Role of inflammation in cancer - Inflammation and cancer

Chapter 2: Literature review

2.3. Inflammation and cancer

2.3.1. Role of inflammation in cancer

Recent studies revealed that cancer and inflammation are connected by two pathways. The first pathway is called extrinsic, in which inflammatory or infectious diseases increase the risk for development of cancer (MANTOVANI et al., 2008). The second pathway is called the intrinsic one. This pathway has been addressed recently when evidence of inflammatory cells and mediators were noted within the tumor microenvironment without the presence of a usual cause of inflammation such as the presence of infectious agents (MANTOVANI et al., 2008).

During this pathway, the genetic alterations that cause neoplasia are responsible for generating the inflammatory changes (MANTOVANI et al., 2008). The two pathways then activate transcription factors, such as nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) which induce the production of inflammatory cytokines and chemokines which recruit inflammatory cell infiltration and augment the inflammation within the tumor microenvironment (MANTOVANI et al., 2008).

The inflammatory reaction and its components within the tumor microenvironment contribute to every aspect of carcinogenesis (GRIVENNIKOV et al., 2010). However, their detailed role in tumor development is still controversial (CHOW et al., 2012). Initial studies found that animals with impaired immune response are more susceptible to virally or chemically induced neoplasms (NOMOTO and TAKEYA, 1969; SANFORD et al., 1973; STUTMAN, 1974).

Furthermore, other investigations demonstrate the importance of cytokines such as interferon γ (IFN- γ) in protecting the host from tumor development (DIGHE et al., 1994). On the other hand, chronic inflammation significantly contributes to tumor development through several mechanisms such as enhancing angiogenesis and tissue invasion (GRIVENNIKOV et al., 2010). However, some surveys suggest that both tumor-suppressing and tumor-induced inflammation can co-exist within the same tumor (SWANN et al., 2008).

21 2.3.2. Inflammatory cell components of tumors

Often a wide range of immune cells, such as monocytes, macrophages, dendritic cells, neutrophils, eosinophils, mast cells and lymphocytes infiltrate a tumor and establish an inflammatory microenvironment (MACARTHUR et al., 2004). The infiltrating immune cells may repress neoplastic growth or may act as tumor promoters (LU et al., 2006). All of these immune cells are capable of producing an assorted array of cytokines, other mediators such as reactive oxygen species, serine and cysteine proteases, MMPs, tumor necrosis factor α (TNF-α), interleukins and interferons which elicit innate immune response, tissue remodeling and angiogenesis (COUSSENS and WERB, 2002). Macrophages represent up to 50% of the tumor mass and are considered as the major players in the connection between inflammation and neoplasms (SOLINAS et al., 2009). Macrophages most often originate from blood-derived monocytes and can be activated in response to cytokines and other signals and differentiate into two distinct types, M1 (classically activated) and M2 (alternatively activated; SCHMID and VARNER, 2012). M1 polarization occurs upon stimulation with IFN-γ and lipopolysaccharides (LPS). M1 macrophages are characterized by production of high levels of IL-12, IL-23, TNF-α and CXC chemokine ligand 10 (CXCL10) and elicit cytotoxicity against microorganisms and neoplastic cells by production of reactive oxygen intermediates (ROI) and antigen presenting cell activity (SOLINAS et al., 2009). On the other hand, when macrophages are stimulated with IL-4, IL-10, IL-13 or corticosteroids, they differentiate into the M2 phenotype which is characterized by the production of IL-10, chemokine 17 ligands (CCL17), CCL22, interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 receptor (IL-1R). M2 macrophages are involved in promotion of wound healing, angiogenesis, tumor progression and control of inflammatory responses (SOLINAS et al., 2009). It is now generally accepted that M2 macrophages are the predominant phenotype of tumor associated macrophages (TAM) within the tumor microenvironment (MANTOVANI and SICA, 2010). The role of TAMs in neoplasms is still controversially discussed in the literature. TAMs in a colorectal cancer model have been reported to act proinflammatory and inhibit the proliferation of tumor cells by producing chemokines and cytokines such as IFN-γ, IL-1, and IL-6 that attract T cells, stimulate proliferation of allogeneic T cells and activate T cells associated with anti-tumor immune responses (ONG et al., 2012). On the other hand, in most tumors such as breast, prostate, ovarian, cervical and lung carcinoma and cutaneous melanoma, TAMs are considered to be anti-inflammatory and correlated with a poor prognosis (HAO et al., 2012). TAMs contribute to tumor development in several ways. They can produce many growth factors which induce the growth and motility of tumor cells

(POLLARD, 2004). Examples of these growth factors are fibroblast growth factor (FGF), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR)-family ligands, platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β;

POLLARD, 2004). TAMs may also release growth factors such as VEGF, PDGF, TGF-β, and members of the FGF family, which can promote angiogenesis in many tumors such as gliomas, squamous cell carcinoma of the esophagus, and breast, bladder and prostate carcinoma (TANAKA et al., 2002; SIVEEN and KUTTAN, 2009; SOLINAS et al., 2009). In addition to TAMs, other inflammatory cells such as neutrophils, mast cells, eosinophils and T lymphocytes similarly have the ability to contribute to tumor progression by releasing proangiogenic factors such as VEGF, IL-8 and proteases such as matrix metalloproteinases (BELLOCQ et al., 1998; SHAMAMIAN et al., 2001; CARUSO et al., 2002; COUSSENS and WERB, 2002; LIN and POLLARD, 2004).

2.3.3. Inflammation as a target for anti-cancer therapy

The best evidence for the role of inflammation in tumor progression originates from studies which showed the preventive and tumor effect of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) on colorectal cancer and probably other cancer types (BARON and SANDLER, 2000; GARCIA-RODRIGUEZ and HUERTA-ALVAREZ, 2001). Sulindac, a member of the NSAIDs is able to regress human adenomatous colorectal polyps via apoptosis (KELLER et al., 1999). In addition, NSAIDs are able to inhibit angiogenesis in cell culture and rodent models (JONES et al., 1999). These findings suggest that NSAIDs represent a promising platform for the development of anticancer agents (THUN et al., 2002).

Tumor necrosis factor (TNF) also represents another candidate for cancer treatment (VAN HORSSEN et al., 2006). The anti-cancer actions of TNF can be due to direct cytotoxic effects and / or indirectly by altering the host stroma namely the neoplastic vasculature or the generation of a specific, cell mediated antitumor immunity (PALLADINO et al., 1987;

SHIMOMURA et al., 1988; UEDA et al., 2013). In addition, TNF can potentiate its antitumor effect through induction of cytotoxicity-related proteins such as IFN-γ (MOCELLIN et al., 2005). Despite the history of TNF and promising pre-clinical studies, TNF needs further therapeutic evaluation due to its biological effects as a mediator of endotoxic shock and endogenous pyrogen (SELBY et al., 1987). Finally, MMPs have long been associated with tumor growth, invasion and metastasis and many studies show their importance in tumor pathogenesis (EGEBLAD and WERB, 2002). Therefore, MMP-inhibitors are also representing other candidates for tumor therapy (PURCELL et al., 2002).

23 2.4. Oncolytic virus therapy

2.4.1. History

The use of viruses in the treatment of cancer came from observations that, occasionally, cancer patients with viral infections went into short periods of clinical remission, for example leukemia patients infected during their disease with influenza virus have shown clinical improvement (DOCK, 1904; PELNER et al., 1958). In 1896, a 42-year-old woman with

‘‘myelogenous leukemia’’ showed remission after a presumed influenza infection. This woman had a greatly enlarged liver and spleen, which shrank to nearly normal size, and highly elevated leukocytes counts, which dropped after the infection (DOCK, 1904). In another case, a 4-year-old boy displayed an enlarged liver, spleen and cervical lymph nodes and severely elevated leukocyte counts. His spleen and liver size returned back to normal and his leucocytes count reverted to the reference range within a few days after he was infected with chickenpox (BIERMAN et al., 1953).

In 1949, clinical trials were performed using hepatitis virus which was administered to patients suffering from Hodgkin’s disease (HOSTER et al., 1949). Some of the patients showed improvement in clinical signs and laboratory findings for at least one month (HOSTER et al., 1949). A few years later, patients with acute monocytic leukemia were injected with Epstein-Barr virus taken from cases of glandular fever (TAYLOR, 1953).

Leukemia cases showed improvement with a sharp drop in leukocyte count (TAYLOR, 1953). After these early clinical studies, many human pathogens were used in therapeutic trials against a wide range of cancers in human patients and also in tumor xenograft models (KELLY and RUSSELL, 2007). Among the viruses used adenoviruses, paramyxoviruses, picornaviruses, and poxviruses gained more attention as viral agents of cancer treatment (KELLY and RUSSELL, 2007). In November 2005, the genetically modified adenovirus H101 was the first drug for viral oncolysis approved by the Chinese authorities (GARBER et al., 2006).

2.4.2. Mechanisms of viral oncolysis

The concept behind using oncolytic viruses for tumor therapy, based on their preferable intratumoral replication as well as their potent anti-tumor effects (PRESTWICH et al., 2008).

Some viruses possess natural inherent tumor selectivity and thus they can specifically target tumor cells. Examples of these viruses include Newcastle disease virus, vesicular stomatitis virus, reovirus and measles virus (ROBERTS et al., 2006). Another group of oncolytic viruses are attenuated viruses with deleted or modified critical genes to restrict their replication to

tumor cells and enhance their oncolytic activity, such as adenovirus and herpes simplex virus type 1 (NETTELBECK and CURIEL, 2003; LIN and NEMUNAITIS, 2004). Following infection, oncolytic viruses mediate the destruction of tumor cells by several mechanisms.

The virus can infect and multiply within the tumor cell with subsequent lysis (SINGH et al., 2012). In addition progeny virions can infect adjacent cells and destroy them (MULLEN and TANABE, 2002; BELL and MCFADDEN, 2015). The mechanisms by which oncolytic viruses induce tumor cell death are poorly understood and may show some variation from the classical schemes of apoptosis and necrosis (WOLLER et al., 2014). A necrosis-like programmed cell death mechanism has been proposed for a class of oncolytic adenoviruses called conditionally replicating adenoviruses (CRAds; ABOU EL HASSAN et al., 2004). In addition, oncolytic vaccinia virus induces necrotic cell death of ovarian cancer cells through a programmed necrosis mechanism which shares limited apoptotic features (WHILDING et al., 2013).

Several oncolytic viruses encode cytotoxic proteins which can induce lysis of infected tumor cells. Examples of these proteins are the adenovirus death protein (ADP) and the adenovirus type 5 E4 open reading frame 4 (E4orf4) protein (TOLLEFSON et al., 1996; SHTRICHMAN and KLEINBERGER, 1998).

Another mechanism is via altering the tumor microenvironment into a milieu that enhances anticancer activity (DE SILVA et al., 2010). Angiogenesis is significantly involved in tumor progression and VEGF is one of the dominant drivers of angiogenesis in neoplasms.

Therefore, inhibition of VEGF signaling represents an attractive cancer treatment (ROSKOSKI, 2007). Some viruses possess inherent antivascular properties. These viruses have the ability to infect both developing and established tumor vasculature without destroying normal vessels (ANGARITA et al., 2013). The mechanism behind this tropism for tumor vasculature is still not clear, but there are three suggested mechanisms by which oncolytic viruses affect tumor vasculature: first through direct infection of endothelial cells of tumor vasculature; second through the induction of virus-mediated immune responses which affect tumor perfusion and third, the expression of viral proteins which have antiangiogenic properties (ANGARITA et al., 2013). Pancreatic cancer cell lines infected with an adenovirus which retained the normal early region 1A (E1A) protein showed an oncolytic effect and inhibited tumor angiogenesis significantly (SAITO et al., 2006). Similarly, intravenous administration of vesicular stomatitis virus (VSV) initiates an inflammatory reaction including a neutrophil-dependent production of microclots within the tumor blood vessels and ultimately tumor vascular collapse (BREITBACH et al., 2011). Another interesting oncolytic

virus, the replication-restricted herpes simplex virus HSV-1716, directly infects tumor endothelium and thereby exerts a direct antiangiogenic effect following intratumoral administration in ovarian carcinoma (BENENCIA et al., 2005). In addition, oncolytic viruses have been genetically engineered to carry antiangiogenic genes that either deliver antiangiogenic factors or target the expression of angiogenic proteins. Interesting examples of this principle are the oncolytic Lister vaccine strain of vaccinia virus which is armed with an endostatin-angiostatin fusion gene, the recombinant human endostatin adenovirus (Ad-hEndo) and the oncolytic herpes simplex virus expressing endostatin (YANG et al., 2010; TYSOME et al., 2011; GOODWIN et al., 2012). Other oncolytic viruses have also been developed to target the VEGF signaling pathway (KANG et al., 2008). Ad-DeltaE1-KOX is an oncolytic adenovirus expressing a VEGF promoter targeted artificial zinc-finger protein that elicited a pronounced antitumor effect against a human glioblastoma xenograft model (KANG et al., 2008).

Another promising therapeutic approach is the use of oncolytic viruses for targeting MMPs, which play an important role in tumor progression as described above (OVERALL and LOPEZ-OTIN, 2002). Infection of glioma cell lines with an oncolytic adenovirus expressing TIMP-3 called AdDelta24TIMP-3, showed enhanced oncolytic activity and significantly reduced tumor growth (LAMFERS et al., 2005). Oncolytic viruses are also able to target the tumors by activating and redirecting functional innate and adaptive immune responses against the transformed cells (CHIOCCA and RABKIN, 2014). Initially, the immune system was seen as a negative factor in oncolytic virus therapy because the immune system limits infection and replication of the virus within the neoplastic tissue (MELCHER et al., 2011).

However, a number of recent studies demonstrate that the immune system plays an important role in the efficacy of intratumoral oncolytic viral therapy, which is sometimes even more important than the direct oncolytic effect of the virus itself (PRESTWICH et al., 2008). This has been shown by using vesicular stomatitis virus (VSV), reovirus and herpes simplex virus (HSV; DIAZ et al., 2007; PRESTWICH et al., 2009; SOBOL et al., 2011; WORKENHE et al., 2014).

2.4.3. Canine distemper virus (CDV)

CDV is an enveloped, non-segmented, single-stranded RNA virus and is a member of the Morbilliviruses, thus closely related to measles virus (LAMB and KOLAKOFSKY, 2001;

VANDEVELDE and ZURBRIGGEN, 2005). CDV possesses a nucleocapsid containing the viral genome, which comprises of the nucleoprotein (N) and the polymerase complex

including the (P) and (L) proteins (DEMETER et al., 2007). The membrane protein (M) is located at the inner surface of the envelope, the attachment protein (H) and the fusion protein (F; VANDEVELDE and ZURBRIGGEN, 2005). CDV belongs to the Morbillivirus genus, which also includes other important viruses like measles virus (MV) and rinderpest virus (CARVALHO et al., 2012). Antigenic differences have been demonstrated serologically among CDV strains, but it’s generally accepted that there is only one serotype (DUIGNAN et al., 2014). Six established CDV lineages are described: lineage Asia 1 (Japan and China), Asia 2 (only in Japan), Arctic, European wildlife, Europe, lineage America 1 and 2 and old CDV strains (Onderstepoort, Convac, Rockborn and Synder Hill; HARDER et al., 1996;

YOSHIDA et al., 1998; HAAS et al., 1999; MARTELLA et al., 2008).

Canine distemper is a highly contagious disease that affects dogs and a wide range of carnivores (DAMIEN et al., 2002; BAUMGÄRTNER et al., 2003). CDV has been reported in dogs, ferrets, wild dogs, foxes, jackals, coyotes, hyenas, lions, tigers, leopards, cheetahs, seals, sea lions, and dolphins (APPEL et al., 1994; KENNEDY et al., 1998; VAN DE BILDT et al., 2002). The disease has also been found in felids including domestic cats (HARDER et al., 1996; WIENER et al., 2013).

CDV transmission mainly occurs by direct animal-to-animal contact or by exposure to infectious aerosols (TERIO and CRAFT, 2013). The virus can be detected at high titers in secretions and excretions, including urine (ELIA et al., 2006). The initial infection occurs in epithelial cells and lymphoid tissue in the nasopharynx and replication takes place primarily in the lymphoid tissue of the respiratory tract (VON MESSLING et al., 2003). The virus replicates within tissue macrophages and spread locally followed by disseminated distribution by lymphatics and blood (APPEL, 1970; BEINEKE et al., 2009). This widespread dissemination of the virus is accompanied by an increase in body temperature and leukopenia (BEINEKE et al., 2009). The virus multiplies within lymphoid tissues including spleen, thymus, lymph nodes, bone marrow, mucosa-associated lymphoid tissues and macrophages in the lamina propria of the gastrointestinal tract and hepatic Kupffer cells (APPEL, 1970;

WRIGHT et al., 1974). The second phase results in spread of the virus into multiple parenchymal organs throughout the body, including respiratory, gastrointestinal and urinary tract, endocrine and central nervous system (BEINEKE et al., 2009). The disease occurs as an acute systemic disease, a chronic nervous disorder or a combination of both. The main manifestations include respiratory and gastrointestinal signs, immunosuppression, demyelinating leukoencephalomyelitis and unusual clinical signs such as enamel defects and hard pad disease (LAPP et al., 2014). Therefore clinical changes include anorexia, depression,

conjunctivitis, hyperkeratosis of digital pads, catarrhal inflammation of bronchi and larynx, vomiting and diarrhea (CARVALHO et al., 2012). Neurological signs include ataxia, paraplegia, quadriplegia, myoclonus, tremor, incontinence, seizures, circling, apathy, coma, muscular atrophy, vocalization, dryness of the retina, and blindness (MARTELLA et al., 2008; BEINEKE et al., 2009).

It’s clear that CDV is a lymphotropic virus and causes severe immunosuppression in dogs depending on the immune status of the animals (KRAKOWKA et al., 1975; BEINEKE et al., 2009)

2.4.4. CDV as an oncolytic virus

As mentioned above, CDV belongs to the morbillivirus genus, which also includes other important infectious viruses like measles virus (MV; CARVALHO et al., 2012). Attenuated measles virus vaccine strains represent a promising approach for oncolytic therapy, having shown significant anti-tumor activity against a broad range of malignant neoplasms (MSAOUEL et al., 2012). Spontaneous regression of tumors was reported following infection of MV in leukemias, Burkitt’s lymphoma and Hodgkin’s disease (BLUMING and ZIEGLER, 1971; GROSS, 1971; MOTA, 1973). In addition, attenuated measles virus strains can selectively infect cancer cells causing their lysis with minimal cytopathic effect on non-transformed cells (MSAOUEL et al., 2012).

CDV which shares many features with MV also represents an interesting candidate as an oncolytic virus (LAPP et al., 2014). Compared to MV which is extensively studied, only very few studies used CDV as an oncolytic agent (LAPP et al., 2014). Previous surveys show that CDV has the ability to induce apoptosis in cell lines derived from human cervical cancer (DEL PUERTO et al., 2011). Moreover, attenuated CDV causes apoptosis in canine lymphoid cell lines and neoplastic B and T lymphocytes collected from dogs with spontaneous lymphoma (SUTER et al., 2005). CDV can also infect CCT and DH82 cells, permanent cell lines originating from canine disseminated histiocytic sarcomas, leading to apoptosis in CCT cells (GRÖNE et al., 2002; YAMAGUCHI et al., 2005; PUFF et al., 2009). Furthermore such an infection induces the expression of pro-inflammatory cytokines such interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) in DH82 cells and results in a down-regulation of MMP-2, TIMP-1 and TIMP-2 while up-regulating RECK (Reversion-inducing-cysteine-rich protein with kazal motifs) in this cell line (GRÖNE et al., 2002; YAMAGUCHI et al., 2005; PUFF et al., 2009).

28 2.5. Aim of the study

The objective of this study is to investigate the inflammatory cell profile and the expression of selected MMPs and TIMPs in spontaneous canine histiocytic sarcoma cases. Furthermore, results will be compared to a murine xenograft model of canine histiocytic sarcomas. In addition to the aforementioned factors, microvessel density in spontaneous canine histiocytic sarcomas will be evaluated and compared to the one in xenotransplanted tumors.

Chapter 3: Inflammatory cell profile, matrix metalloproteinase expression and its correlation with microvessel density in spontaneous canine histiocytic sarcomas and a canine histiocytic sarcoma xenograft model

Adnan Fayyad¹, Stefanie Lapp¹, Engy Risha¹, Karl Rohn², Yvonne Barthel¹, Dirk Schaudien³, Wolfgang Baumgärtner^1,*, Christina Puff¹

1 Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany

2 Institute for Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Bünteweg 2, 30559 Hannover, Germany

3 Fraunhofer Institute for Toxicology and Experimental Medicine, Nikolai-Fuchs-Straße 1, 30625 Hannover, Germany

* corresponding author:

Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Department of Pathology

University of Veterinary Medicine Hannover Bünteweg 17

30559 Hannover Germany

Tel: +49-511-953-8620 Fax: +49-511-953-8675

e-mail: Wolfgang.Baumgaertner@tiho-hannover.de

Abstract

Canine histiocytic sarcoma (HS) represents a malignant neoplastic disorder with a rapid and progressive clinical course. Due to its invasiveness, high rate of relapses as well as the common occurrence of metastases especially the disseminated form of the disease carries a grave prognosis. A better understanding of the interaction between tumor cells and the local microenvironment may provide new insights into mechanisms of tumor growth and metastases. The influence of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) on tumor angiogenesis, invasion and metastasis has been detailed in previous studies. In addition, inflammatory cells infiltrating neoplasms especially tumor associated macrophages (TAM) may contribute significantly to tumor progression. Due to the high variability of canine HS, standardized models are highly required to investigate tumor progression and interaction with the surrounding microenvironment. Therefore, the present study comparatively characterizes the intratumoral immune response as well as the expression of MMP-2, MMP-9, MMP-14 and TIMP-1 in spontaneous canine HS and its murine xenotransplantation model. In spontaneous canine HS, an intratumoral predominance of macrophages and T lymphocytes was observed while B lymphocytes were only found in low numbers. Interestingly, morphometric results demonstrated that MMP-2, MMP-9, MMP-14 and TIMP-1 were mainly expressed at the invasive front of the neoplasms while the tumor centers exhibited a significantly smaller immunoreactive area. Similar findings were obtained

Im Dokument Comparative evaluation of the tumor microenvironment in spontaneous canine histiocytic sarcomas and a canine histiocytic sarcoma xenotransplantation model (Seite 30-0)