THE ROLE OF TUMOR-INFILTRATING IMMUNE CELLS AND SERUM CYTOKINES IN NON-SMALL CELL LUNG CANCER

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Jurgita

Jackutė

THE ROLE OF TUMOR-INFILTRATING

IMMUNE CELLS AND SERUM

CYTOKINES IN NON-SMALL

CELL LUNG CANCER

Doctoral Dissertation Biomedical Sciences,

Medicine (06B)

Kaunas, 2017

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Dissertation has been prepared at the Department of Pulmonology, Medical Academy of Lithuanian University of Health Sciences during the period of 2012–2017.

Scientific Supervisor

Assoc. Prof. Dr. Marius Žemaitis (Lithuanian University of Health Scien-ces, Biomedical ScienScien-ces, Medicine – 06B)

Dissertation is defended at the Medicine Research Council of the Me-dical Academy of Lithuanian University of Health Sciences:

Chairperson

Prof. Habil. Dr. Albinas Naudžiūnas (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Members:

Prof. Dr. Elona Juozaitytė (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Prof. Dr. Saulius Vaitkus (Lithuanian University of Health Sciences, Bio-medical Sciences, Medicine – 06B)

Prof. Dr. Saulius Šatkauskas (Vytautas Magnus University, Biomedical Sciences, Biophysics – 02B)

Dr. Osvaldas Pranevičius (Cornell University Medical College, Biome-dical Sciences, Medicine – 06B)

Dissertation will be defended at the open session of the Medicine Research Council on June 28, 2017, at 1:00 pm in the Large Auditorium at the Hospital of Lithuanian University of Health Sciences Kauno Klinikos.

Address: Eivenių 2, LT-50161 Kaunas, Lithuania. 2

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LIETUVOS SVEKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Jurgita Jackutė

NAVIKINĮ AUDINĮ INFILTRUOJANČIŲ

IMUNINIŲ LĄSTELIŲ IR SERUMO

CITOKINŲ REIKŠMĖ SERGANT

NESMULKIŲJŲ LĄSTELIŲ

PLAUČIŲ VĖŽIU

Daktaro disertacija Biomedicinos mokslai, Medicina (06B) Kaunas, 2017 3

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Disertacija rengta 2012–2017 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijos Pulmonologijos klinikoje.

Mokslinis vadovas

Doc. dr. Marius Žemaitis (Lietuvos sveikatos mokslų universitetas, Bio-medicinos mokslai, Medicina – 06B).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijos biomedicinos mokslų srities medicinos krypties taryboje: Pirmininkas

prof. habil. dr. Albinas Naudžiūnas (Lietuvos sveikatos mokslo universi-tetas, biomedicinos mokslai, medicina – 06B)

Nariai:

prof. dr. Elona Juozaitytė (Lietuvos sveikatos mokslo universitetas, biomedicinos mokslai, medicina – 06B)

prof. dr. Saulius Vaitkus (Lietuvos sveikatos mokslo universitetas, bio-medicinos mokslai, medicina – 06B)

prof. dr. Saulius Šatkauskas (Vytauto Didžiojo universitetas, biome-dicinos mokslai, biofizika – 01B)

dr. Osvaldas Pranevičius (Kornelio universiteto Medicinos koledžas, biomedicinos mokslai, medicina – 06B)

Disertacija ginama viešame Lietuvos sveikatos mokslų universiteto Me-dicinos akademijos MeMe-dicinos krypties tarybos posėdyje 2017 m. birželio 28 d. 13 val. Lietuvos sveikatos mokslų universiteto ligoninės Kauno kli-nikų Didžiojoje auditorijoje.

Disertacijos gynimo vietos adresas: Eivenių g. 2, LT-50161 Kaunas, Lietuva.

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ABBREVIATIONS

Ab – antibody

APCs – antigen-presenting cells CK5 – cytokeratin 5

CK7 – cytokeratin 7

CCL2 – monocyte chemotactic protein-2 COPD – chronic obstructive pulmonary disease CD4+ T cells – helper T cells

CD8+ T cells – cytotoxic T cells DC – dendritic cells

DNA – deoxyribonucleic acid ECC – European Cancer Congress ECM – extracellular matrix

ELCC – European lung cancer conference ELISA – enzyme-linked immunosorbent assay ERS – European Respiratory Society

ESMO – European Society for Medical Oncology FEV1 – forced expiratory volume in 1 sec. FVC – forced vital capacity

Foxp3 – forkhead box P3

Foxp3+CD4+ T cells – regulatory T cells expressing the transcription factor forkhead box P3

GM-CFUs – granulocyte/macrophage colony-forming units IARC – International Agency for Research on Cancer IHC – immunohistochemistry

IFN-γ – interferon gamma IL-10 – interleukin 10 IL-17A – interleukin 17A

iNOS – inducible form of nitric oxide synthase IPF – idiopathic pulmonary fibrosis

LPS – lipopolysaccharide MMP – matrix metalloproteinase

MHC – major histocompatibility complex min. – minute

NK – natural killer

NSCLC – non-small cell lung cancer PGE2 – prostaglandin E2

SCLC – small cell lung cancer TCR – T cell receptor

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TGF-β – transforming growth factor TNF-α – tumor necrosis factor alpha TTF-1 – thyroid transcription factor-1

Th17 cells – T helper cells producing interleukin 17A VEGF – vascular endothelial growth factor WHO – World Health Organization

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INTRODUCTION

Lung cancer is most frequent carcinoma and leading cause of cancer-related mortality. There are more than 1.6 million new lung cancer cases yearly [1]. Despite the newest anticancer treatment the prognosis of NSCLC remains very poor with less than 15% of the total survival [2]. Lung cancer has highly complex mutational landscape and treatment strategies against lung cancer remains largely ineffective. The immune system plays an essential role in prevention of tumors, including the specific identification and elimination on the basis of their expression of tumor-specific antigens or molecules induced by cellular stress, thereby employing both innate and adaptive immune mechanisms. Immune evasion is recognized as a key strategy for cancer survival and progression [3]. The lung immune system consists not only of large numbers of immune cells with a complex cytokine network, but also structural elements such as endothelial, epithelial, and mesenchymal cells with different functions. The tumor microenvironment comprises a wide variety of cells including malignant and non-malignant populations [4]. Crosstalk between tumor cells and other tumor-associated cells may lead to either inhibition of tumor formation or enhancement of tumor growth and progression, and this double-edged sword characteristic of many tumor-infiltrating immune cells, such as macrophages, T cells, and dendritic cells, has been recognized [5-7]. The prognostic markers are required to distinguish the patients with poor and good prognosis in order to choose the better treatment and follow-up strategy.

Study aim

The aim of this study is to evaluate the role of tumor-infiltrating immune cells (CD4+, CD8+, Foxp3+CD4+, IL-17A+CD4+ T cells, M1 and M2 macro-phages) and serum cytokine (interferon-γ, tumor necrosis factor-α, inter-leukin-10 and interleukin-17A) levels in non-small cell lung cancer patients.

Study objectives

1. To investigate non-small cell lung cancer patients’ tumor-infiltrating immune cell (CD4+, CD8+, Foxp3+CD4+, IL-17A+CD4+ T cells, M1 and M2 macrophages) count and compare to control group as well as to evaluate the distribution of immune cells in tumor islets and stroma. 2. To investigate the associations between tumor-infiltrating immune cells

and non-small cell lung cancer patients’ clinicopathological data.

3. To evaluate non-small cell lung cancer patients’ serum cytokine (inter-feron-γ, tumor necrosis factor-α, interleukin-10 and interleukin-17A)

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levels and compare to control group and investigate the associations with non-small cell lung cancer patients’ clinicopathological data.

4. To determine the associations of tumor-infiltrating immune cells, serum cytokine levels and non-small cell lung cancer patients’ prognostic data.

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1. REVIEW OF LITERATURE

1.1. Lung cancer epidemiology

Cancer is a disease that presents with numerous faces and manifests itself in different ways in various organs and tissue types. This thesis focuses on non-small cell lung cancer, one of the most common as well as most deadly cancer types.

The epidemiology of lung cancer has evolved impressively. Lung cancer was a rare disease at the start of 20th century however, since 1980 it has been the most common cancer worldwide. In 2012 approximately 1.8 million people received a new diagnosis of lung cancer. It accounts for about 13% of all cancer diagnoses both Europe and the USA. Lung cancer was the most frequently diagnosed cancer and the leading cause of cancer death among males in 2012 [8]. Among females, lung cancer was the leading cause of cancer death in more developed countries, and the second leading cause of cancer death in less developed countries. In more developed countries, however, prostate cancer was the most frequently diagnosed cancer among men [8]. Recent data have shown that Europe and Northern America have the highest incidence of lung cancer. In men, the highest lung cancer incidence rates were in Europe, Eastern Asia, and Northern America, and the lowest rates were in Africa. Among women, the highest lung cancer rates were in Northern America, Northern and Western Europe, Australia and Eastern Asia [8].

Lung cancer is estimated to be responsible for nearly 1.59 million deaths worldwide, which accounts for 19.4% of all cancers in total [8]. The European Union average mortality is 35.2 deaths per 100 000 person-years as reported by European Network of cancer registries. Hungary has the highest mortality rates for males and Denmark has the highest mortality rates for females. In Lithuania lung cancer mortality rates per 100.000 population were 49.8 for men and 5.7 for women, lung cancer morbidity rates per 100.000 population 55.6 for men and 6.5 for women.

Worldwide data for lung cancer confirm that it still remains lethal in the majority geographic areas, although there are some countries reporting more encouraging survival rates. Between 2005 and 2009, Japan and Israel reported 5-year survival rates of 30% and 24%, respectively. Lithuania, Bulgaria, Mongolia and Thailand presented survival rates less than 10% and Libya 2%. Moreover, 2-4% decrease in lung cancer survival was noted in Lithuania, Cyprus, Croatia and Malaysia [9].

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1.2. Factors affecting lung cancer development Smoking

The World Health Organization (WHO) estimates that lung cancer deaths worldwide will continue to rise, largely as a result of an increase in global tobacco use, especially in Asia [2]. European incidence rates of lung cancer seem to be linked to European patterns of incidence of smoking. In several countries, such as Denmark and the United Kingdom, where the tobacco epidemic began earliest and peaked around the middle of the last century, lung cancer mortality rates have been decreasing in men and plateauing in women [10, 11]. Lung cancer is one of the most preventable cancers. Most lung cancers could be avoided by eliminating smoking initiation and increasing smoking cessation among current smokers. Sweden had one of the lowest incidences of lung cancer in men in Europe and only 14% of the adult population were daily smokers. This is the lowest smoking rate in Europe and can be used to justify the comparatively lower incidence of disease in the country [10].

There is no doubt that tobacco smoking remains the most important modifiable risk factor for lung cancer. Pisani and colleagues estimated that up to 20% of all cancer deaths worldwide could be prevented by the elimination of tobacco smoking [12]. It is clear that individual susceptibility is also a factor in carcinogenesis. Although more than 80% of lung cancers occur in persons with tobacco exposure, fewer than 20% of smokers will ever develop lung cancer [2].

Cigarette smoke is a complex aerosol composed of gaseous and particulate compounds. The nicotine is the principal addictive component of tobacco, and tar is the total particulate matter of cigarette smoke minus nicotine and water content. Tar exposure seems to be the major link to lung cancer risk [2]. Over 5000 compounds have been identified in cigarette smoke, including 73 compounds which are considered carcinogenic to either laboratory animals or humans by the International Agency for Research on Cancer (IARC). Cigarette smoke contains many potential carcinogens such as aromatic amines, polycyclic aromatic hydrocarbons (PAHs), N-nitrosa-mines, and other organic and inorganic compounds. Radioactive materials, such as radon, bismuth, and polonium, are also present in tobacco smoke [13]. The cumulative lung cancer risk among heavy smokers may be as high as 30% compared with a lifetime risk of 1% or less in non-smokers. The lung cancer risk is proportional to the quantity of cigarette consumption, because factors, such as the number of packs per day smoked, the age of onset of smoking, the degree of inhalation, the tar and nicotine content of cigarettes, and use of unfiltered cigarettes, become important [14]. Although

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the use of filter tips decreases the amount of nicotine and tar in mainstream smoke, the effect of filter tips also varies because the compression of the tips by lips or fingers and the depth of inhalation of the smoker. Moreover, the effect of pipe and cigar use on the risk of lung cancer is similar to that of light cigarette smoking [15].

A number of studies have demonstrated that risk for lung cancer decrea-ses with smoking cessation, most recently described in the Lung Health Study, where the efficacy of smoking cessation interventions in decreasing lung cancer deaths was demonstrated in a prospective, controlled trial [16]. Another study by Peto et al. showed that for men who stopped smoking at 60, 50, 40 and 30 years of age the cumulative risk of lung cancer by age 75 were 10%, 6%, 3% and 2%, respectively [17]. The residual effects of smoking on lung cancer risk remain most notable in former smokers and a significant proportion of lung cancer is now diagnosed in former smokers.

The causal association that has been well established between second-hand tobacco smoking and lung cancer, which may be responsible for 1.6% of lung cancers [18]. Results from a comprehensive review by Whitrow et al. showed a relative risk between 1.14 to 5.20 in people who had never smoked but who lived with a smoker. Although there has been no predo-minant causal factor that can fully explain lung cancer in never smokers, the risk factors considered important for never smokers include secondhand smoke; radon exposure; environmental exposures, such as indoor air pollution, asbestos, and arsenic; history of lung disease; and genetic factors [19]. A study in non-smoking women showed a dose-response relationship of the risk for lung cancer with both the number of cigarettes smoked by the spouse and the duration of exposure [20]. A more recent study by Vineis et al. reported that passive smoking during childhood increased lung cancer risk in adulthood by 3.6 fold [21].

Genetic factors

Despite the fact that tobacco smoke is implicated in most cases of lung cancer, only a proportion of smokers develop lung cancer. Clearly, there are other important factors operating apart from the simple inhalation of tobacco smoke. It has been suggested, that individuals may exhibit genetic polymorphism in carcinogen metabolizing pathways, leading to inherited differences in the risk of lung cancer associated with tobacco smoking [22]. Truong et al. observed, that most frequent genetic association for lung cancer in smokers was with nicotine receptor single nucleotide polymor-phisms in chromosomal region15q25 [23].

Spitz et al. have developed a lung cancer risk prediction model, that incorporates multiple variables, such as smoking history, exposure to

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environmental tobacco smoke, environmental exposures (dusts and asbes-tos), and family history of cancer. Their analysis showed the influence of a family history of cancer on the risk for lung cancer in never smokers, former smokers, and current smokers [24]. Cassidy and colleagues also highlighted the significantly increased risk for lung cancer specifically for persons with a family history of early-onset lung cancer (<60 years of age) [25]. Studies on familial aggregation have supported the hypothesis that there is a hereditary component to the risk for lung cancer. A meta-analysis involving 32 studies showed a 2 fold increased risk for lung cancer in persons with a family history of lung cancer with an increased risk also present in non-smokers [26].

An increased genetic susceptibility to lung cancer is associated with the status of the TP53 germline carrier, germline epidermal growth factor receptor (EGFR) T790M sequence mutation, 6q23–25p. The carriers of TP53 germline who smoked cigarettes are more than 3 times more likely to develop lung cancer than carriers who did not smoke [27]. Bailey-Wilson and colleagues, using family linkage approaches, reported the first association of familial lung cancer to the region on chromosome 6q23–25 [15]. The germline EGFR T790M mutation was found in a family with mul-tiple cases of NSCLC [28].

Lung diseases

The increased risk of lung cancer is associated with some non-malignant diseases, the strongest association being with chronic obstructive pulmonary disease (COPD). Tobacco smoking is the primary cause of both lung cancer and COPDand there is increasing evidence linking the two diseases beyond a common etiology. COPD has been reported to be a risk factor for lung cancer despite the smoking status [29]. A study of non-smoker females with lung cancer showed a statistically significant association between the presence of airflow obstruction and the development of lung cancer [30]. Lung cancer is up to five times more likely to occur in smokers with airflow obstruction than those with normal lung functions, suggesting that COPD itself is an important independent risk factor with potential relationship to the pathogenesis of lung cancer [31]. The annual incidence of lung cancer arising from COPD has been reported to be 0.8%–1.7% [32, 33].

The mechanisms by which COPD increases the risk of development of lung cancer as well as the influence of COPD on the prognosis of patients with lung cancer are not clear. There are certain pathogenic pathways in COPD and emphysema (increased apoptosis, matrix degradation and repair, activation of immune response) that are essentially opposed to those asso-ciated with lung cancer (decreased apoptosis, genome instability, invasion

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angiogenesis). However, it has been hypothesized that several mechanisms (chronic inflammation, retention of airborne carcinogens, genetic factors) link both disease [34]. Chronic airway obstruction causes air retention and suppression of the clearance of secretions, therefore was hypothesized that it causes chronic exposure to carcinogens and repetitive stimulation of bronchoalveolar stem cells, a potential initial step to carcinogenesis [35]. Cellular damage can active the induction of cell death, which recruits more inflammatory cells; and the loss of tissue integrity which triggers a regene-rative response with the recruitment of tissue stem cells. In this tissue repair phase small percentage of cells escape the pro-apoptotic safety switches and accumulate genetic, epigenetic changes to progress towards the path of dysplasia, in situ carcinoma and invasive cancer [36].

Genome-wide studies have demonstrated common chromosomal loci and candidate genes that might be implicated in the shared genetic susceptibility for COPD and lung cancer [37]. Moreover, deoxyribonucleic acid (DNA) hypermethylation has been described in COPD and has been associated with quicker lung function decline. Also it has been described in association with progression from cell hyperplasia to squamous metaplasia and carcinoma in situ [38].

There seems to be increased risk of lung cancer present in several interstitial lung diseases, with more evident link in idiopathic pulmonary fibrosis (IPF), systemic sclerosis and some forms of pneumoconiosis, such as asbestosis. A study Hubbard et al. showed that the incidence of lung cancer in patients with IPF was greatly increased compared to control group patients, even after adjustment for smoking [39]. The study by Le Jeune et al. showed that the incidence of lung cancer markedly increased in patients with IPF compared with the general population [40]. The population based cohort study by Hill et al. reported that scleroderma was associated with cancer, and in particular, lung cancer [41]. Even though the mechanisms by which pulmonary interstitial disease may predispose lung cancer remains unclear, various hypotheses have been suggested, including malignant transformation, impaired clearance of carcinogens, epithelial hyperplasia, and infections.

Air pollution

Air pollution (both indoor and outdoor) is equally considered to be risk factor for lung cancer. The risk depends on the level of air pollution people are regularly exposed to, but due to variability of air pollution levels it is hard to specify how the risk is affected for the people living in certain area [42]. Interestingly, the proportion of lung cancer cases in women never-smokers was particularly high in East and South Asia. Also, lung cancer

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rates in Chinese women were higher than rates among women in some European countries despite a lower prevalence of smoking. This is thought to reflect indoor air pollution from unventilated coal-fueled stoves and cooking fumes [43]. Lissowska et al. study showed a modest increased risk of lung cancer related to solid fuel use for cooking rather than heating [44]. Air pollution has become a worldwide problem given the current staggering rate of globalization and industrialization. Outdoor air pollution has long been thought to increase the risk for lung cancers. A case-control study in Sweden by Nyberg and colleagues showed a relative risk for lung cancer of 1.44 for persons exposed to more than 29.3 µg/m3 of nitric oxide (as a measure of traffic air pollutant) over 21 to 30 years compared with exposures to lower than 12.8 µg/m3 of nitric oxide [45]. Boffetta et al. reported that the proportion of lung cancers attributable to urban air pollution in Europe is estimated to be 11% [18]. It is believed, that cumulative exposure to ambient air pollution, such as emissions rich in various polycyclic aromatic hydrocarbon compounds, may cause lung cancer.

Occupational exposure

Several workplace substances have been suggested to be or have been proved carcinogens in the lung. The IARC has identified arsenic, asbestos, beryllium, cadmium, chloromethyl ethers, chromium, nickel, radon, silica, and vinyl chloride as carcinogens [46]. Asbestos is the most widely known and most common occupational cause of lung cancer. Recent studies have found that asbestos exposure was associated with a relative risk for lung cancer of 3.5 after adjusting for age, smoking, and vitamin intake [47]. This risk for lung cancer associated with asbestos exposure is dose-dependent but varied with the type of asbestos fiber exposure. Chrysotile fibers do not accumulate within lung tissue to the same extent as amphiboles because of faster clearance rates therefore the risk for lung cancer seems higher for workers exposed to amphibole fibers than for those exposed to chrysotile fibers. [47]. It is really difficult to tell asbestos-related cancers apart from those due to other causes such as smoking therefore, there are no precise data reporting overall scale of asbestos-related lung cancer incidence.

Darby et al. published an analysis of 13 case-control studies of residential radon and lung cancer from nine European countries and concluded that radon is responsible for about 2 % of all death from cancer in Europe [48]. Uranium miners and nuclear plant workers also have an increased cancer risk due to exposure to radioactive particulate mass [2]. Many work settings could have exposed workers to carcinogens, leading to an increased risk of lung and other cancers.

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1.3. Lung cancer classification and staging

Lung cancer has been classified by conventional morphology into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [49]. In 2015 the new WHO classification of lung cancer was published [50]. SCLC is one of the most aggressive and rapidly growing lung cancers comprising 20% of all lung cancers [51]. This type of cancer is strongly related to cigarette smoking. More than 90% of SCLC patients are heavy smokers [52, 53]. SCLC is sensitive to chemotherapy, but a majority of patients rapidly develop treatment resistance and survival beyond five years is rare. Metastatic disease is seen at the time of diagnosis in a majority of SCLCs [54]. NSCLC is the most common type of lung cancers and accounts for about 80% of all lung cancers NSCLC can be divided into three main histo-logical types: squamous cell carcinoma, adenocarcinoma and large cell lung carcinoma [55]. Other NSCLC subtypes are very rare. NSCLC histologic groups are defined based on differences in cellular morphology and to make this distinction has become increasingly important as therapy choice today is influenced by histology [56, 57].

The TNM classification system is used to subgroup the patients ac-cording to the extent of the disease. Assessment of the extent of the tumor burden in the individual patient is based on the size of the tumor, the invasion of organ structures and lymph nodes, and the presence of distant metastasis. The TNM system is the most widely used scheme for catego-rization of these parameters and the 7th edition was applied [58]. The 8th edition of the TNM classification is announced in January, 2017 [59]. The T component of classification represents the size of the primary tumor and its growth into neighbouring organs. N describes the existence and degree of regional lymph node involvement. The M component describes the presence or absence of metastasis. The TNM components define the cancer stage.

1.4. Immunosurveillance and immunoediting

Carcinogenesis is a multistep process that reflects dysregulation of oncogenes, tumor suppression and pro-apoptotic signals. Accumulation of genetic and epigenetic changes may cause uncontrolled cell growth, in-creased proliferation and altered differentiation leading to development of tumors.

In 2000, Hanahan and Weinberg suggested that malignant growth is accomplished through the different cancer cell genotypes and six essential alterations in cell physiology. Each of these capabilities is acquired by the tumors to escape the recognition by the host cells [60]. The hallmarks of

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cancer comprise six biological capabilities acquired during the multistep development of human tumors. They include: (a) sustaining proliferative signaling, (b) evading anti-growth signals, (c) evading programmed cell death, (d) evading replicative immortality, (e) sustaining angiogenesis, and (f) activating tissue invasion and metastasis.

a) Sustaining proliferative signaling. Normal cells ells of the body re-quire hormones and other molecules that act as signals for them to grow and divide. Tumor cells acquire the ability to generate their own growth factors, and they can grow without external signals. Also, tumor cells may stimulate normal cells within tumor stroma to supply tumor cells with various growth factors [61].

b) Evading anti-growth signals. To maintain tissue homeostasis multiple anti-proliferative signals are established within a normal tissue. These processes are orchestrated by proteins known as tumor suppressor genes. Many cancer cells are generally insensitive to growth preventing signals [62].

c) Evading programmed cell death. Cells are programmed to die in the event they become damaged. Programmed cell death by apoptosis serves as a natural barrier to cancer development. Tumor cells evolve a variety of strategies to limit or circumvent apoptosis [62].

d) Evading replicative immortality. Normal cells are able to pass through only a limited number of successive cell growth and division cycles. Tumor cells may escape the normal limits of cell replication. This in turn permits unlimited multiplication of descendant cells [60].

e) Sustaining angiogenesis. Normal tissues of the body have blood vessels running through them that deliver oxygen from the lungs. Cells must be close to the blood vessels to get enough oxygen for them to survive. Secretion of enhanced levels of the vascular endothelial growth factor (VEGF) to promote angiogenesis is one of the most common features of tumor cells [60].

f) Activating tissue invasion and metastasis. During tumor progression tumor cells escape the primary tumor mass and invade distant tissues. This invasion requires alterations of proteins involved in cell-cell adhesion, as well as in the secretion of proteases like matrix metalloproteinases (MMPs) which degrade extracellular matrix (ECM) proteins [60].

In 2010 Hanahan and Weinberg proposed four new hallmarks. They include: genetic instability and mutations, reprogramming energy meta-bolism, tumor promoting inflammation and evading immune destruction [62].

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Cancer immunoediting

Cancer immunoediting is considered to be an important host protection process to inhibit carcinogenesis and to maintain cellular homeostasis. The concept of cancer immunoediting predicts that the immune system can recognize neoplastic cells. By eliminating high immunogenic tumor cells, new tumor cell variants with reduced immunogenicity may be selected and therefore favor the generation of tumors that show increased resistance to immune attack or that have acquired mechanisms that suppress immune effector functions [63]. In the interaction of host and tumor cells, three essential phases have been proposed: elimination, equilibrium and escape (Fig. 1.4.1), which are designated the ‘three E’s' [64].

Fig. 1.4.1. Cancer immunoediting

Adapted according Bremes RM et al., Thoracic Oncology, 2016 and Swann JB, Smyth MJ, Clinical Investigation 2007

Elimination: in the elimination phase the innate and adaptive immune systems work together to detect the presence of a developing tumor and destroy it before it becomes clinically apparent [65]. However, the

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nisms by which the immune system is alerted to the presence of a de-veloping tumor are not fully understood.

The process of elimination includes innate and adaptive immune res-ponses to tumor cells. For the innate immune response, several effector cells such as natural killers (NK), and T cells are activated by the inflammatory cytokines, which are released by the growing tumor cells, macrophages and stromal cells surrounding the tumor cells. The secreted cytokines recruit more immune cells, which produce other pro-inflammatory cytokines such as IL-12 and IFN-γ [64]. It is thought that cellular transformation did not provide sufficient pro-inflammatory signals to activate the immune system in response to a developing tumor. In the absence of such signals, there is often no immune response and tolerance may develop [66].

The elimination phase can be complete, when all tumor cells are cleared, or incomplete, when only a portion of tumor cells are eliminated. In the case of partial tumor elimination, the theory of immunoediting is that a tempo-rary state of equilibrium can then develop between the immune system and the developing tumor [3].

Equilibrium: in this phase, the immune system and tumor cells that have survived the elimination phase enter into equilibrium. In this process, lymphocytes and IFN-γ play a critical role in exerting immune selection pressure on tumor cells that is enough to limit but not to fully suppress genetically unstable and mutating tumor cells. Since the equilibrium phase involves the continuous elimination of tumor cells and the production of resistant tumor variants, Dunn and colleagues hypothesized that equilibrium may be the longest of the three processes in cancer immunoediting and may occur over a period of many years perhaps extending throughout the life of the host in human [67].

This process leads to the immune selection of tumor cells with reduced immunogenicity. During this period tumor cells either remain dormant or continue to evolve, accumulating further changes (such as DNA mutations or changes in gene expression) that can modulate the tumor-specific antigens and stress-induced antigens that they express [64]. The immune system during this phase is sufficient to control tumor progression, but eventually, if the immune response still fails to completely eliminate the tumor, the process results in the selection of tumor cell variants that are able to resist, avoid, or suppress the antitumor immune response, leading to the escape phase [68].

Escape: in the escape phase, tumor cells that have acquired the ability to circumvent immune recognition or destruction arise as progressively growing, visible tumors. Progression from equilibrium to the escape phase can occur because the tumor cell population changes in response to the

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immune system’s editing functions or because the host immune system changes in response to increased cancer induced immunosuppression or immune system deterioration [65]. During the escape phase the immune system is no longer able to contain tumor cells growth, and a progressively growing tumor.

Tumors evolve mechanisms to escape immune control by a process called immunoediting. A variety of tumor derived soluble factors contribute to the emergence of complex local and regional immunosuppressive networks [69]. Although deposited at the primary tumor site, these secreted factors can extend immunosuppressive effects into local lymph nodes and the spleen, thereby promoting invasion and metastasis [70]. It seems that the immune system is incapable of influencing tumor progression, but although the immune system may prevent or delay tumor growth.

1.5. Tumor microenvironment

In the local tissue environment, malignant cells are surrounded by cells of the tumor stroma and the various cell types are involved in complicated and continuous interplay. Today it is generally accepted that many aspects of tumor formation and growth are influenced by non-malignant cell types in the tumor microenvironment [71]. The tumor stroma basically consists of the non-malignant cells of the tumor such as fibroblasts, mesenchymal cell distinctive to each tissue environment, immune and inflammatory cells, and vasculature with endothelial cells, pericytes and the ECM; also growth fac-tors, cytokines and other proteins that are locally or systemically produced. Although none of these stromal cells are tumorigenic, they may either stimulate or inhibit cancer cell proliferation and malignancy depending on the tumor microenvironment and the various interactions they may have with the cancer cells [72]. The schema of tumor microenvironment is shown in Fig. 1.5.1.

In physiological conditions, tissues bear the large number of cells which work in harmony to perform the normal functioning of the body. Due to mutations in oncogenes or tumor suppressor genes or genes related to the growth and survival of the cells, some of these normal cells lose the cons-traint and become cancerous cells [73]. Although cancers have altered identity it does not loses interaction with surrounding environment. These interactions may lead to the infiltration of the different immune cells through chemokine and cytokine cocktail. In some cases, the defense signals exerted by the immune system are circumvented by tumors to exploit the surrounding cells, proliferate and finally invade to metastasize [73].

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Fig. 1.5.1. Tumor microenvironment

Adapted according Koontongkaew S, Cancer 2013

Immunohistochemistry, gene expression, and the clinical techniques have helped to study the presence of the various cell substrates infiltrating the different areas of tumors. Lymphocytes are not randomly distributed but are specifically localized in tumor microenvironment [74]. Immune cell infiltra-tion is guided by various events in the tumor microenvironment. Complex crosstalk among the receptors and chemokines expressed by the cells is responsible for the build of tumor microenvironment. If this balance is lost, it will result into the loss of co-ordination and inefficiency of the immune system in controlling the tumor [75].

Fibroblasts present locally in the tumor tissue are thought to be the pre-decessors of their cancer associated counterparts [76]. There is considerable evidence that stromal inflammation contributes to the proliferation and survival of malignant cells, facilitates genomic instability, stimulates angio-genesis and metastasis, and alters the response to anti-cancer therapies [77].

In general, tumor microenvironment consists of immune cells with dif-ferent functional properties. It consists of the antigen-presenting cells (B cells, DCs, and macrophages), T cell subsets, NK cells, neutrophils, and

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mast cells. Even within individual cell types, different subsets may have adverse functions. For example different subsets of CD4+ T cells, NK cells and macrophages may have either tumor suppressing or tumor promoting properties [78]. From patient to patient and from tumor to tumor, hetero-geneity can be observed with respect to the numbers, localization of the tumor-infiltrating immune cells. These immune cells can be located either in the stroma or in the tumor islets. Analysis of this immune contexture allows the determination of the beneficial or deleterious effects on the cancer patients. Distribution of the immune cells in different areas of the tumors suggests that these cells may have different role in tumor control. Corre-lation between the immune infiltration and clinical outcome was investi-gated in the large number of cases in literature. The type, density and loca-lization of immune cells within tumors define the immune contexture, which has proved to be the major determinant of tumor development and patients’ outcome [78].

The capacity of tumor cells to avoid detection and elimination by the host immune system is important for tumor initiation and has been proposed to be included among the characteristics that constitute the cancer hallmarks [62]. On the other hand, it is apparent that concurrent inflammation in the early stages of tumor formation may endorse tumor development [79]. These two aspects highlight the two-sided role of the immune system in cancer, counteracting as well as promoting the tumorigenic process.

Until recently, the principal focus in cancer research has primarily been the malignant cell. As cancers are not solely neoplastic cells but harbor tumor microenvironments, the lack of research interest in the tumor microenvironment has led to a significant discrepancy between the profound knowledge on cancer cell biology and more limited knowledge on which roles the tumor microenvironment plays.

1.6. Cancer and inflammation

Inflammation is a normal and essential process of infection and wound healing, but when it is unresolved and becomes chronic, the resulting tissue damage can be extensive and disastrous. Persistent exposure to many factors, including smoking, obesity, and viral infections, increases risk of chronic inflammation and consequently diseases such as chronic obstructive pulmonary disease, diabetes, and cancer [80]. It is now evident that inflam-mation is involved in all stages of tumorigenesis, from malignant transfor-mation and tumor initiation to invasion and metastasis of established tu-mors. In fact, it is estimated that underlying infections and inflammatory responses are linked to 15 to 20% of all cancer-related deaths worldwide

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[81]. However, the mechanisms underlying cancer-associated inflammation have not been well understood.

Chronic inflammation is associated with malignant transformation and an increased incidence of local cancer in such a way how reflux esophagitis is associated with esophageal carcinoma; Helicobacter pylori gastric mation – with stomach cancer; viral hepatitis – with liver cancer; inflam-matory bowel diseases (chronic ulcerative colitis and Crohn’s disease) – with colon carcinoma [82-84]. Allavena et al. indicated that tumors that arise at sites of chronic inflammation are characterized by the presence of infiltrating leukocytes (dominated by macrophages), cytokines, chemokines, growth factors, and matrix-degrading enzymes [85]. Tumor-infiltrating immune cells can have either tumor-suppressing or tumor-promoting ef-fects, depending on the type of immune cell infiltrate and the nature of the tumor microenvironment. Tumor promoting factors, such as protein and DNA damage through oxidative stress, as well as, angiogenesis and tissue remodeling, are induced by chronic inflammation [86].

Mantovani et al. summarize the yet unrevealed molecular pathways between inflammation and cancer in a review published in 2008. In some types of cancer, inflammatory conditions can be found before a malignant change occurs. In other types of cancer, the oncogenic change induces an inflammatory microenvironment that promotes the development of tumors [87]. Tumor-associated inflammation is a chronic process, which is not beneficial, but often unfavorable for the host. Many immune cells of the tumor microenvironment may be associated with the tissue disruption caused by inflammatory agents or be a response to tumor growth. However, there is no clear association between the presence of any individual immune cell type and a defined outcome in prognosis across various tumors [88]. Even within individual immune cell types, there are opposing functions as CD4+, CD8+, Foxp3+CD4+, IL-17A+CD4+ T cells, macrophages have either tumor-suppressive or tumor-promoting properties, depending on tissue context.

1.6.1. Immune cells and non-small cell lung cancer T cells

T cells develop in the thymus from a common lymphoid progenitor and are defined by expression of a T cell receptor (TCR) that is responsible for recognizing antigens presented by major histocompatibility complex (MHC) family of genes (also called human leukocyte antigen or HLA) [89]. Tumor infiltrating lymphocytes mainly consists of T cells. As other leucocytes, T cells are derived from progenitor cells in the bone marrow from which they

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migrate to the thymus where they mature. During maturation in thymus T cells are differentiated into CD4+ and CD8+ T cells defined by their ex-pression of the co-receptor molecules CD4 and CD8 [7]. Both T cells recog-nize antigens that have been processed and presented on the surface of antigen-presenting cells (APCs). CD4+ T cells recognize antigens presented on the MHC class II molecules, present on APCs, which mainly constitute of monocytes or macrophages and DCs. CD8+ T cells recognize antigens presented by MHC class I, present on all cells in the body. MHC class I presents intracellular antigens or tumor proteins [90]. Tumors that can be associated with significant expression of MHC class II are melanoma, lung cancer, breast, and osteosarcomas. Expression in lung cancer is dependent on the subtype. While squamous cell and small cell lung cancer consistently have low level expression, approximately 60% of adenocarcinomas and large cell carcinomas express some level of the molecules [91].

CD4+ T cells

CD4+ T cells are central to the development of immune responses, for protection against infection and possibly malignancy, by activating antigen-specific effector cells and recruiting cells of the innate immune system such as macrophages, eosinophils and mast cells [91]. Naive CD4+ T cells are activated by recognition through the TCR of MHC class II molecule-peptide complexes on professional APCs. Full CD4+ T cell activation requires sig-nals by costimulatory molecules expressed on APCs and differentiation toward different CD4+ T cell subsets depends on the environmental cytokine milieu in which priming occurs [92]. Tumor cells release tumor antigens that are up-taken by resident immature DCs, which then traffic to draining lymph nodes where they present tumor antigens in the form of processed peptides in the context of MHC class I and class II molecules to CD8+ and CD4+ T cells, respectively. Priming of CD8+ T cells requires full activation of DCs that is provided by CD4+ T cells [93]. Furthermore, CD4+ T cells recruited to the tumor site may exert their functions both directly on MHC class II expressing tumor cells possibly by tumor cell killing and indirectly by releasing cytokines that in turn activate immune cells with tumoricidal functions (i.e. through release of reactive oxygen intermediates and granule contents by macrophages and eosinophils, respectively) [93].

Examination of T cell infiltrates in tumors reveals cells that can display activation markers and are able to recognize tumor antigens, indicating that some tumors are indeed immunogenic and can induce an anti-tumor immu-ne response [89]. A long list of tumor antigens and CD4+ T cell tumor epi-topes is now available. As anticipated by the first tumor antigen identified, most tumor antigens recognized by CD4+ T cells belong to the same

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gories of those recognized by cytotoxic CD8+ T cells [92]. Activation of CD8+ T cells has long been regarded as a major antitumor mechanism of the immune system. Emerging evidence suggests that CD4+ T cells are required for the generation and maintenance of effective CD8+ T cells, a pheno-menon known as CD4+ T cell help [94]. Alternatively, CD4+ T cells may influence DCs to increase their ability to stimulate CD8+ T cells [95]. In the absence of CD4+ T cell help, specific CD8+ T cells can become lethargic or be deleted [96]. CD4+ T cells are essential for CD8+ T cell transformation into long-lived functional effector cells.

CD4+ T cells may mediate tumor rejection through other mechanisms. The anti-tumor effects of CD4+ T cells are dependent on cytokine signaling, especially IFN-γ and TNF-α. These cytokines, produced by CD4+ T cells, have cytotoxic effect on tumor cells [97]. Furthermore, CD4+ T cells inhibit tumor angiogenesis through a combined action of IFN-γ and TNF-α, which induces DCs to produce potent antiangiogenic chemokines, CXCL10 and CXCL9 [98]. In addition, CD4+ T cells can inhibit tumor growth in the absence of CD8+ T cells by direct lyses or recruiting other cells [99]. CD4+ T cells represent an essential component of adaptive immunity since they are absolutely necessary to regulate CD8+ T cells and B cells responses and to induce late recruitment of innate immune cells at inflammatory sites [99]. The presence of CD4+ T cells in NSCLC has provided contradictory re-sults regarding prognosis. Al-Shibli et al. reported, that high density of stro-mal CD4+ T cells was a favorable independent prognostic factor in patients with resected stages I to IIIA NSCLC [96]. Nakamura et al. found, that neit-her CD4+ T cells nor CD8+ T cells had any prognostic relevance in NSCLC [100]. Moreover, Hiraoka et al. study revealed, that simultaneously infiltra-tion of both CD4+ and CD8+ T cells had favorable effect in NSCLC [101].

Foxp3+CD4+ T cells

Tumor progression, which often also is seen in the presence of subs-tantial lymphocytic infiltration, suggests that T cells are not capable provide effective immune responses to control tumor growth [102]. Recently Foxp3+CD4+ T cells became an object of great interest in studies of different cancer types as well as in NSCLC. In 2003, Forkhead Box P3 (FoxP3) was identified as unique marker for T regulatory cells as it was predominantly expressed within CD25+CD4+ T cells [103]. Foxp3+CD4+ T cells are ge-nerally considered to be significant contributors to tumor escape from the host immune system. Emerging evidence suggests, however, that in some human cancers, Foxp3+CD4+ T cells are necessary to control chronic in-flammation, prevent tissue damage, and limit inflammation-associated can-cer development [104].

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It is thought that Foxp3+CD4+ T cells have the ability to suppress or regulate cell-mediate immunity [105]. In the presence of tumor-derived chemokines, Foxp3+CD4+ T cells accumulate in the tumor, and once in place, proceed to prevent or blunt antitumor responses of immune cells infiltrating the tumor. Thus, Foxp3+CD4+ T cells which accumulate in situ and in the peripheral circulation of cancer patients can be viewed as one of multiple attempts by the tumor to promote its own escape from the host immune system by silencing antitumor immune effector cells. However, different mechanisms by which Foxp3+CD4+ T cells suppress antitumor immune response exist, but still not fully understood yet [106]. In a recent study, Ganesan et al. demonstrated that tumor-infiltrating Foxp3+CD4+ T cells partially repressed CD8+ T cell responses in murine models of lung adenocarcinoma [107]. It seems equally likely that in tumors characterized by extensive inflammatory infiltrates, such as colon or breast cancers, Foxp3+CD4+ T cells are necessary for control of chronic inflammation, prevention of tissue damage, and limiting of tumor development associated with inflammation [108, 109]. Because chronic inflammation is one of the critical processes promoting carcinogenesis and tumor growth, Foxp3+CD4+ T cells are able to down-regulate the pro-tumorigenic inflammatory res-ponses.

In many human cancers and in most mouse models of tumor growth, the frequency of Foxp3+CD4+ T cells and their suppressor functions are in-creased as compared to those reported for healthy subjects [110, 111]. Whelan et al. study showed that elimination of Foxp3+CD4+ T cells and concomitant stimulation of effector T cells result in tumor rejection in 90% of sarcoma-bearing mice [112]. Despite the general perception that Foxp3+CD4+ T cells accumulations in cancer predict poor outcome [113-115], in human colorectal cancer and in breast cancer, the presence and density of Foxp3+CD4+ T cells have been reported to predict favorable outcome and a better locoregional control of the tumor [108, 116]. Also, in human lymphomas, elevated circulating Foxp3+CD4+ T cells predict better outcome [117, 118]. Shimizu et al. demonstrated that patients with NSCLC (stages I-IIIB) containing three or more infiltrating Foxp3+CD4+ T cells/10 HPFs in the tumor tissue had significantly worse recurrence-free survival, and among patients with node-negative NSCLC, Foxp3+CD4+ T cells were an independent poor prognostic factor [119]. An increased Foxp3+CD4+ T cells count was also found to be associated with worse overall survival in another study with I-IIIA stage NSCLC [120]. However, et al. NSCLC study showed that in patients with N2 disease was a significantly improved disease free survival with high FOXP3 infiltrate [121]. Further studies are needed for better understanding the role of Foxp3+CD4+ T cells in NSCLC.

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IL-17A+CD4+ T cells

Th17 cells were first characterized in 2005 as a Th cell lineage inde-pendent from Th1 and Th2 subsets [122]. Th17 cells are defined by their production of 17 (also known as 17A), although they also produce IL-17F, IL-21, GM-CSF, and IL-22 [123]. Engagement of naive CD4+ T cells into the Th17 subset depends on different cytokines including TGF-β, IL-6, IL-1β, or IL-21 [123]. Th17 cells participate in antimicrobial immunity at mucosal and epithelial barriers and particularly fight against extracellular bacteria and fungi. While a role for Th17 cells in promoting inflammation and autoimmune disorders has been extensively and elegantly demonstrated, it is still controversial whether and how Th17 cells influence tumor im-munity. Although Th17 cells specifically accumulate in many different types of tumors compared to healthy tissues, the outcome might however differ from a tumor type to another. Th17 cells were consequently asso-ciated with both good and bad prognoses. On the one hand, Th17 cells promote tumor growth by inducing angiogenesis (via IL-17) and by exerting themselves immunosuppressive functions. On the other hand, Th17 cells drive antitumor immune responses by recruiting immune cells into tumors, activating effector CD8+ T cells, or even directly by converting toward Th1 phenotype and producing IFN-γ [124]. IL-17-producing T cells have dis-played both antitumor and protumor functions, due to their plasticity and functions in the tumor microenvironments [125]. On the contrary, data have shown that Th17 cells elicit antitumor effects, by promoting cytotoxic activities, enhancing Th1 response, and augmenting the expression of MHC antigens [126, 127]. In a murine model of lung cancer, enhanced Th17 cells and overexpression of IL-17A stimulated tumor growth in the lungs [128, 129]. Similarly, an increased number of intratumoral IL-17-positive cells in patients with lung cancer correlated with poor prognosis [130]. However, Th17 cells are associated with inflammatory and autoimmune diseases in mice and human. Notably, antigen-specific Th17 cells and their related cytokines are highly pathogenic and exhibit detrimental roles in multiple sclerosis, psoriasis, systemic lupus erythematosus, rheumatoid arthritis, in-flammatory bowel disease, and asthma [123].

Tumor-infiltrating Th17 cells were reported for many cancers in mice and humans, including melanoma, breast, colon, hepatocellular, ovarian, pancreatic, prostate, and renal tumors [131]. Moreover, Th17 cells accu-mulate specifically in many different tumors (esophageal carcinomas, breast, colon cancers, and melanoma) compared to healthy tissues [132], demonstrating a specific recruitment of Th17 cells by the tumor microen-vironment itself. However, it is still unknown whether Th17 cells are induced, recruited, expanded, or converted from Foxp3+CD4+ T cells in

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tumors. It is likely that all of these processes coexist [124]. Intratumoral recruitment of Th17 cells was proposed to rely on various chemokines depending on the tumor context, produced by immature myeloid cells [133]. Moreover, cancer cells, tumor-derived fibroblasts, and antigen-presenting cells secrete several key cytokines for Th17 differentiation such as IL-1β, IL-6, IL-23, and TGF-β. In the tumor, IL-1β, probably produced by tumor-associated macrophages, was shown to be critical for the expansion of memory Th17 cells in ovarian and breast cancers [132]. Intratumoral Th17 cell infiltration has been associated with both good and bad prognoses. Indeed, Th17 cell infiltration in human tumors was correlated with better survival in ovarian cancer patients [132], prostate cancer patients [134], lung carcinoma, and squamous cell carcinoma patients [135] or with bad prognosis in hepatocellular [130], colorectal [136], pancreatic [137], and hormone resistant prostate carcinoma patients [138]. However, Th17 cells role in cancer is still under debate.

CD8+ T cells

CD8+ T cell responses are essential for the control and clearance of viral infections as well as for the elimination of transformed and tumorigenic cells. CD8+ T cell protection is mediated by its ability to specifically target host cells compromised by microbial infection or oncogenic transformation. After antigen stimulation of naive CD8+ T cells follows a tri-phasic res-ponse: an initial activation phase characterized by a clonal expansion of antigen-specific cells and concurrent acquisition of peripheral tissue-homing capabilities, effector cytokine release, and cytolytic activity; a death phase characterized by a rapid, apoptosis-induced contraction of antigen-specific effector T cells; and formation of a persistent population of antigen-ex-perienced cells that represent immunologic memory [139]. The activation of specific CD8+ T cells by CD4+ T cells may be most efficient when cells recognize the same antigen presented by a DC, in the context of both MHC class I and class II molecules. Recognition of DC by CD4+ T cells can lead to IL-2-production resulting in proliferative effects on proximate CD8+ T cells. Perhaps more importantly, CD4+ T cells activate DC, enhancing their ability to stimulate naive CD8+ T cells [140]. Following exposure to anti-gens by DCs in an appropriate inflammatory environment, CD8+ T cells undergo a period of massive expansion, activation, and differentiation to terminally differentiated cells with effector functions. Once the pathogenic process is resolved, most effector CD8+ T cells undergo apoptosis, leaving a long-lived subset of memory cells. These cells possess an enhanced ability to control secondary exposures to antigens [139], which is attributed to their increased frequency, rapid acquisition of effector functions, and recruitment

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to the tumor sites. In both animal models and humans, CD8+ T cells have been shown to play an important role in the host’s defense against ma-lignancies [141].

One of the most common evasion mechanisms against CD8+ T cells in cancer is loss or down-regulation of HLA molecules expression. Lung cancer cells have been shown to downregulate HLA I expression which may lead to develop cancer [142]. However, lung tumors express tumor asso-ciated antigens, which can be recognized by the CD8+ T cells of the host immune system. Several studies have reported that cytotoxic T cell clones can be established; these clones are MHC class I restricted and show spe-cific cytotoxicity against autologous target cells [86]. Thus, the poor immu-ne response observed against lung cancer may be attributed to the evasion mechanisms presented by lung tumor cells.

In lung cancer, the essential role in the immune response to cancer cells is played by tumor-infiltrating CD4+ and CD8+ T cells [101, 143]. CD8+ T cells represent a major arm of the antitumor response because they have cytotoxic cell-mediated activity toward tumor cells expressing tumor-associated antigens [144]. It is thought that CD4+ T cells also play a sig-nificant role in antitumor response by allowing CD8+ T cells to entry into the tumor site [145] and infected mucosa [146], and they also are required for angiogenesis inhibition at the tumor site [147]. Contradictory data have been published about the influence of the different infiltration patterns of immune cells in lung cancer on the prognosis of these patients [84].

As CD8+ T cells exhibit marked cytotoxic capacities that may induce tumor cell death [148], by releasing perforins and granzymes in acquired immune responses, thereby playing a critical role in antitumor immunity [149]. CD8+ T cells are most likely functionally relevant in NSCLC, as the number of apoptotic tumor cells was significantly higher in tumors with a high number of CD3+ and CD8+ T cells [150] therefore CD8+ T cells com-prise a well-established group of effector T cells with potent cytotoxic ef-fects in cancer [151]. However, the presence of CD8+ T cells in NSCLC has been identified as positive, neutral or negative prognostic marker [143, 152, 153]. Another important factor affecting clinical outcomes in NSCLC might be tumor infiltrating CD4+ T cells. These cells produce immunoregulatory cytokines such IFN-γ and TNF-α that may induce cytolytic CD8+ T cell res-ponses in lung cancer [149, 154]. Another important factor effecting clinical outcomes in NSCLC might be relationship between T cells and macro-phages.

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Macrophages

Macrophages exist in almost all tissues and play important roles in the maintenance of tissue homeostasis. In mature adults, macrophages differen-tiate from peripheral blood monocytes, which develop from common myeloid progenitor cells [155]. These cells are identified as granulocyte/ macrophage colony-forming units (GM-CFUs) in the bone marrow. In res-ponse to a macrophage colony-forming factor, GM-CFUs sequentially give rise to macrophage colony-forming units (M-CFUs), monoblasts, and pro-monocytes. Subsequently, they move into the peripheral blood and differentiate into monocytes. Finally, the monocytes migrate into different tissues and supplement the populations of long-lived tissue-specific macro-phages, such as alveolar macrophages and Kupffer cells [156]. However, not all tissue macrophages are differentiated from monocytes. It has been reported that Langerhans cells in the skin and microglial cells in the brain, which are tissue-resident macrophage populations, seem to be maintained through local proliferation, and recent studies indicate that these cells ini-tially develop from M-CFU in the yolk sac of the developing embryo [155]. During inflammation or infection blood monocytes are recruited into the tissues, where they differentiate into macrophages or DCs [157]. Depending on the microenvironment, macrophages can be polarized into different phenotypes: either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages.M1 macrophages are induced with IFN-γ alone or together with microbial stimuli such as lipopolysaccharide (LPS) or cytokines such as TNF. M2 macrophages differentiate when monocytes are stimulated with IL-4 and IL-13, with immune complexes, or with IL-10 and glucocorticoids [158]. M1 macrophages are tumoricidal and they have the ability to kill pa-thogens. M2 macrophages participate in wound healing where they downre-gulate the inflammatory reactions, promote angiogenesis, recruit and regu-late connective tissue remodeling [159]. Most importantly, M1 and M2 phenotypes might not be stably differentiated subsets. Foster et al. study showed, that in vitro, LPS-activated macrophages after a few hours become unable to reactivate a large fraction of pro-inflammatory genes following restimulation [160]. Moreover, previous studies indicated that in the absence of M1 macrophages orienting signals M2 macrophages rather promoted tumor cell growth in vitro as well as in experimental murine models [161]. These numerous roles, ranging from host defense against infectious agents, to tissue development, wound healing, and immune system regulation, are reflected in the wide spectrum of possible phenotypes. Defining and diffe-rentiating distinct protumoral and antitumoral subsets of macrophages re-mains a challenging work.

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Macrophages are particularly abundant among the innate and adaptive immune cells recruited to the tumor site and are present at all stages of tu-mor progression. Previous in vitro studies with IFN-γ stimulated macro-phages have indicated that under certain conditions these cells display cytotoxic functions against tumor cells [162]. Macrophages derived from healthy tissues may present tumor-associated antigens to T cells, lyse tumor cells, and express cytokines to stimulate the proliferation and antitumor functions of NK cells and T cells in vitro. In contrast, macrophages from human tumors had greatly diminished levels of these activities [163]. One possible explanation is that something alters or prevents macrophages from killing tumor cells, such as an immunosuppressive microenvironment. Tumor cells coax macrophages to a M2 phenotype via chemokines and po-larizing cytokines, aiding their own escape from destruction, and promoting their development [164].

The role of macrophages in NSCLC patients remains controversial. A significant portion of the available evidence points to a positive correlation between macrophage infiltration and good prognosis. A study by Welsh et al. revealed that in patients with surgically resected NSCLC, high CD68+ macrophage density in tumor islets was independent predictor of increased survival [165]. In contrast, high stromal macrophage density was an independent predictor of reduced survival [165]. Furthermore, two inde-pendent groups found that patients with a high tumor islet macrophage density but incomplete resection survived significantly longer than patients with a low tumor islet macrophage density but complete resection [166, 167]. However, some studies have shown no correlation with prognosis in NSCLC [143, 168, 169]. Other authors found correlations between macro-phages and poor NSCLC prognosis. A study by Chen et al. found that CD68+ macrophage density correlated negatively with prognosis in patients with NSCLC [170]. In another study by Zhang at al. higher M2 macrophage density was associated with poor prognosis when compared to low M2 mac-rophage density [171]. Similarly, Ohtaki et al. found that M2 macmac-rophages significantly correlated with poor outcome in patients with adenocarcinoma [172].

1.6.2. Cytokines and non-small cell lung cancer

Importantly, immune cells do not act in isolation, and their effector functions are largely dependent upon the release of cytokines and binding of inhibitory and activating receptors to ligands expressed by other immune cells, stromal cells, and even tumor cells [173]. Cytokines are small secreted proteins released by cells have a specific effect on the interactions and

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communications between cells. Cytokines participate in the induction and effector phases of all immune and inflammatory responses. They are the-refore obvious tools and targets for strategies designed to promote, inhibit or redirect these responses [174]. Immune and stromal cells, such as fibroblasts and endothelial cells, synthesize them and they regulate proliferation, cell survival, differentiation, immune cell activation, cell migration, and death. A number of cytokines have been described as possessing dual roles in NSCLC [175]. Depending on the tumor microenvironment, cytokines can modulate an antitumoral response, but during chronic inflammation, they can also induce cell transformation and malignancy, conditional on the balance of pro- and anti-inflammatory cytokines, their relative concentra-tions, cytokine receptor expression content, and the activation state of surrounding cells [176].

IL-10

Interleukin 10 (IL-10) is known to be a potent anti-inflammatory cyto-kine. Stimulation of antigen-presenting cells with LPS induces IL-10 re-lease, which is further increased by the presence of damaged cells. Almost all immune cells, including T cells, B cells, monocytes, macrophages, mast cells, granulocytes, DCs, and keratinocytes, produce IL-10. Tumor cells can also secrete IL-10 [177].

IL-10, by reducing or inhibiting antigen presentation via downregulation of MHC class II expression in APCs as well as MHC class I in tumor cells, is thought contribute to an immunosuppressive environment and thus facilitate tumor escape. The issue is complex, however, due to the in vivo relationship between inflammation and tumor progression [178]. Due to its immunosuppressive effect on DCs and macrophages, IL-10 can dampen antigen presentation, cell maturation, and differentiation, allowing tumor cells to evade immune surveillance mechanisms [179]. IL-10 can also directly promote DCs apoptosis and skew the differentiation of monocytes from DCs to macrophages [180]. Another negative effect of tumor-derived IL-10 on DCs might be to prevent them from being recruited and accumu-lating within the tumor, as suggested by an experimental mouse model using granulocyte-macrophage colony-stimulating factor gene transfer [181].

Despite the findings presented above, there is a large amount of evidence that supports IL-10 having potent antitumor effects as well. Murine tumor models engineered to express IL-10 showed rapid tumor rejection that increased with IL-10 secretion [182]. The beneficial effect of IL-10 on CD8+ T cell memory may occur either by direct or indirect mechanisms. Consistent with a direct role, IL-10 has been shown to increase the proli-feration, recruitment, and cytotoxicity of CD8+ T cells in vitro [183]. Also, a

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possible mechanism that has been proposed to explain the findings that IL-10 has anti-tumor activity is IL-IL-10-mediated stimulation of NK cells. As well as direct NK stimulation, IL-10 might indirectly stimulate NK activity in vivo [178]. IL-10 has also been shown to modulate apoptosis and supp-ress angiogenesis during tumor regsupp-ression [184]. In addition, by inhibiting MMP secretion, IL-10 could also decrease the invasive potential of tumor cells [181]. IL-10 is most likely not acting alone, as it is probable that there are other cytokines in this microenvironment that affect the outcome of IL-10 stimulation [178].

Data concerning the role of IL-10 for tumor progression are partially contradictory. IL-10 is commonly regarded as an anti-inflammatory, immu-nosuppressive cytokine that favors tumor escape from immune surveillance. However, some authors indicated some immunostimulating properties of IL-10 and its role in antitumor response [185, 186]. Hatanaka et al. found that NSCLC patients have elevated IL-10 serum levels compared to healthy controls which has been associated with poorer prognosis [187]. In contrast to these results, some studies suggest that IL-10 is important for tumor rejection [188, 189]. Therefore, it is necessary to further define the role of IL-10 in lung cancer.

IFN-γ

IFN-γ is a necessary cytokine in the innate and adaptive immune res-ponses that protect against tumor development. Several immunological and non-immunological mechanisms have been proposed to explain the effect of IFN-γ in tumor immunity [190]. The source of this cytokine early after tumor challenge has not been identified. NK cells and T cells are considered to be important for bridging the innate and adaptive immune responses and they might provide the initial source of IFN-γ in tumor protection. This in turn could facilitate the cascade of the adaptive immune response and enhance later IFN-γ production [191].

It is now well recognized that IFN-γ exerts its biologic effects by inter-acting with an IFN-γ receptor that is expressed on nearly all cells [192]. IFN-γ may activate, recruit, or enhance the production of cells such as NK cells, macrophages, and neutrophils that may promote innate antitumor responses. Moreover, IFN-γ may have anti-metabolic or anti-proliferative effects on certain types of tumor cells. Also, IFN-γ may promote elimination of transformed cells either through nonimmune mechanisms such as those that restrict tumor growth by interfering with the development of a blood supply to the growing tumor [193]. Developing solid tumors require new blood vessel formation in order to grow. This neovascularization process comes as a result of angiogenesis, and the ultimate angiogenic status of a