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Adnan Fayyad Abedalkhader
Hannover 2016
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Comparative evaluation of the tumor microenvironment in spontaneous canine histiocytic sarcomas and a canine histiocytic
sarcoma xenotransplantation model
INAUGURAL –DISSERTATION
in partial fulfillment of the requirements of the degree of Doctor of Veterinary Medicine
- Doctor medicinae veterinariae - (Dr. med. vet.)
submitted by
Adnan Fayyad Abedalkhader Nablus
Hannover 2016
Scientific supervision: Univ.-Prof. Dr. Wolfgang Baumgärtner, Ph.D.
Department of Pathology
University of Veterinary Medicine Hannover, Germany
1st Supervisor: Univ.-Prof. Dr. Wolfgang Baumgärtner, Ph.D.
Department of Pathology
University of Veterinary Medicine Hannover, Germany
2nd Supervisor: Prof. Dr. Ludwig Haas Department of Virology
University of Veterinary Medicine Hannover, Germany
Day of the oral examination: 18.11.2016
This work was supported by the “German Academic Exchange Service”.
To my family
II
Chapter 1: Introduction... 1
Chapter 2: Literature review ... 3
2.1. Canine histiocytic proliferative disorders / diseases ... 3
2.1.1. Canine reactive histiocytoses ... 3
2.1.2. Canine cutaneous histiocytoma ... 5
2.1.3. Histiocytic sarcoma complex ... 6
2.1.4. DH82 cells ... 7
2.2. Matrix Metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) ... 8
2.2.1. MMP classification ... 8
2.2.2. MMP structure ... 9
2.2.3. Regulation of MMP activity ... 10
2.2.4. TIMPs ... 14
2.2.5. Role of MMPs and TIMPs in tumor progression ... 14
2.2.6. Role of MMPs and TIMPs in invasion and metastasis ... 16
2.2.7. Angiogenesis and MMPs / TIMPs ... 16
2.3. Inflammation and cancer ... 20
2.3.1. Role of inflammation in cancer ... 20
2.3.2. Inflammatory cell components of tumors ... 21
2.3.3. Inflammation as a target for anti-cancer therapy ... 22
2.4. Oncolytic virus therapy ... 23
2.4.1. History ... 23
2.4.2. Mechanisms of viral oncolysis ... 23
2.4.3. Canine distemper virus (CDV) ... 25
2.4.4. CDV as an oncolytic virus ... 27
2.5. Aim of the study ... 28
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 ...29
Chapter 4: Discussion ...61
Chapter 5: Summary ...65
Chapter 6: Zusammenfassung ...67
Chapter 7: References ...69
Chapter 8: Appendix ...95
III
Chapter 9: Acknowledgments ...187
1
Chapter 1: Introduction
Canine histiocytic proliferative disorders represent a group of diseases which occur in dogs and less frequently in cats (KRAJE et al., 2001; MOORE, 2014). They include canine cutaneous histiocytomas, canine reactive histiocytoses, hemophagocytic histiocytic sarcomas and the histiocytic sarcoma complex (HS) (MOORE, 2014). The HS complex comprises two distinct types of malignant neoplasms and is characterized by proliferation of tumor cells mostly arising from activated interstitial dendritic cells forming poorly demarcated sheets of large pleomorphic cells with marked atypia and a high mitotic rate with variable numbers of infiltrating inflammatory cells (CONSTANTINO-CASAS et al., 2011; MOORE, 2014;
PAZDZIOR-CZAPULA et al., 2015). HS is a highly aggressive neoplasm and is characterized by a rapid and progressive clinical course and a poor prognosis (MOORE and ROSIN, 1986). In addition, HS shows a poor response to the current therapeutic approaches (AFFOLTER and MOORE, 2002; FIDEL SCHILLER et al., 2006). The limited therapeutic efficiency of HS have led to an increasing interest in more effective alternative options to control the aggressiveness of this disease and to improve cancer therapy by targeting the tumor microenvironment; including the tumor adjacent extracellular matrix compartment and tumor-related inflammation (SOUNNI and NOEL, 2013).
The influence of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) on tumor angiogenesis, invasion and metastasis has been outlined in previous studies in detail (WESTERMARCK and KAHARI, 1999; MARTIN et al., 1999; LI et al., 2001; YOSHIDA et al., 2002). In addition, several studies described that tumor infiltrating inflammatory cells especially tumor associated macrophages (TAM) may contribute significantly to tumor progression (TANAKA et al., 2002; POLLARD, 2004). Therefore a comprehensive understanding of the interaction between tumor cells and the tumor microenvironment may provide new insights into mechanisms of tumor growth and metastasis.
Therefore, the aim of this thesis is:
(i) to characterize the intratumoral immune response in conjunction with analysis of selected MMPs and TIMPs in spontaneous canine histiocytic sarcomas and xenotransplanted canine histiocytic sarcomas in a mouse model.
(i) to compare the obtained data from spontaneous canine histiocytic sarcomas with that of xenotransplanted HS in a mouse model.
(iii) to determine the correlation of MMP expression and microvessel density in xenotransplanted HS.
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The obtained information will give an insight into the pathogenesis of tumor development in canine histiocytic sarcoma and might offer an opportunity to evaluate a new therapeutic option for one of the most aggressive neoplastic diseases affecting the canine population.
3
Chapter 2: Literature review
2.1. Canine histiocytic proliferative disorders / diseases
The first reports about histiocytic disorders in dogs originated in the late 1970s. Described tumors consisted of solitary histiocytic lesions, which came to be known as canine cutaneous histiocytomas (TAYLOR et al., 1969; GLICK et al., 1976). These reports were soon followed by description of the clinical and pathological features of a newly recognized, distinctive, histiocytic proliferative disorder in the Bernese mountain dog breed called systemic histiocytosis (MOORE, 1984).
The histiocytic disorders occur more commonly in dogs than in cats (KRAJE et al., 2001) (Tab. 1). In dogs, histiocytic disorders are classified into three major categories (MOORE, 2014):
- The canine cutaneous histiocytoma which exhibits evidence of Langerhans cell (LC) differentiation and presented as sequestered tumors in young dogs and usually regresses spontaneously.
- The canine reactive histiocytoses including both cutaneous and systemic forms arising from activated interstitial dendritic cells (iDC). These disorders are histiocyte- and lymphocyte-rich reactive lesions.
- The histiocytic sarcoma complex which includes both localized and disseminated histiocytic sarcomas. These tumors mostly arise from activated interstitial dendritic cells.
2.1.1. Canine reactive histiocytoses
These diseases are described in dogs and include cutaneous histiocytosis (CH) which affects the skin and may extend into draining lymph nodes and systemic histiocytosis (SH) which has similar skin lesions, but can extend into other organs (MOORE, 2014). Both CH and SH are related diseases with similar clinical behavior and they share lesion topography and immunophenotype of histiocytic cells (AFFOLTER and MOORE, 2000). Cutaneous and subcutaneous lesions in cutaneous and systemic histiocytosis have identical histopathologic features characterized by multinodular infiltrates predominantly observed in the deep dermis and panniculus consisting of mainly activated dermal interstitial DCs, T cells (most often CD8+ T-cells) and neutrophils, accumulated around vessels and forming perivascular cuffs (MOORE, 2014). Pronounced angiocentric infiltration with vasoinvasion is more consistently seen in cases with systemic involvement (AFFOLTER and MOORE, 2002).
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The etiology and pathogenesis of canine reactive histiocytosis is unknown, but canine reactive histiocytoses regress with systemic immunosuppressive therapy (AFFOLTER and MOORE 2000). Therefore, mechanisms of immune impairment are anticipated in the pathogenesis of this disease (AFFOLTER and MOORE 2000).
2.1.1.1. Cutaneous reactive histiocytosis
Cutaneous histiocytosis (CH) is an inflammatory, lymphohistiocytic, proliferative, reactive disorder that predominately affects skin, subcutis and local lymph nodes with no evidence of local lymph node metastases (MAYS and BERGERON, 1986; MOORE, 2014). CH was first reported in dogs in 1986 as a proliferative disorder of histiocytic cells (MAYS and BERGERON, 1986). Lesions consist of multiple cutaneous and subcutaneous nodules that predominantly affect the middle/deep dermis and subcutis with common ulceration of the overlying skin. The infiltrate is composed predominantly of histiocytic cells and lymphocytes with variable numbers of neutrophils, eosinophils and plasma cells (PALMEIRO et al., 2007).
Lesions usually regress spontaneously and appear at new sites simultaneously (MOORE, 2014). The most common locations of these lesions are face, nose, neck, trunk, extremities, perineum, and scrotum (MOORE, 2014).
2.1.1.2. Systemic reactive histiocytosis
Systemic histiocytosis (SH) was first described in closely related Bernese Mountain Dogs (MOORE, 1984), other breeds such as Rottweiler, Labrador Retriever, Basset Hound, Irish Wolfhound, and others are less commonly affected (MOORE, 2014). The disease occurs as multiple cutaneous nodules affecting large areas of the body (MOORE, 2014). Lesions consist of angiocentric infiltrates of large histiocytic cells and fewer lymphocytes, neutrophils, and eosinophils (MOORE, 1984). The disease course is characterized by periods of remission and relapses (MOORE, 2014).
2.1.2. Canine cutaneous histiocytoma
Canine cutaneous histiocytoma (CCH) appears as a benign, cutaneous neoplasm (TAYLOR et al., 1969; GLICK et al., 1976; COCKERELL and SLAUSON, 1979; KAIM et al., 2006).
Although it occurs in all breeds, boxers and bulldogs, Scottish terriers, Doberman pinschers, cocker spaniels and dachshunds are reported to have a significantly higher risk than all other breeds (TAYLOR et al., 1969; FULMER and MAULDIN, 2007).
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CCH usually occurs as a solitary lesion anywhere on the body, but commonly arises on the head especially on the pinna (TAYLOR et al., 1969). It is a well circumscribed, circular dome-shaped lesion in the skin and ranges from 0.5 to 4 cm in diameter (TAYLOR et al., 1969; KAIM et al., 2006). The lesion has a benign behavior and usually undergoes spontaneous regression within 1 to 2 months (FULMER and MAULDIN, 2007; PUFF et al., 2013).
Microscopically, neoplastic histiocytes appear as uniform sheets of cells within the superficial dermis and epidermis (TAYLOR et al., 1969; MOORE, 2014). Tumor cells usually show different cytological features with round to oval or indented nuclei and abundant eosinophilic cytoplasm (TAYLOR et al., 1969; MOORE, 2014). Neoplastic cells in CCH are considered to originate from Langerhans cells (LCs; MARCHAL et al., 1995). They express CD1a, CD11a/CD18, CD11c/CD18, CD44, CD45, MHC class II and high levels of E-cadherin (MOORE et al., 1996; MOORE, 2014).
2.1.3. Histiocytic sarcoma complex
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 (FULMER and MAULDIN, 2007). Localized histiocytic sarcomas originate at a single tissue site or in a single organ whereas disseminated histiocytic sarcomas involve several organs or tissues (MOORE, 2014). The latter form has formerly been termed
“malignant histiocytosis” and was initially observed in Bernese Mountain Dogs (MOORE and ROSIN, 1986; PADGETT et al., 1995). Beside Bernese Mountain Dogs, Rottweilers, Flat- Coated Retrievers, Golden Retrievers, and Pembroke Welsh Corgis are at increased risk for developing the disease (AFFOLTER and MOORE, 2002; IDE et al., 2011).
2.1.3.1. Clinical and pathological features of canine histiocytic sarcomas
Canine histiocytic sarcoma is a highly malignant neoplasm with a rapid and progressive clinical course (MOORE and ROSIN, 1986). The disease has a poor prognosis, even with aggressive therapy (FULMER and MAULDIN, 2007).
The most commonly reported systemic clinical symptoms include: anorexia, weakness, hyperthermia, weight loss and lethargy (ABADIE et al., 2009). Other clinical signs depend upon the organs involved, such as dyspnea, cough, shortness of breath and abnormal pulmonary sound when the lungs are involved and seizures, progressive incoordination and paralysis when the central nervous system (CNS) is affected (MOORE, 2014). In addition,
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mild non regenerative to severe regenerative anemia, thrombocytopenia and other hematologic abnormalities were reported in dogs with HS (ABADIE et al., 2009).
Typically HS in dogs are found in spleen, liver, lymph nodes, lung, bone marrow, central nervous system, skin, subcutis, and in periarticular and articular tissues of the limbs (FULMER and MAULDIN, 2007). Grossly, lesions occur as destructive, infiltratively growing, solitary or multiple masses with a uniform, smooth cut surface and a white/cream to tan color (MOORE, 2014). When joints are involved, lesions occur as multiple tan nodules located beneath the synovial lining (MOORE, 2014).
Microscopically, neoplasms consist 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 cells including lymphocytes and neutrophils (MOORE, 2014).
Localized HS are usually treated using surgery along with radiation therapy compared to systemic chemotherapy which is used for treatment of disseminated HS (FULMER and MAULDIN, 2007). Several chemotherapeutic drugs have been applied for the treatment of HS, such as doxorubicin and paclitaxel (POIRIER et al., 2004). Dogs affected by the localized form have a more favorable prognosis than those suffering from a disseminated histiocytic sarcoma (FULMER and MAULDIN, 2007). However, both localized and disseminated HS are characterized by a highly aggressive clinical behavior and most dogs are euthanized early in the disease course (FULMER and MAULDIN, 2007).
2.1.4. DH82 cells
DH82 cells are initially isolated from a dog with a disseminated histiocytic sarcoma and therefore represent a macrophage/monocytic tumor cell line (WELLMAN et al., 1988). DH82 cells can grow as a loosely adherent monolayer (WELLMAN et al., 1988). In addition, they share many similarities with histiocytic cells in that they possess a round morphology with a diameter ranging from 25-50 µm and abundant basophilic cytoplasm, irregular eosinophilic granules, cytoplasmic vacuoles and pseudopods (WELLMAN et al., 1988). The nuclei are large, round to irregular in shape and eccentrically located with a delicate chromatin pattern and many nuclei contain multiple nucleoli (WELLMAN et al., 1988). Since DH82 cells are derived from a disseminated canine histiocytic sarcoma, this cell line can be used to study the molecular mechanisms of tumor growth, invasion and metastasis in vitro (PUFF et al., 2009;
HEINRICH et al., 2014) and in murine xenograft models of canine histiocytic sarcoma.
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2.2. Matrix Metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent endopeptidases, which have important roles in the responses of cells to their microenvironment (LEE and MURPHY, 2004; LAGENTE et al., 2005). MMPs can be secreted, membrane-bound or intracellularly located (ALI et al., 2012). They have been identified as the major enzymes responsible for extracellular matrix (ECM) turnover and degradation of ECM components such as collagen and proteoglycans (WOESSNER, 1991).
MMPs are found to be produced by many types of cells for example, fibroblasts, keratinocytes, chondrocytes, osteoblasts, in addition to inflammatory cells including monocytes / macrophages, lymphocytes and neutrophils (WESTERMARCK and KAHARI, 1999). In addition to normal cells, MMPs can also be expressed by a large variety of tumor cells (ROOMI et al., 2009). Numerous studies have demonstrated that MMPs can play key roles in many physiological processes, such as embryonic growth and tissue morphogenesis, bone elongation, angiogenesis, ovulation, sperm maturation, uterine involution, mammary gland development, wound healing, tissue repair and remodeling in response to injury (PAGE-MCCAW et al., 2007; MURPHY and NAGASE, 2011). Moreover, impaired expression of MMPs can lead to many pathological diseases such as arthritis, cancer, cirrhosis, multiple sclerosis and cardiovascular disease (WEBSTER and CROWE, 2006;
MURPHY and NAGASE, 2008).
2.2.1. MMP classification
The enzymatic activity of MMPs was first identified by Jerome Gross and Charles Lapiere in 1962 when they demonstrated the enzymatic activity of MMP-1 during the metamorphosis of the tadpole tail (GROSS and LAPIERE, 1962). Currently, 24 different vertebrate MMPs have been identified, of which 23 are found in humans (VISSE and NAGASE, 2003). In addition, a number of nonvertebrate MMPs has been described in hydra (LEONTOVICH et al., 2000), sea urchin (LEPAGE and GACHE, 1990), and Arabidopsis (MAIDMENT et al., 1999).
MMPs can be classified based on the organization of their domains, their ability to recognize and bind specific substrates and the degree of sequence identity (SBARDELLA et al., 2012).
Six groups of MMPs can be distinguished (Tab. 2; SNOEK-VAN BEURDEN and VON DEN HOFF, 2005):
(1.) The collagenase group which includes MMP-1, MMP-8, and MMP-13. These enzymes are able to cleave the interstitial collagens I, II, III and also certain other ECM and non-ECM proteins (VISSE and NAGASE, 2003).
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(2.) The gelatinase group, including MMP-2 and MMP-9, mainly digests gelatin (VISSE and NAGASE, 2003). MMP-2 can also digest type I, II, and III collagens (AIMES and QUIGLEY, 1995; PATTERSON et al., 2001).
(3.) The stromelysins, which consist of MMP-3 and MMP-10, digest ECM components such as collagen IV and fibronectin (VISSE and NAGASE, 2003). MMP-11 known as stromelysin- 3, but its sequence and substrate specificity are different from that of MMP-3 and MMP-10.
Thus, MMP-11 is classified in the heterogeneous subgroup (see subgroup 6) (VISSE and NAGASE, 2003).
(4.) The matrilysins, which consist of MMP-7 and MMP-26, characterized by lacking a hemopexin domain. This group of MMPs can degrade a number of ECM proteins, such as fibronectin and gelatin (BRUSCHI and PINTO, 2013).
(5.) The membrane-type matrix metalloproteinases (MT-MMP) that have been shown to readily degrade a number of ECM proteins such as gelatin, fibronectin, and laminin. Six different membrane-type MMPs have been identified (JONES et al., 2003; VISSE and NAGASE, 2003)
(6.) Other MMPs, which show a different substrate specificity, amino acid sequence, or domain organization, are gathered in a more heterogeneous subgroup. This group includes MMP-12, MMP-19, MMP-20, MMP-21, MMP-23, MMP-27, and MMP-28, which cleave substrates such as elastin and aggrecan (SNOEK-VAN BEURDEN et al., 2005).
2.2.2. MMP structure
MMPs are multi-domain enzymes which show similarity in their domain structure and also sequence homologies (BODE et al., 1999). In general a MMP consists of (1) a propeptide (pro-domain) with a zinc-interacting thiol (SH) group, which contains about 80 amino acids with a N-terminal hydrophobic residue (it plays an important role in the regulation of MMP activity; NAGASE et al., 2006), (2) an amino-terminal signal sequence (Pre) which directs them to the endoplasmic reticulum (EGEBLAD and WERB, 2002) and (3) a catalytic domain with a zinc-binding site (Zn). The latter consists of about 160-170 amino acids and mediates interactions with tissue inhibitors of metalloproteinases, cell-surface molecules and proteolytic substrates (KIM and JOH, 2012). In addition to the above mentioned structures, some MMPs contain a hemopexin-like domain, which is attached to the catalytic domain by a hinge (H) region (EGEBLAD and WERB, 2002). The first and last repeats in the hemopexin- like domain are linked by a disulphide bond (S-S; EGEBLAD and WERB, 2002). Other MMP structural groups contain inserts that resemble collagen-binding type II repeats of
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fibronectin (Fi), a furin-like serine proteinase (Fu) and vitronectine-like inserts (Vn) which are located between their propeptide and catalytic domains (EGEBLAD and WERB, 2002; Fig.
1).
2.2.3. Regulation of MMP activity
The expression of MMPs is usually low in normal tissue under normal conditions and is induced when ECM remodeling is needed (LOFFEK et al., 2011). The biological activity of MMPs is regulated tightly at three levels: (1) gene transcription and synthesis of inactive zymogens, (2) posttranslational activation of zymogens, and (3) interactions of secreted MMPs with TIMPs (BREW et al., 2000).
MMP gene expression is mainly regulated at the transcriptional level (FANJUL- FERNANDEZ et al., 2010). In addition, many of the MMP promoters are similar, in that they share common Cis-elements for the regulation of MMP gene expression, as a result to this, most MMPs are co-expressed in response to multiple stimuli, such as growth factors or inflammatory cytokines (YAN and BOYD, 2007). Several hours after exposure to stimuli, early response genes are induced and encode signaling proteins that phosphorylate different transcription factors, which are later bind to MMP genes promoters (FANJUL-FERNANDEZ et al., 2010). These signaling intermediates involved in activation of transcription factors include nuclear factor kappa B (NF-κB), the mitogen activated protein kinases (MAPK), the signal transducers and activators of transcription (STAT) and the Smad family of proteins (FANJUL-FERNANDEZ et al., 2010).
The second level of MMP regulation is activation from the latent, inactive form (VAN WART and BIRKEDAL-HANSEN, 1990). At this level, the inactive MMP proenzymes or zymogens are activated extracellularly after their secretion by the so-called cysteine switch mechanism of activation, where the interaction between the cysteine residue and the Zn2+ ion is disrupted and this switches the role of zinc from a noncatalytic to a catalytic one (SPRINGMAN et al., 1990; VAN WART and BIRKEDAL-HANSEN, 1990).
Despite the fact that MMPs are initially secreted as zymogens and activated extracellularly following their secretion, some MMPs like MMP-11, MMP-28 and transmembrane MMPs contain an RXK/RR furin-like enzyme recognition motif between their propeptide and the catalytic domain, which allows them to be activated intracellularly before they are secreted (PEI and WEISS, 1995). In addition, most MMPs can be activated by other activated MMPs or by other serine proteases which can cleave peptide bonds within MMP pro-domains (STERNLICHT and WERB, 2001). ProMMP-1 was found to be activated directly by MMP-3
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(NAGASE et al., 1992). MMP-13 can be activated by MT1-MMP and activated MMP-13 can also activate MMP-9 (COWELL et al., 1998; KNAUPER et al., 2002). MMP-3 may also be responsible for the activation of MMP-9 (SOBRIN et al., 2000). MMP-2 can be activated by the membrane-type MMPs through a unique multistep pathway involving MT1-MMP and TIMP-2 (DERYUGINA et al., 2001; FILLMORE et al., 2001).
The third level in the regulation of MMP activity is inhibition of the active enzyme by physiological inhibitors (GOMEZ et al., 1997). Almost all MMPs are inhibited by endogenous tissue inhibitors (TIMPs) which block MMP activity by binding non-covalently to the target, inhibiting functioning of the active site by interacting with the Zn2+ (NAGASE et al., 2006). Differences in the ability to inhibit MMPs are found among TIMPs. TIMP-1 has the ability to inhibit MMP-3, MMP-9 and MMP-1 more effectively than TIMP-2 (O'CONNELL et al., 1994). On the other hand, TIMP-2 can inhibit MMP-2 more effective than TIMP-1 (STETLER-STEVENSON et al., 1989; HOWARD et al., 1991). However, TIMP-2 can mediate pro-MMP-2 activation when the concentration of TIMP-2 is low, whereas a greater concentration of TIMP-2 inhibits MMP-2 (BUTLER et al., 1998). TIMP-3 was found to inhibit both MMP-2 and MMP-9 (BUTLER et al., 1999), whereas TIMP-4 has the ability to inhibit all classes of MMPs (STRATMANN et al., 2001). In addition to TIMPs, other physiological MMP inhibitors have been recognized, such as procollagen C-terminal proteinase enhancer (PCPE), reversion-inducing cysteine-rich protein with Kazal motifs (RECK) which inhibits MMP-9, MMP-2 and MT1-MMP, beta-amyloid precursor protein (APP) and thrombospondin-2 (TSP-2), both proteins can inhibit MMP-2 (MOTT et al., 2000;
OH et al., 2001b; YANG et al., 2001b; HIGASHI and MIYAZAKI, 2003).
12 Fig. 1: Domain structure of the MMPs
(Modified from EGEBLAD and WERB, 2002; POLIMENI and PRATO, 2014)
MMP, matrix metalloproteinase; Pre, signal sequence; Pro, propeptide with a free zinc- ligating thiol (SH) group; Zn, zinc-binding site; Fi, collagen-binding fibronectin type II inserts H, hinge region; Cy, very short cytoplasmic tail; GPI, glycophosphatidyl inositol-anchoring domain; Fu, furin-susceptible site; Vn, vitronectin-like domain; TM, carboxy-terminal single transmembrane domain; Ig, immunoglobulin-like domain.
13 Tab. 2: Members of the MMP family
(modified from SNOEK-VAN BEURDEN and VON DEN HOFF, 2005) Subtype of MMPs MMP no. Substrates
Collagenases
Interstitial collagenase MMP-1 Col I, II, III, VII, VIII, X, gelatin
Neutrophil collagenase MMP-8 Col I, II, III, VII, VIII, X, aggrecan, gelatin Collagenase 3 MMP-13 Col I, II, III, IV, IX, X, XIV, gelatin Gelatinases
Gelatinase A MMP-2 Gelatin, Col I, II, III, IV, VII, X Gelatinase B MMP-9 Gelatin, Col IV, V
Stromelysins
Stromelysin-1 MMP-3 Col II, IV, IX, X, XI, gelatin Stromelysin-2 MMP-10 Col IV, laminin, fibronectin, elastin Stromelysin-3 MMP-11 Col IV, fibronectin, laminin, aggrecan Matrilysins
Matrilysin-1 MMP-7 Fibronectin, laminin, Col IV, gelatin Matrilysin-2 MMP-26 Fibrinogen, fibronectin, gelatin MT-MMP
MT1-MMP MMP-14 Gelatin, fibronectin, laminin MT2-MMP MMP-15 Gelatin, fibronectin, laminin MT3-MMP MMP-16 Gelatin, fibronectin, laminin
MT4-MMP MMP-17 Fibrinogen, fibrin
MT5-MMP MMP-24 Gelatin, fibronectin, laminin
MT6-MMP MMP-25 Gelatin
Others
Macrophage metalloelastase MMP-12 Elastin, fibronectin, Col IV
MMP-19 Aggrecan, elastin, fibrillin, Col IV, gelatin Enamelysin MMP-20 Aggrecan, cartilage oligomeric matrix protein
MMP-21 Aggrecan
MMP-23 Gelatin, casein, fibronectin
CMMP MMP-27 Unknown
Epilysin MMP-28 Unknown
MMP, matrix metalloproteinase; MT-MMP, membrane-type matrix metalloproteinase; Col, collagen; CMMP, chicken MMP
14 2.2.4. TIMPs
Tissue inhibitors of metalloproteinases (TIMPs) are specific endogenous inhibitors of matrix metalloproteinases (BREW and NAGASE, 2010). Therefore, they regulate ECM turnover, and tissue remodeling (BREW and NAGASE, 2010). Four mammalian TIMPs have been characterized (TIMP-1, -2, -3 and -4; BREW and NAGASE, 2010). In 1975 the inhibitory activity of TIMP-1 was first reported in a media of cultured human skin fibroblasts (BAUER et al., 1975).
TIMPs are consisting of 184–194 amino acids and contain two domains (one N-terminal domain of about 125 amino acid residues and a C-terminal domain with about 65 residues) (XU et al., 2011). The conformation of each domain is stabilized by three disulfide bonds (NAGASE et al., 2006). Although the mammalian TIMPs share many basic similarities, they exhibit distinctive structural features, biochemical properties and expression patterns and this may contribute to the different roles of TIMPs in vivo (BAKER et al., 2002).
2.2.5. Role of MMPs and TIMPs in tumor progression
In several studies MMPs are strongly implicated in cancer progression and MMP-mediated degradation of ECM is one of the principal alterations observed in the cancer microenvironment (GIALELI et al., 2014). The strong association of MMP expression with neoplastic progression is well documented in both in vitro and in vivo systems (JACOB and PREKERIS, 2015). In many animal transplantation assays, tumor cells increase their invasive behavior and metastatic rate when MMP expression is up-regulated. In contrast, down- regulation of MMPs reduces the aggressive behavior of tumor cells (COUSSENS and WERB, 1996; EGEBLAD and WERB, 2002). Moreover, it has been found that the expression of MMPs is increased in many types of cancers and their expression correlates with advanced tumor stage and increased invasion and metastasis (COUSSENS and WERB, 1996;
EGEBLAD and WERB, 2002). A negative association between MMP expression and tumor prognosis has been found in most studies (EGEBLAD and WERB, 2002). However, there are a few reports in which increased expression of specific MMPs reflects a favorable prognosis;
for example, MMP-12, also referred as human macrophage metalloelastase (HME), has been shown to play an important role in the inhibition of tumor progression and its overexpression is associated with a better prognosis in patients with colorectal carcinoma (YANG et al., 2001a). This can be attributed to the ability of MMP-12 to convert plasminogen into angiostatin, an inhibitor of tumor angiogenesis (SHI et al., 2006).
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MMPs are not only involved in the invasion and metastasis of neoplastic cells, but are also implicated in several steps of cancer development, for example regulating growth and survival of tumor cells, promoting or inhibiting angiogenesis, promoting the epithelial-to- mesenchymal transition and regulating the immune response against neoplastic cells (EGEBLAD and WERB, 2002).
MMP-9 knockout mice exhibited a reduced tumor cell proliferation and invasion compared to wild-type animals (COUSSENS et al., 2000). There are three main mechanisms through which MMPs can promote tumor cell proliferation. First through releasing precursors of growth factors, for example, TGF-α (PESCHON et al., 1998), second, through liberating peptide growth factors like insulin-like growth factor which is released under the influence of MMP-9 (MANES et al., 1999) and third, through ECM remodeling which may cause the expression of cell receptors like alpha v beta integrin 6 (αvß6), which enhances the proliferation of a human colon carcinoma cell line (AGREZ et al., 1994; Fig 2 a).
MMPs like MMP-3, -7, -9 and -11 have the ability to regulate apoptosis (EGEBLAD and WERB, 2002). MMP-11, expressed by fibroblastic cells surrounding tumor cells, was found to increase tumorigenesis by decreasing tumor cell death through regulating both necrotic and apoptotic processes (BOULAY et al., 2001). Moreover, MMP-7 can promote tumor cell survival by cleaving Fas (apoptosis stimulating fragment) ligand and reducing its effectiveness in triggering Fas-mediated apoptosis (MITSIADES et al., 2001). On the other hand, an increase in the number of apoptotic cells was detected in MMP-3 transgenic mice with mammary tumors compared to control animals (WITTY et al., 1995).
The functions of TIMPs are not restricted to MMP inhibition. Numerous studies report a wide variety of other functions. Moreover, conflicting data of the role of TIMPs in tumor progression have been shown (HENRIET et al., 1999). Some studies demonstrate an increase of TIMP-1, -2 and -3 expression in malignant tumor tissue compared to the non-malignant counterpart and a low expression of TIMP-1 was associated with an increased tumor grade (YOSHIJI et al., 1996; HEWITT et al., 2000; SOGAWA et al., 2003; AABERG-JESSEN et al., 2009; KORNFELD et al., 2011). However, other studies showed that downregulation of TIMP-1 and TIMP-2 leads to increase tumor invasiveness compared to overexpression which leads to reduce tumor growth and metastasis (KHOKHA et al., 1989; ALBINI et al., 1991;
DECLERCK et al., 1992). The negative association between TIMP expression and tumor progression can be attributed to the known inhibiting effect of TIMPs on MMPs, on the other hand, the positive association between TIMP expression and tumor progression can be attributed to (1) the growth factor like activity of specific TIMPs, such as TIMP-1 on a variety
16
of normal and neoplastic cell types (HAYAKAWA et al., 1992), (2) its positive role in angiogenesis through the up-regulation of vascular endothelial growth factor (VEGF) expression (YOSHIJI et al., 1998) and (3) the role of TIMP-2 in the activation of MMP-2 (KINOSHITA et al., 1998).
2.2.6. Role of MMPs and TIMPs in invasion and metastasis
Metastases are the major cause of death from cancer whereas local lesions due to the primary tumor are a minor issue (YOON et al., 2003). In addition, studies performed over the last decades reveal the pivotal role of MMPs in tumor metastasis (WESTERMARCK and KAHARI, 1999).
The first evidence for a role of MMPs in metastasis came from the antimetastatic effect of TIMPs on murine melanoma cells (SCHULTZ et al., 1988). Among the previously reported human MMPs, MMP-2 and MMP-9 are the key proteins involved in tumor invasion and metastasis and they are abundantly expressed in various malignant tumors (JOHNSEN et al., 1998). Metastasis is an extremely complex process and involves detachment of the cell from the neoplastic tissue, invasion of the surrounding stroma and spread through blood vessels or lymphatics (EGEBLAD and WERB, 2002). Cleavage of laminin-5 (ln-5), an extracellular matrix substrate for cell adhesion found in epithelial basement membranes, by MMP-14 and MMP-2 can trigger cell migration and thus may play a role in the early phases of tumor invasion (KOSHIKAWA et al., 2000). MMP-14 promotes cell migration through the cleavage of CD44, a major receptor for hyaluronan (KAJITA et al., 2001). Moreover, CD44 also binds MMP-9 on the surface of mouse mammary carcinoma and human melanoma cells. This localization promotes collagen IV degradation and mediates tumor cell invasion (YU and STAMENKOVIC, 1999). MMPs have also important roles in the late events of metastasis which involve the intravasation into blood vessels or lymphatics, extravasation and establishment of a secondary growth site (CHAMBERS and MATRISIAN, 1997). MMP-9 is required for the intravasation of tumor cells and MMP-14 enhances the survival rate of tumor cells in an experimental pulmonary metastasis assay (TSUNEZUKA et al., 1996; KIM et al., 1998) (Fig.2 b).
2.2.7. Angiogenesis and MMPs / TIMPs
Pathological angiogenesis is a hallmark of cancer and it is necessary for persistent tumor growth, because it provides the tumor cells with the necessary gas exchange and nutrients (CHANG and WERB, 2001). Many studies demonstrate the significant role of MMPs in the
17
induction of angiogenesis by targeting these MMPs using endogenous or synthetic MMP inhibitors (MARTIN et al., 1999; LI et al., 2001; OH et al., 2001a; RODRIGUEZ- MANZANEQUE et al., 2001; YOSHIDA et al., 2002). In addition, MMP-14- and MMP-9- deficient mice have severe defects in angiogenesis, demonstrating that MMP-14 and MMP-9 are implicated directly in the regulation of angiogenesis (VU et al., 1998; ZHOU et al., 2000).
MMPs may contribute in the angiogenesis process through the cleavage of individual matrix components, such as type I collagen (SEANDEL et al., 2001). In a rat corneal micropocket angiogenesis assay, cleavage of collagen type IV results in the exposure of an important cryptic site within its triple helical structure and blockage of this site disrupts integrin- dependent endothelial cell interaction and inhibits angiogenesis (XU et al., 2001). Another possibility is that MMPs, specifically MMP-9, are important for angiogenesis by releasing pro-angiogenic growth factors, such as VEGF during carcinogenesis of pancreatic islets in mice (BERGERS et al., 2000). Using an in vitro wound healing migration model, MMP-14 has been shown to play an important role in angiogenesis through regulating endothelial migration, invasion and formation of capillary tubes during angiogenic response and blocking the catalytic domain of MMP-14 inhibits these processes (GALVEZ et al., 2001). Moreover, MMP-14 can regulate neovascularization by acting as a pericellular fibrinolysin, thereby potentially allowing endothelial cells to invade further into the tumor tissue (HIRAOKA et al., 1998) (Fig. 2 c).
On the other hand, MMPs, specifically MMP-2, MMP-9 and MMP-12, may act anti- angiogenetically through cleavage of plasminogen to generate angiostatin which inhibits endothelial cell proliferation and thus diminishes metastatic tumor cell growth (DONG et al., 1997; CORNELIUS et al., 1998). In murine hemangioendothelioma cell cultures, MMP-3, MMP-9, MMP-13 and MMP-20 were found to have important roles in the generation of endostatin, a C-terminal fragment of the basement membrane collagen type XVIII, which is able to inhibit endothelial cell proliferation, migration in addition to angiogenesis and tumor growth (FERRERAS et al., 2000) (Fig. 2 c).
The TIMP family plays an important role in angiogenesis (SANG, 1998). TIMPs can prevent angiogenesis by the inhibition of MMPs (HANDSLEY and EDWARDS, 2005). Another possibility is the activation of MMP inhibitors, such as RECK, which in turn, inhibits angiogenesis (OH et al., 2004). In addition, TIMPs exhibit anti-angiogenic activity that is independent of their MMP inhibitory activities. Using in vitro cell culture and a syngeneic murine tumor model of colon cancer, the over-expression of TIMP-2 can inhibit tumor growth and angiogenesis by up-regulating mitogen-activated protein kinase phosphatase 1 which
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dephosphorylates p38 mitogen-activated protein kinase, a molecule involved in endothelial cell proliferation and migration (FELDMAN et al., 2004). Moreover, TIMP-1 inhibits endothelial cell invasion of human amniotic membranes (MIGNATTI et al., 1986). A TIMP- related protein isolated from bovine articular cartilage blocks endothelial cell proliferation and angiogenesis (MOSES et al., 1990). In addition, TIMP-1 and TIMP-2 inhibit chick yolk sac vessel morphogenesis in response to polyamines (TAKIGAWA et al., 1990).
TIMPs may also enhance tumor angiogenesis by inhibiting MMPs which play a critical role in the generation of potent inhibitors of angiogenesis. For example MMP-12 can generate angiostatin by cancer-mediated proteolysis of plasminogen (CORNELIUS et al., 1998). In addition, other members of the MMP family, such as MMP-3, -7, -9, -13, -20 can release endostatin, another well-known anti-angiogenic factor from collagen XVIII (HELJASVAARA et al., 2005). Therefore, by inhibiting these MMPs, TIMPs may enhance tumor angiogenesis (CORNELIUS et al., 1998; WEN et al., 1999). Supporting this idea, mammary carcinoma over-expressing TIMP-1, showed enhanced VEGF expression and enhanced growth rate (YOSHIJI et al., 1998).
19 Fig. 2: Role of MMPs in tumor progression (modified from EGEBLAD and WERB, 2002)
a. Matrix metalloproteinases (MMPs) promote tumor growth by cleaving insulin-growth- factor-binding protein (IGF-BP), thereby liberating IGF, by shedding precursors of growth factors including transforming growth factor-α (TGF-α) and by regulating extracellular matrix (ECM), which promotes growth indirectly through interactions between ECM molecules and integrins.
b. MMPs promote invasion and migration by degrading structural ECM components, such as laminin 5 (Lam-5). In addition, MMPs promote cell migration through cleavage of CD44.
c. MMPs promote angiogenesis by increasing the bioavailability of pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF). In addition, MMPs promote invasion of endothelial cells by cleaving structural components of the ECM, such as collagen types I (Col-I) and IV (Col-IV) and fibrin. Cleaved Col-IV acts pro- angiogenetically by binding to αvβ3 integrin. MMPs may act anti-angiogenetically through cleavage of plasminogen and Col-XVIII resulting in the generation of anti-angiogenic factors such as angiostatin and endostatin.
20 2.3. Inflammation and cancer
In 1863, Rudolf Virchow was the first to describe the association of inflammation and cancer when he noted leukocytes in neoplastic tissue and suggested that the ‘‘lymphoreticular infiltrate’’ reflected the origin of cancer at sites of chronic inflammation (VIDAL- VANACLOCHA, 2009). In addition, previous epidemiological studies in humans have shown that chronic inflammation is a cofactor in many carcinogeneses, such as gastric cancer and gastric mucosal lymphoma which is linked to Helicobacter pylori, and other malignancies caused by chemical and physical agents and autoimmune reactions (EKBOM et al., 1990;
MANTOVANI et al., 2008).
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
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(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 anti-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
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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
25
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
26
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,
27
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.
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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 Baumgärtner1,*, 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
