Inflammation as a target for anti-cancer therapy

Chapter 2: Literature review

2.3. Inflammation and cancer

2.3.3. Inflammation as a target for anti-cancer therapy

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 for the MMP and TIMP-1 distribution in subcutaneously xenotransplanted HS, rendering this model highly suitable to investigate HS under standardized conditions. Moreover, tumor associated macrophages (TAM) strongly express MMPs and TIMP-1 and MMP-14 showed a strong correlation with microvessel density in xenotransplanted HS. These results indicate that MMP expression contributed to tumor progression and invasion and TAM seem to be major players in the interaction between neoplastic cells and the local microenvironment rendering therapeutic approaches modulating these molecules and cells promising treatment options for increasing life expectancy and/or quality of affected individuals.

Key words: canine histiocytic sarcoma; MMP; murine xenotransplantation model;

spontaneous neoplasm; tumor associated macrophages, microvessel density; TIMP

1. Introduction

Canine proliferative histiocytic diseases represent a range of well-defined disorders with marked differences in their clinical behavior and pathologic features [1,2]. This group of diseases, which are common in dogs and less frequently observed in cats, include cutaneous histiocytoma, the histiocytic sarcoma (HS) complex, reactive histiocytoses (cutaneous and systemic forms) and hemophagocytic histiocytic sarcomas [3-6]. The histiocytic sarcoma (HS) complex of dogs comprises two distinct types of malignant proliferative disorders, characterized by infiltration of neoplastic cells arising from interstitial dendritic cells [7]. The localized HS most often involves a single tissue or organ, usually the skin, whereas the disseminated HS spreads beyond the local draining lymph nodes to distant sites, usually lungs, lymph nodes, liver, spleen and central nervous system [2,8]. HS are typically composed of poorly demarcated sheets of large, pleomorphic cells with one or multiple nuclei, marked cellular atypia and a high mitotic index with variable numbers of inflammatory cell infiltrates consisting mainly of T lymphocytes [2,8,9]. HS is a highly aggressive neoplasm in dogs with a rapid and progressive clinical course leading to a poor prognosis [5].

Several studies have provided evidence that increased expression of matrix metalloproteinases (MMPs) is associated with invasion, metastasis and poor prognosis in numerous human and animal malignancies, including lung and oral neoplasms, breast carcinoma and esophageal squamous cell carcinoma [10-15]. An increased MMP expression is often accompanied by an enhanced invasion and metastasis rate and therefore a poor prognosis [16,17]. However, little is known about the importance and distribution of MMPs and their inhibitors in HS.

MMPs are a family of zinc-dependent endoproteinases whose enzymatic activity is directed mainly against components of the extracellular matrix (ECM) [18]. These proteinases are linked by a core of common domain structures and by their relationship to a family of proteinase inhibitors called tissue inhibitors of metalloproteinases (TIMPs) [19]. MMPs play important roles in tumor angiogenesis, invasion and metastasis through degradation of the stromal connective tissue and basement membrane components which permit the migration of tumor cells and secretion of growth factors, cytokines and vascular growth factors necessary for tumor development [20-24]. In addition, MMPs are also able to influence the tumor microenvironment by regulating innate and acquired immunity through modulating the function of cytokines and chemokines and increase the infiltration of inflammatory cells [25,26]. Notably, MMPs and their inhibitors are secreted by different cell types, including stromal fibroblasts in the vicinity of the neoplasm, tumor infiltrating macrophages or from the tumor cells themselves [27-30]. Inflammatory cells infiltrating tumors include macrophages,

dendritic cells, myeloid-derived suppressor cells and T-cells which contribute either positively or negatively to tumor invasion, growth and metastasis [31]. Analysis of cellular phenotypes of inflammatory cells infiltrating the neoplasm seems to be of critical importance for predicting tumor outcome [31]. Tumor-associated macrophages (TAM) are considered as the major players of tumor-related inflammation and are one important source of MMPs [32,33].

It’s suggested that tumor cells use MMPs produced by these macrophages, fibroblasts and other stromal cells for invasion [34] and tumor progression, including metastasis, which is only possible through close interaction between neoplastic and stromal cells [35]. However, the functional interactions between tumor and surrounding stromal cells are not completely understood [36] and are often unpredictable in spontaneously arising neoplasms. These difficulties highlight the importance of a well characterized, standardized model for detailed investigation of specific questions and analysis of the effectiveness of novel treatment options especially in relatively rare tumor types. To overcome these limitations and allow detailed investigations of the tumor microenvironment in histiocytic sarcomas, the aim of the present study was to comparatively analyze spontaneous canine histiocytic sarcomas and xenotransplanted neoplasms in a murine model to verify the usability of this model. Therefore the intratumoral immune response in spontaneous canine histiocytic sarcomas in combination

Im Dokument Comparative evaluation of the tumor microenvironment in spontaneous canine histiocytic sarcomas and a canine histiocytic sarcoma xenotransplantation model (Seite 32-0)