression in tumor-associated macrophages (TAMs) and reductions in IL-10 expression in TAMs, dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) [
51]. In the current study, although the populations of MDSCs and TAMs were significantly increased in 2cKO tumor bearing mice, the A2AR antagonist was unable to decrease the population of these cells. These results indicated that A2AR blockade probably did not affect the expansion of immunosuppressive myeloid cells in HNSCC. However, the effect of A2AR signaling on the functions of myeloid cells needed additional studies.
Cytotoxic chemotherapeutic agents are widely employed in the war for fighting against cancer [
52]. Nevertheless, emerging evidence has indicated cytotoxic agents altered the local immune state, interfering the response of treatment [
53]. Several cytotoxic chemotherapeutic agents, including 5-FU, appear to produce an in situ vaccination as a consequence of their initial cytotoxic effect and to facilitate an immunogenic cell death (ICD) [
52]. During this process, the release of ATP has been identified as a critical mediator [
54]. ATP was eventually catabolized to immunosuppressive adenosine by CD39 and CD73 pathway, which are frequently activated by hypoxia in tumor microenvironment, and subsequently changing the immune status in tumor microenvironment. Of interest, we detected a significant up-regulation of A2AR in the HNSCC tissues with induction chemotherapy, indicating that A2AR may facilitate drug resistance probably by altering the immune status in tumor microenvironment. This phenomenon may reflect the potential therapeutic value of combining use of A2AR antagonist and conventional chemotherapeutic reagents in the treatment for HNSCC. In addition, inhibition of immune checkpoints still leaves T cells vulnerable to multi-faceted and powerful immunosuppression by hypoxia-adenosine pathway [
55]. Inhibitor of hypoxia-A2-adenosinergic pathway may decrease the intensity of other immunosuppressive factors including CTLA-4 or TGF-β1. This hypothesis was supported by recent studies indicating that CTLA-4 or PD-1 blockade combined with the inhibition of the extracellular adenosine or A2AR/A2BR signaling resulted in a stronger anti-tumor effect [
56,
57]. A phase I clinical trial (NCT02655822) of A2AR antagonist (PBF-509 and CPI-444) alone or with immune checkpoint inhibitor (atezolizumab, a PD-L1 inhibitor) is currently recruiting participants to study the clinical efficiency of A2AR blockade for solid tumor including HNSCC.
Funding
This work was supported by National Natural Science Foundation of China (81672668, 81472529, 81672667 and 81472528).