Altered purinergic signaling in the tumor associated immunologic microenvironment in metastasized non-small-cell lung cancer

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Altered purinergic signaling in the tumor associated immunologic microenvironment in metastasized non-small-cell lung cancer Severin Schmid a,∗ , Markus Kübler a , C. Korcan Ayata b , Zsofia Lazar c , Benedikt Haager a , Madelon Hoßfeld b , Anja Meyer b , Sanja Cicko b , Mirjam Elze a , Sebastian Wiesemann a , Gernot Zissel b , Bernward Passlick a , Marco Idzko b a

University Medical Center Freiburg, Department of Thoracic Surgery, Freiburg, Germany University Medical Center Freiburg, Department of Pneumology, Freiburg, Germany c Semmelweis University, Department of Pneumology, Budapest, Hungary b

a r t i c l e

i n f o

Article history: Received 5 August 2015 Received in revised form 13 September 2015 Accepted 4 October 2015 Keywords: Lung cancer Purinergic signaling P2-receptor Tumor microenvironment CD39

a b s t r a c t Objectives: Purines are well-known as intracellular sources for energy but they also act as extracellular signaling molecules. In the recent years, there has been a growing interest in the therapeutic potential of purinergic signaling for cancer treatment. This is the first study to analyze lung purine levels and purinergic receptors in non-small-cell lung cancer (NSCLC) patients. Materials and methods: In this prospective clinical trial we enrolled 26 patients with NSCLC and 21 patients with chronic obstructive pulmonary disease (COPD) without signs of malignancy. The purine concentrations were analyzed in bronchoalveolar lavage fluid (BALF) using fluorescent/luminescent assays. Expression of purinergic receptors and ectonucleotidases were analyzed using real time quantitative polymerase chain reaction (RT-qPCR). Results: Patients with NSCLC have significantly lower ATP and ADP concentrations in BALF than patients with COPD (p = 0.006 and p = 0.009). Expression of the ectonucleotidase CD39 is significantly higher in BAL cells from cancer patients compared to COPD (p = 0.001) as well as in metastasized tumors compared to non-metastasized tumors (p = 0.009). Receptor-analysis revealed a higher expression of P2X4 (p = 0.03), P2X7 (p = 0.001) and P2Y1 (p = 0.003) in BAL cells of tumors with distant metastasis. Conclusion: Our data suggests a role for CD39 in lung cancer tumor microenvironment, influencing tumor invasiveness and metastasization. Potentially the increased degradation of ATP and ADP leads to a subversion of their anti-neoplastic effects. Furthermore P2Y1, P2X4 and P2X7 receptors are upregulated in BAL cells in metastatic disease. Our findings might facilitate the identification of new therapeutic targets for cancer immunotherapy. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer-related death worldwide and is usually divided into the two large entities of small-cell (SCLC) and non-small-cell lung carcinoma (NSCLC), the latter accounting for about 87% of cases. Only in about 34 percent of patients with NSCLC the disease is diagnosed at an early stage where a complete resection is the treatment of choice and a cure is possible. Due to the aggressive nature even in early stage NSCLC recurrence and metastasis are relatively common, lead-

∗ Corresponding author at : Department of Thoracic Surgery, University Hospital Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany. Fax: +49 0 761 270 90701. E-mail address: [email protected] (S. Schmid).

ing to a 5-year survival rate of approximately 20 percent [1–3]. Besides known cytotoxic agents there have been advances in individualized therapy for treatment of systemic disease focusing on molecular targets such as the epidermal growth factor receptor (EGFR) and the anaplastic lymphoma kinase (ALK) [4–6]. The importance of tumor microenvironment for growth and invasiveness is widely acknowledged offering a variety of potential new therapeutic targets. The host’s immune system is subverted by the tumor as cytotoxic effector cells are suppressed by dysfunctional regulatory cells such as the regulatory T-cells (Treg), tumor associated macrophages (TAM) and myeloid-derived suppressor cells. Tumor aggressiveness was shown to be dependent on the ratio of these suppressor cells and cytotoxic effector cells [7,8].

http://dx.doi.org/10.1016/j.lungcan.2015.10.005 0169-5002/© 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: S. Schmid, et al., Altered purinergic signaling in the tumor associated immunologic microenvironment in metastasized non-small-cell lung cancer, Lung Cancer (2015), http://dx.doi.org/10.1016/j.lungcan.2015.10.005

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Purines are not only well-known as intracellular sources for energy but also as extracellular signaling molecules. In the recent years there has been a growing interest in the therapeutic potential of purinergic signaling for cancer treatment. The effects carried out depend on the concentration, expression pattern of purinergic receptors and general dynamics of synthesis and degradation [9–11]. Anti-neoplastic as well as growth-promoting effects have previously been shown for extracellular purine-based nucleotides represented by adenosine, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) in different tumor types [10,12–14]. Effects are carried out via the activation of P2-purinergic receptor family (P2R), divided into the ion-channel linked P2X- and the G-protein coupled P2Y-receptors. In lung cancer cell lines the expression of P2X4, P2Y2, P2Y4 and P2Y6 receptors was shown previously [15]. Schäfer and coworkers found growth promoting effects of ATP and UTP, which were mediated by P2Y2 and P2Y6 receptors [16]. However, also ATP induced dose-dependent growth inhibition and enhanced cytotoxic effects of chemotherapeutic agents in combination with ATP have been described in various NSCLC cell lines [14,17]. In a series of clinical studies the therapeutic value of intravenously administered ATP in lung cancer patients has been evaluated. In advanced NSCLC ATP treatment had positive effects on cancer cachexia, muscle strength and overall survival but did not influence tumor growth [18–22]. Positive effects on nutritional status of cancer patients might be due to lower glucose turnover between infusions with ATP [21]. Furthermore, Zanini and colleagues showed that increased occurrence of thromboembolic events in lung cancer patients might be due to alterations in purine metabolization [23]. To our knowledge, to date there is no data on purinergic signaling in the context of the tumor associated immunologic microenvironment in NSCLC. In this study we analyzed the expression of different purines and purinergic receptors in bronchoalveolar lavage (BAL) in patients with NSCLC to provide further insight into possible effects on tumor growth and metastasization in this context.

2. Materials and methods In this prospective observational study we enrolled 26 patients with proven NSCLC and 21 patients with chronic obstructive pulmonary disease (COPD). The study was approved by our local ethics committee and registered as a clinical trial in the German Registry for Clinical Trials (DRKS-ID: DRKS00005415). All patients signed an informed consent before participating in the study. Inclusion criteria were the presence of a pulmonary nodule suspicious of primary lung cancer and age above 18 years. Exclusion criteria were clinical or radiologic signs of pneumonia, centrally located tumors, presence of other malignancies, previous radiation or chemotherapy and inadequate quality of the BAL sample (presence of ciliated epithelium, bloody samples and low recovery). BAL was performed using flexible bronchoscopes (Olympus, Hamburg, Germany). In patients with a suspicious nodule the bronchoscope was wedged into the subsegmental bronchus where the tumor was present and a total of 300 ml sterile saline at room temperature was instilled. The BAL was recovered by gentle aspiration. BAL samples were centrifuged for 10 min at 500 × g at 4 ◦ C. Supernatants were aliquoted and the cells were resuspended in phosphate buffered saline (pH 7.4). Citrate buffer was added to BAL fluid (BALF) samples for analysis of purinergic metabolites to prevent further degradation. BAL differential cell counts were determined. Samples that were not immediately analyzed were stored at −80 ◦ C. Nucleotides (ATP, ADP, adenosine monophosphate (AMP), adenosine and inosine) were measured in BALF supernatant

Table 1 Demographics and BAL characteristics.

N Age, yr Female gender, N Histology Adeno-carcinoma, N Squamous-cell-carcinom, N Pulmonary function FEV1 , % predicted FEV1 , % FVC BAL characteristics 106 Cells/100 ml BALF Macrophages, % Lymphocytes, % Neutrophils, % Eosinophils, %

NSCLC

COPD

26 60.2 (±8.3) 10 (38%)

21 64.7 (±8.4) 8 (38%)

20 (77%) 6 (23%)

N/A N/A

63.8 (±12.5) 63.9 (±12.6)

59.8 (±24.7) 61.3 (±19.8)

11.4 (±15) 65 (±31) 18 (±21) 15 (±27) 2 (±4)

31.4 (±30.5) 67 (±31) 14 (±16) 18 (±29) 1 (±2)

Results are presented as mean with standard deviation.

using fluorescent/luminescent assays as described before in NSCLC (n = 26) and COPD (n = 21) [24]. Expression of purinergic receptors and ectonucleotidases (P2X1, P2X4, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2Y13, P2Y14, CD39, CD73) were analyzed in BALF cell pellet using real-time quantitative polymerase-chain-reaction (RT-qPCR) in NSCLC (n = 25) and COPD (n = 10) [25]. Total RNA was isolated using Qiazol (Qiagen), complementary DNA synthesis was performed with the First Strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA). TaqMan qPCR was performed using 2× Takyon qPCR Mastermix (Eurogentec, Cologne, Germany), on a Lightcycler 480 (Roche, Basel Switzerland). Primers and dual-labeled hybridization probes were designed as previously described, sequences are available on request [26]. ß2-microglobulin (ß2M) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as reference genes (RG). Percent RG values were calculated using the formula: % RG = 100 × 2(−Ct) . Standard deviations (SD) of RG and gene of interest (GOI) were calculated using the formula: SD = 100 × 2(−Ct) × ((ln2 × SDRG )2 + (ln2 × SDGOI )2 )1/2 [27]. Data were recorded in a database designed in Microsoft Office Excel (Microsoft, Redmond, WA, USA) and GraphPad Prism 6.03 (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis. The D’Agostino–Pearson omnibus normality test was performed. Normally distributed datasets were compared using the student’s t-test. In non-normally distributed data Mann-Whitney test was employed. Results were considered significant if the p value was less than 0.05. 3. Results 26 patients with suspicion of NSCLC and 21 patients with COPD were enrolled in the study. The age and lung function of patients was comparable in both groups. Histologic analysis of lung biopsy tissue verified lung adenocarcinoma in 20 and squamous cell carcinoma in 6 cases. Clinical and BAL characteristics are summarized in Table 1. 3.1. ATP and ADP concentrations in BALF supernatant are lower in NSCLC compared to COPD Depending on the concentrations ATP and ADP are known to carry out anti-proliferative and cytotoxic effects, hence purine metabolites were measured in BALF from NSCLC and compared to a well matched COPD cohort. Our group previously studied purines and the related receptors in COPD compared to healthy subjects (HC) [28]. HC showed very low purine concentrations and very little expression of P2-receptors. Also, forced expiratory volume in 1 s (FEV1 ) correlated well with ATP concentrations. As NSCLC patients

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Fig. 1. Comparison of concentrations of purines in bronchoalveolar lavage fluid from NSCLC and COPD patients. Patients with NSCLC have significantly lower ATP and ADP concentrations in BALF than patients with COPD without signs of malignancy (p = 0.006 and p = 0.009). Inosine concentrations however are higher in patients with malignant disease (p = 0.01). AMP and Adenosine concentrations show no significant differences. * p < 0.05, ** p < 0.01.

are mostly smokers with impaired lung functions, COPD patients with comparable FEV1 are the most adequate control group. Patients with NSCLC had significantly lower ATP and ADP concentrations in BALF than patients with COPD without signs of malignancy (p = 0.006 and p = 0.009). Inosine concentrations however were higher in patients with malignant disease (p = 0.01). AMP and Adenosine concentrations showed no significant differences (Fig. 1). Interestingly, purine metabolite comparison in subgroup analysis of early stage NSCLC to late stage disease as well as metastatic diseases showed no significant differences of ATP, ADP, AMP and Adenosine concentrations. However, there was a trend towards higher Inosine concentrations in late stage disease (p = 0.08).

3.2. The ectonucleotidase CD39 is overexpressed in NSCLC, particularly in metastatic disease In tumor microenvironment ATP is released from immune cells, endothelial cells as well as from dying tumor cells and is considered to be an important danger signal. ATP concentrations were lower in malignant disease thus we analyzed expression of the ectonucleotidases CD39 and CD73 which are responsible for purine degradation. CD39 mRNA expression was markedly elevated in BAL cells from NSCLC compared to COPD patients (p = 0.001) (Fig. 2A). Furthermore analysis of CD39 expression in NSCLC with presence of distant metastasis at the time of sampling to nonmetastasized NSCLC revealed an overexpression in metastatic disease (p = 0.007) (Fig. 2B). CD73 showed no significant differences in the respective groups.

Fig. 2. Expression of CD39 in tumor microenvironment. CD39 expression is markedly elevated in NSCLC tumor microenvironment compared to COPD (p = 0.001) (A). Analysis of CD39 expression in NSCLC with presence of distant metastasis at the time of sampling to non-metastasized NSCLC revealed an overexpression in metastatic disease (p = 0.007) (B). ** p < 0.01.

3.3. P2X4, P2X7 and P2Y1 are overexpressed in tumors with distant metastasis To evaluate association of purinergic receptors with tumor aggressiveness and metastatic potential, we compared purinergic receptors in the respective groups. Receptor-analysis revealed a higher expression of P2X4 (p = 0.03), P2X7 (p = 0.001) and P2Y1 (p = 0.003) in metastasized compared to non-metastasized tumors (Fig. 3). Also expression of purinergic receptors P2X7 and P2X4 is elevated in NSCLC compared to COPD, however not statistically significant (p = 0.17 and p = 0.46) (Fig. 4). Comparison of other P2 receptors showed no statistically significant differences between the respective groups.

Fig. 3. Purinergic receptor expression in metastasized NSCLC. Receptor-analysis revealed a higher expression of P2X4 (p = 0.03), P2X7 (p = 0.001) and P2Y1 (p = 0.003) in metastasized compared to non-metastasized tumors. * p < 0.05, ** p < 0.01.

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Fig. 4. Comparison of purinergic receptor expression in NSCLC and COPD. P2X7 and P2X4 are overexpressed in NSCLC compared to COPD, however not statistically significant (p = 0.17 and p =0.46).

4. Discussion Previous preclinical findings provide promising results on the therapeutic potential of purinergic signaling in malignant disease. Depending on metabolite kinetics and receptor expression patterns different tumor models have shown anti-neoplastic as well as tumor growth-promoting and pro-metastatic effects of purine based nucleotides on various tumor types. This is the first study to analyze purinergic signaling in the tumor associated immunologic microenvironment in NSCLC. In particular extracellular ATP and ADP have been reported to carry out anti-neoplastic effects by directly inhibiting cell proliferation and promoting cancer cell death. In lung cancer cell lines ATP was shown to induce a dose-dependent growth inhibition and to enhance anti-tumor effects of chemotherapeutic agents [17,29]. Furthermore, low ATP-concentrations lead to an increased tumor growth and increased invasiveness of malignancies [30,31]. In tumor microenvironment ATP is released from dying tumor cells and immune cells and is considered as an important danger signal promoting immune response [32–34]. In the present study ATP and ADP concentrations are lower in patients with NSCLC compared to COPD. ATP concentrations are known to be elevated in COPD compared to healthy individuals and to be positively correlated with disease stage [28]. However, the underlying study cohorts are well matched thus ruling out an influence of lung function or emphysema on nucleotide comparison. Based on these findings the authors suggest that increased release of ATP is antagonized in tumor microenvironment of NSCLC and potential immunologic anti-neoplastic effects are thus undermined. Bolstering this hypothesis the ectonucleotidase CD39/ENTPD1 which is responsible for degradation of ATP and ADP, is overexpressed in NSCLC compared to COPD as well as in the subgroup analysis of metastasized versus non-metastasized tumors. Thus presence of NSCLC as well as tumor invasiveness is linked to increased expression of CD39 with highest expression in late stage disease. In tumor microenvironment CD39 is expressed by immunologic cells, especially Tregs, as well as endothelial and tumor cells. Jackson and colleagues have shown a retardation of lung cancer tumor growth as well as a reduction of tumor metastases in CD39 null-mice [35]. Also Feng and coworkers showed that CD39 expression stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP using knock-out mice. It was suggested that expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP [12]. In a hepatic tumor model, deletion of CD39 reduced Treg-mediated immunosuppression, resulting in increased natural killer cell infiltration and cytotoxic activity [36]. Based on these findings it was sug-

gested that blockage of CD39 would have beneficial effects on tumor growth by restoring the immune antitumor response and consequently selective inhibition of CD39 has been suggested as a therapeutic target in malignant diseases. Recently there have been promising developments in the field of selective inhibitors of ectonucleotidases [37–40]. CD39 is responsible for degradation of ATP to ADP and AMP, which is later degraded to adenosine by CD73. Adenosine was shown to inhibit tumor infiltrating cytotoxic T-cells hence contributing to the tumor associated immunosuppression [41–43]. Also targeting the CD39/CD73 ectonucleotidase pair has been described to effectively antagonize this immunosuppressive effect in various tumors leading to a favorable effect on tumor progression and metastasis [44–46]. The ectonucleotidase CD73 is not overexpressed in our analysis and adenosine concentrations are only slightly elevated in NSCLC patients compared to COPD, thus our findings do not support relevance of the adenosinergic pathway in NSCLC microenvironment. Potentially, it depends on the tumor type whether antagonization of cytotoxic effects of ATP and ADP are regulated by CD39 or immunosuppressive effects of adenosine mediated by CD39/CD73 enzyme pair influence tumor growth or not. Additionally our findings revealed an association of certain P2R with metastatic disease. Increased RNA expression of P2X4, P2Y1 and particularly P2X7 were associated with presence of distant metastasis. P2X7 was also elevated in in BAL cells from NSCLC compared to COPD without presence of malignancy, however not statistically significant. P2X7 is expressed on different immune cells but mainly on macrophages and is overexpressed in various tumor types [47–50]. Most data existing on P2R expression in tumor microenvironment focuses on tumor cells but not on tumor associated immune cells. It has to be emphasized that purinergic receptor-expression in BAL does not represent the tumor cells but the tumor associated immunologic microenvironment represented by the immune cells. Alveolar immune cells are known to express a variety of P2-receptors and previous studies have shown their involvement in the pathogenesis of pulmonary inflammatory diseases [51]. Furthermore, P2R mediate chemotactic functions on neutrophil granulocytes and thus contribute to the cigarette smoke induced inflammation and development of emphysema [28,52]. Besides other factors, in malignant diseases the effects of P2R vary depending on their expression site. One of the most studied and hence best understood P2-receptors is P2X7. On the one hand P2X7 is expressed on many cancer cells including lung cancer and was shown to promote tumor invasiveness and metastasization. [53–55]. Consequently, antagonization of P2X7 in cancer cell lines lead to a beneficial impact on cell invasiveness [56]. On the other hand expression of P2X7 on immune cells has been shown to have anticancer effects and whilst blockage of the P2X7-receptor dampened inflammation, it lead to an increased tumor incidence a mouse model of colitis associated cancer [57]. Also, P2X7 on dendritic cells is activated by ATP released from dying tumor cells and thus is part of a pathway which activates the innate and adaptive immunosystem against tumor cells [58]. Taken together, previous data suggests beneficial effects on malignant tumors of P2X7 on immune cells. So at first glance results from the study at hand seem contradictory to these findings, as we found higher expression of P2X7 on BAL cells to be associated with late stage disease. However, multiple factors might influence the functions of P2X7 in this context. The effects that are carried out by P2X7 in tumor microenvironment also depend on ATP concentrations and whilst very high concentrations result in pro-inflammatory and potentially cytotoxic effects, low concentrations mediate growth-promoting, pro metastastic functions [30,31]. In the underlying study we show relatively low ATP concentrations in combination with overexpression of P2X7 in BAL cells in metastasized NSCLC in particular, which could be one

Please cite this article in press as: S. Schmid, et al., Altered purinergic signaling in the tumor associated immunologic microenvironment in metastasized non-small-cell lung cancer, Lung Cancer (2015), http://dx.doi.org/10.1016/j.lungcan.2015.10.005

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factor contributing to pro-tumorigenic and pro-metastatic functions of P2X7 in NSCLC tumor microenvironment. Nevertheless, the interpretation of these results remains difficult since exact functions of these receptors in immunologic interactions in tumor microenvironment are still lacking. P2R analysis in cancer mostly focuses on expression on tumor cell lines and data from clinical studies are very limited. Despite an abundance of data on purinergic signaling, the lack of specimen representing tumor microenvironment or the associated immune cells in human subjects leave many questions unanswered. BAL in lung cancer provides an excellent opportunity to create insight on these interactions and similar analysis are not possible in all tumor types, emphasizing the significance of the presented data. In conclusion, our findings suggest important functions of CD39 in tumor associated immunologic microenvironment influencing tumor growth and metastasization. Potentially the increased degradation of ATP and ADP by CD39 leads to a subversion of their anti-neoplastic effects. Also, we observed an association of certain purinergic receptors, P2X7, P2X4 and P2Y1, with distant metastases in NSCLC. These findings might facilitate the identification of new therapeutic targets for cancer immunotherapy, however before the therapeutic potential can be valued, exact mechanisms need to be addressed in future studies.

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