Interleukin-27 re-educates intratumoral myeloid cells and down-regulates stemness genes in non-small cell lung cancer.

Current therapies for Non-Small Cell Lung Cancer (NSCLC) still fail to significantly increase its survival rate. Here we asked whether Interleukin(IL)-27, which has revealed powerful antitumor activity and is toxicity-free in humans, is a promising therapeutic choice for NSCLC patients. IL-27's effects were tested on Adenocarcinoma (AC) and Squamous Cell Carcinoma (SCC) cell lines and xenograft models. IL-27Receptor(R) expression was assessed in lung tissues from 78 NSCLC patients. In vitro, IL-27 was ineffective on cancer cell proliferation or apoptosis, but fostered CXCL3/GROγ/MIP2β expression. In vitro and in vivo, IL-27 down-regulated stemness-related genes, namely SONIC HEDGEHOG in AC cells, and OCT4A, SOX2, NOTCH1, KLF4 along with Nestin, SNAI1/SNAIL, SNAI2/SLUG and ZEB1, in SCC cells. In vivo, IL-27 hampered both AC and SCC tumor growth in association with a prominent granulocyte- and macrophage-driven colliquative necrosis, CXCL3 production, and a reduced pluripotency- and EMT-related gene expression. Myeloablation of tumor-bearing hosts mostly abolished IL-27's antitumor effects. In clinical samples, IL-27R expression was found in AC, SCC, pre-cancerous lesions and tumor infiltrating myeloid cells, and correlated with advanced stages of disease. Our data suggest that even immunocompromised or advancer NSCLC patients may benefit from IL-27's antitumor properties based on its ability to drive myeloid cells towards antitumor activities, and down-regulate stemness- and EMT-related genes in cancer cells.


INTRODUCTION
Lung cancer is the leading cause of cancer induced mortality worldwide [1,2]. Non-small cell lung cancers (NSCLC), particularly, adenocarcinoma (AC) and squamous cell carcinoma (SCC), are the most frequently diagnosed histotypes [1,3]. New targeted therapies have been developed and some are currently used to treat advanced AC, but are unsuitable for SCC [4,5]. Moreover, not all AC patients benefit from these treatments [5]. Development of effective and well tolerable immunotherapies to replace or be combined with surgery, radiotherapy or personalized treatments may be of great value. In the last few years, Interleukin(IL)-27, a member of the IL-12 family of cytokines, with important roles in both innate and adaptive immunity [6,7], has revealed potent antitumor effects in the form of anti-proliferation, anti-angiogenesis, and immune system stimulation in a variety of tumors [8][9][10]. Its overexpression in murine Lewis lung carcinoma line 1 (LLC1) cells induces a specific cytotoxic T cell and antibody response in vivo, and also activates non-immunological mechanisms reducing cancer cell motility and migration [11]. Inhibition of AC cell migration, together with downregulation of pro-angiogenesis genes by IL-27 has also been reported in the human A549 AC cell line [12]. Moreover, murine IL-27 gene-transfected LLC1 cells have been used to generate an autologous cell vaccine boosting an efficient T lymphocyte activation and IFNγ production [13]. However, definitive in vivo proof of IL-27's efficacy in pre-clinical models of human lung AC and SCC is still lacking.
We here investigate the in vitro and in vivo effects of IL-27 on the regulation of angiogenesis-, stemness-and epitelial-mesenchymal transition (EMT)-related genes in human AC and SCC cell lines and lung tumors grown in B/T cell deficient mice. Furthermore, by means of molecular biology and immunohistochemical studies, we have assessed IL-27Receptor(R) expression in lung cancer samples and analyzed the rationale for a future IL-27 application in the clinical setting of NSCLC.

Human lung AC and SCC cell lines express IL-27R and respond to IL-27 up-regulating CXCL3 expression and down-modulating stemness-and EMT-related genes
Since AC and SCC are the most common histotypes of lung cancers, ~85% of NSCLC [1,3], we looked to see whether IL-27 acts as an antitumor agent in these forms.
Expression of both chains of the IL-27R, gp130 and WSX-1 (TCCR, IL-27Rα), was investigated, by flow cytometry, in a series of cell lines derived from human lung AC, namely A549, GLC82, Calu-6 or from SCC, namely Calu-1 and SK-MES. As shown in Figure 1A and 1B, Calu-6 and SK-MES lines expressed the highest levels of both chains (gp130: 68% and 65%; WSX-1: 97% and 70% respectively) and were therefore chosen as representative of AC and SCC histotypes for the subsequent experiments.
We began by determining whether IL-27 affects the in vitro proliferation or apoptosis of these lines by culturing them with or without human (h) recombinant (r) hrIL-27 for 120 hours, and harvesting an aliquot every 24 hours to be analyzed for CFSE intracellular staining and for apoptosis. In both lines hrIL-27 was unable to directly modulate proliferation or apoptosis (not shown).
We next investigated whether IL-27 regulated, in both lines, sets of genes shaping tumor malignancy and specifically related to angiogenesis, stemness and invasiveness.

IL-27 hinders tumour growth in pre-clinical xenograft models of lung cancer in association with a remarkable colliquative necrosis and apoptotic events
In vivo studies using pre-clinical models of severe combined immunodeficient SCID/NOD and T-cell deficient athymic-nude mice, s.c. injected with Calu-6 and SK-MES cell lines respectively, showed that hrIL-27 considerably reduced tumor growth in both models. In particular, the mean tumor volume (mtv) ± standard error (SE) of Calu-6 tumors grown www.impactjournals.com/oncotarget Results are representative of three independent experiments. *P < 0.05. www.impactjournals.com/oncotarget in hrIL-27-treated mice was 54.22 ± 24.2 mm 3 versus 241 ± 69 mm 3 of tumors from controls (P = 0.0336) (Figure 2A), whereas the mtv ± SE of SK-MES tumors was 59.08 ± 13.1 mm 3 versus 123.3 ± 24.5 mm 3 of tumors from controls (P = 0.0469) ( Figure 2B).
To get an insight into the mechanisms underlying the in vivo antitumor effects of IL-27, tumor growth/ suppression areas were histopathologically analyzed. The histologic features of Calu-6 and SK-MES tumors from control mice, recalled human poorly-differentiated AC and SSC, respectively. Tumors harvested from hrIL-27treated mice displayed wide areas of colliquative necrosis characterized by a prominent reactive cell infiltrate, ( Figure 2C and 2D), in addition, SK-MES tumors also presented evident alterations in cancer cell morphology ranging from a spindle to a polygonal-round phenotype ( Figure 2D). Inflammatory infiltrates were wider in both Calu-6 and SK-MES tumors from hrIL-27-treated mice than in control tumors, because of the significant (P < 0.05) increase in their granulocyte and macrophage content, along with cancer cell expression of CXCL3 ( Figure 2C and 2D) ( Table 3). In addition to the areas of colliquative necrosis, frequent apoptotic features were evidenced, by the TUNEL assay, close to granulocytes identified by their segmented nuclei, ( Figure 2C and 2D), in both Calu-6 and SK-MES tumors from hrIL-27-treated mice ( Figure 2C and 2D) ( Table 3).
To understand the molecular mechanism underlying apoptotic events in vivo, we next assessed in tumors from hrIL-27-treated and control mice the expression of apoptosis-inducing proteins Tumor Necrosis Factor (TNF)α and TNF-Related Apoptosis Inducing Ligand (TRAIL). Immunostainings revealed that TRAIL was almost undetectable, while TNFα expression was distinct to strong, in the foci of reactive infiltrates, in both tumor types harvested from hrIL-27-treated mice ( Figure 2C and 2D) ( Table 3).
Lastly immunohistochemical analyses revealed that the microvascular network of Calu-6 tumors from hrIL-27-treated mice was similar to that of the controls whereas that of SK-MES tumors from hrIL-27-treated mice was clearly impaired ( Figure 2C and 2D) ( Table 3) in association with a weakened laminin network and a faint, but distinct cancer cell expression of IFNγ ( Figure 3A).

IL-27 down-modulates stemness-and EMTrelated genes, particularly in SCC tumors
To assess whether IL-27 regulation of pluripotencyand EMT-related genes also occurs in vivo at protein level, we carried out immunohistochemical analyses of tumors from hrIL-27-and PBS-treated mice.
In Calu-6 tumors from hrIL-27-treated animals, the percentage of cancer cells displaying a distinct to strong SHH staining was decreased compared with control tumors ( Figure 3B), whereas in SK-MES tumors from treated animals, the percentage of cells endowed with a distinct to strong nuclear staining for OCT4A, NOTCH1, and KLF4 was clearly decreased (Table 3) as was reduced the percentage of tumor cells displaying both nuclear and cytoplasmic SOX2 positivity and nuclear SOX9 positivity ( Figure 3C) ( Table 3). Expression of Nestin was weakened in cancer cells forming SK-MES tumors from IL-27treated mice ( Figure 3D). Nuclear and cytoplasmic SNAI1 stainings were dramatically and moderately reduced respectively, and the percentage of cancer cells endowed with a distinct to strong nuclear SNAI1, SNAI2, and ZEB1 staining also significantly (P < 0.05) decreased following hrIL-27 treatment ( Figure 3D), whereas E-Cadherin expression was reinforced in tumors from hrIL27-treated mice ( Figure 3D) ( Table 3).

Myeloablation by treosulfan thwarts the in vivo anti-lung cancer effects of IL-27
To assess whether granulocytes and macrophages may, as suggested by the morphological data, account for the anti-lung cancer effects of IL-27 in vivo, we next repeated tumor growth experiments in mice pre-treated with myeloablative doses of treosulfan to obtain a severe or complete depletion of bone marrow cells [18].
Histological features of both Calu-6 and SK-MES tumors developed in myeloablated hrIL-27-treated mice were similar to those from myeloablated controls. However, both histotypes, developed in myeloablated mice, independently from hrIL-27-treatment, presented small ischaemic necrotic foci ( Figure 4C) in association with an evident (vessel counts: 3 ± 2 in Calu-6, and 5 ± 2 in SK-MES tumors from myeloablated controls, versus 9 ± 3 in Calu-6, and 12 ± 4 in SK-MES tumors from nonmyeloablated controls; P < 0.05) decrease of the whole vascular supply ( Figure 4C). These tumours were almost devoid of granulocyte and macrophage content ( Figure  4C). CXCL3 was still firmly expressed by tumor cells in both Calu-6 and SK-MES tumors from myeloablated and hrIL-27-treated mice ( Figure 4C), while a faint IFNγ production was only detected in the latter. Results are representative of three independent experiments. *P < 0.05. Histological and immunohistochemical analyses (C, D) revealed that Calu-6 and SK-MES tumors grown in PBS treated mice had the features of poorly-differentiated human lung AC (rare glandular lumen) and SSC (epithelial cells with spindle morphology, inset), and displayed frequent mitotic figures, whereas both tumors from hrIL-27-treated mice showed wide areas of colliquative necrosis (N) and, particularly for SK-MES tumor cells, the acquisition of a more polygonal-round morphology (inset). Both histotypes from hrIL-27-treated mice showed a prominent granulocyte (PMN) and macrophage (CD11b/CD18) infiltrate, along with a strong CXCL3 production by tumor cells in comparison with control tumors. Following hrIL-27 treatment apoptotic events, close to cells endowed with segmented nuclei (inset), were frequent, as assessed by the TUNEL assay, in both histotypes, whereas they were almost absent in control tumors, and a distinct to strong expression of TNFα was detected at the sites of reactive infiltrates, whereas it was lacking in control tumors. The microvascular network supplying Calu-6 tumors from hrIL-27-treated mice was similar to that of control tumors. By contrast, in SK-MES tumors from hrIL-27-treated mice it was evidently impoverished, in comparison with control tumors. (C and D: H&E, PMN, CD11b/CD18, TNFα, and CD31 at X400; C and D: CXCL3, Apoptosis, and H&E insets at X630; D: Apoptosis inset: X1000).

Human lung AC and SCC, and their precursor lesions express IL-27R in neoplastic and dysplastic cells, microvessels and tumor-associated reactive cells
To determine whether lung cancer patients could benefit from IL-27's antitumor effects, we next immunohistochemically evaluated the expression and distribution of IL-27R in AC and SCC tissue sections. Since expression of gp130 has been documented in lung cancer [24,25], we only assessed IL-27Rα expression in both cancerous and normal lung samples (from both cancer and control patients). In the normal tissue, it was basically found in mononuclear/macrophage-like cells fluctuating within alveolar walls ( Figure 5D). Normal bronchial epithelia firmly expressed IL-27Rα mRNA, whereas in neoplastic samples it was expressed by the majority of AC, 90%, and SCC, 84% (Table 2) ( Figure 5A and 5D). Furthermore, within AC, metastatic tumors revealed significantly (P < 0.05) higher expression levels of IL-27Rα mRNA than normal bronchial epithelia ( Figure 5B). Notably, IL-27Rα expression in microdissected bronchial epithelium from normal samples of patients with lung cancer was analogous to that in control patients. These molecular data showed a good correlation (ρ = 0.82) with the immunohistochemical findings.
IL-27Rα expression by the primary tumor was significantly associated with lymph node status (P = 0,001), and advanced stages of disease (P = 0,02) as assessed by Fisher's exact test, whereas no significant association was observed with patient age or smoking history.
Immunohistochemistry also revealed IL-27Rα expression in AC and SCC precursor lesions, namely atypical adenomatous hyperplasia (AAH) and severe dysplasia, squamous metaplasia (SM), squamous cell carcinoma in situ (SCIS) ( Figure 5D), respectively (Table 1). IL-27Rα expression may also be found in microvessels and infiltrating immune cells ( Figure 5C), mostly identifiable as CD68 + monocytes/macrophages ( Figure 5D) and CD11c + myeloid dendritic cells. They were found in the stroma of both AC and SCC, scattered or within the lymph node-like structures, known as tertiary lymphoid structures (TLS) ( Figure 5D) [26,27]. CD15 + granulocytes that may be found in necrotic foci of rapidly growing tumors also expressed IL-27Rα ( Figure 5D).

DISCUSSION
AC and SCC constitute the commonly diagnosed lung cancer histotypes [1,2], but their management still results in low overall cure rates, suggesting the need for novel therapeutic approaches. The idea of strengthening patient's immune system to fight cancer is of growing interest for oncologists [28]. Our data indicate that IL-27, a well-tolerated and toxicity-free cytokine [9] may provide a new therapeutic option in NSCLC. Previous studies assessing IL-27's effects in lung cancer have used mouse autograft models [11,13] or, in vitro experiments with both murine and human lung AC cell lines [11,12]. Our data confirm, in pre-clinical xenograft models of human lung AC and SCC, the anti-lung cancer effects of IL-27. They have also revealed unforeseen implications for its immunological antitumor capability in the form of I. boosting the potent granulocyte/macrophage chemoattractant CXCL3, in both AC and SCC cells, leading to intratumoral myeloid cell recruitment and activation, and II. re-education of these immune cells from the status of "cancer feeder", and host-detrimental [29,30] towards that of "cancer killer", thus host-beneficial [30][31][32].
Tumor destruction by the prominent neutrophil and macrophage influx mediated by CXCL3 [15][16][17] overcomes, at least in our setting, its well-known proangiogenic effects [14], since microvessel density remained unaltered in AC and slightly decreased in SCC tumors from IL-27-treated animals, when compared with controls. Anti-angiogenesis has a marginal or nil role in IL-27's anti-lung cancer efficacy, and apart from a slight IFNγ induction and LAMININ-α5 down-regulation observed in SCC cells, IL-27 even down-regulates the angiogenesis inhibitor THROMBOSPONDIN-1 [33] in SK-MES cells, and up-regulates VE-CADHERIN [34] and the pro-angiogenesis gene TYMP-1, encoding for ECGF1 [35], in Calu-6 cells, but without significant in vivo consequences for tumor vascularity.
Granulocytes and macrophages are endowed with IL-27R, and may respond to IL-27 by increasing their oxidative burst and cytokine production [36][37][38], suggesting that the range of action for this cytokine is not restricted to T cells, the key mediators of its antitumor effect in an immune-intact host [9]. Our results provide the first evidence that intratumor recruited and activated myeloid cells may, in a B/T cell-deficient host, take the place of T lymphocytes in mediating IL-27's antitumor activity, leading to a dramatic colliquative necrosis, and TNFα-associated apoptotic events in both AC and SCC.
While extending to human SCC in vivo the finding of IL-27's down-modulation of EMT transcription factors such as SNAI1, SNAI2 and ZEB1 [20,21], our discoveries identify a novel role for IL-27 as a negative regulator of pluripotency genes such as SHH in human AC cells, and SOX2, OCT4A, NOTCH1, KLF4, SOX9 and Nestin [18,19] in human SCC cells, both in vitro and in vivo. EMT, a key event during the early phases of invasion and metastatization, selects for stem cell property [39], which, in turn, may condition the self-renewal capability of a cancer and correlate with its aggressiveness [40,41]. Down-regulation of critical pluripotency genes by IL-27 increases the propensity of cells to differentiate towards a less aggressive phenotype [42][43][44] as shown by their transition from a fibroblast-like to a polygonal-round E-Cadherin-positive phenotype. www.impactjournals.com/oncotarget Tumor responsiveness to IL-27, however, is the prerequisite for its entry into clinical trials. Most AC and SCC express IL-27R, and those with N1 involvement are often endowed with higher expression levels. Dibra et al. observed that WSX-1 expression in tumors induces immune tolerance exerting pro-tumorigenic functions, independently from IL-27 [45]. Our data, drawn from patient samples, suggest that IL-27 may be used to overcome this drawback and be of particular benefit in advanced lung cancer stages. The myeloid cell mediated anti-lung cancer effects of IL-27 may be exerted in humans, since in our samples IL-27R is not only expressed by cancer cells, and microvessels, but also by CD15 + granulocytes, CD68 + monocytes/ macrophages and CD11c + myeloid dendritic cells scattered and SCC (X630) and frequently in dysplastic cells forming AAH (arrows) (X630), severe dysplasia (X630) and in situ SCC (X400). It may also be found in stromal small vessels (X400) and infiltrating reactive cells (X630), particularly in CD68 + macrophages (red) (X630, inset at X1000) and CD11c + myeloid dendritic cells (red) (X630, inset at X1000) scattered in the stroma (in the bottom left panel) or intermingled with lymphocytes in TLS (in the bottom middle panels). CD15 + granulocytes (red) (in the bottom right panel), which may be found in the small necrotic foci of rapidly developing SCC, also express IL-27Rα (brown dots) (X630, inset at X1000).
in the stroma or arranged in TLS. TLS have been associated with a favorable clinical outcome in NSCLC [26,27] and IL-27 may promote, in these lymphoid structures, an efficient adaptive antitumor immunity.
Altogether, our results highlight novel aspects of IL-27's antitumor potential, specifically in NSCLC, such as the ability I. to drive myeloid cells towards antitumor activities, and II. down-regulate stemness genes, particularly in SCC cells, thus suppressing their selfrenewal potential. IL-27 may thus be proposed for clinical trials with the prospect of its clinical use in immunedefective or advanced NSCLC patients.

Ethics statement
Animal experiments were performed at the IRCCS "San Martino" National Institute for Cancer Research, Genoa, in keeping with the National and International regulations (Italian Legislative Order 27/01/1992, n.116, European Economic Community Council Directive 86/609, OJL 358, Dec. 1,1987). For studies on human tissues, written informed consent was obtained from patients. The study was approved by the Ethical Committee of the "G. d'Annunzio" University of Chieti (Italy), and Local Health Authority No.2, in the report n.14 of 19/07/2012, and performed in accordance with the principles outlined in the Declaration of Helsinki.

Mouse studies
Four-to six-week-old athymic-nude and SCID/ NOD mice (Harlan Laboratories, Udine, Italy) were housed under specific pathogen-free conditions. Details on Tumor growth experiments in mice pre-treated or not with the myeloablative agent treosulfan (Medac) [50], and schedule of hrIL-27 administration are provided in the Supplementary information.

Histopathological and morphometric analyses
Methods for histology, immunohistochemistry, TUNEL assay, and the list of Abs used are reported in the Supplementary information.

Statistical analysis
Tumor volumes were reported in mm 3 versus time. Differences in tumor volume, microvessel density and counts of immune cells or percentage of apoptotic cells or stemness and EMT-gene expressing cells between tumors from hrIL-27-and PBS-treated mice were assessed by Student's t test, and data were reported as mean ± standard deviation (SD). Between-group differences in the relative expression of IL-27Rα mRNA, by real-time RT-PCR, were Assessment of cytokine, Nestin and E-cadherin expression, and counts of microvessels, immune cells, TUNEL positive cells, stemness and EMT-related gene expressing cells were performed at X400 in a 0.180 mm 2 field. At least 3 samples (three sections/sample), and 6-8 (depending on the tumor width) randomly chosen fields/section were evaluated. Results are expressed as mean ± SD of CD31 positive microvessels per field; or RB6-8C5 (granulocytes) or CD11b/CD18 (macrophages) positive cells per field; or as mean ± SD percentage of TUNEL positive cells, or stemness-or EMT-related gene expressing cells evaluated on paraffin embedded sections by immunohistochemistry. *The expression of cytokines, Nestin and E-Cadherin was defined as absent (─); scarce (±); distinct (+) or strong (++) on paraffin embedded (CXCL3, IFNγ, Nestin and E-Cadherin) or frozen (TNFα) sections stained with the corresponding Ab. †Values significantly different (P < 0.05) from corresponding values in tumors developed in PBS-treated mice. ‡ND, not detected.
assessed by one-way analysis of variance (ANOVA) and the difference between each pair of means was evaluated with the Tukey's HSD test.
Fisher's exact test was used to examine the association between IL-27Rα protein expression, evaluated by immunohistochemistry, in primary lung tumors, and clinic-pathological characteristics. The Spearman rank correlation coefficient (ρ) was used to examine the correlation between immunohistochemical staining and real-time RT-PCR for IL-27Rα expression. The SPSS software, version 11.0 (IBM, Armonk, NY, USA) was employed, with P < 0.05 as the significance cut-off.