Cancer is considered as one of the leading causes of death among humans and small animals causing about 20% of deaths in dogs at 5 years of age and increases to approximately 40% to 50% in individuals between 10 to 16 years of age (BRONSON, 1982; ADAMS et al., 2010;
JEMAL et al., 2011). Canine histiocytic sarcoma (HS) is a highly aggressive neoplastic disease affecting dogs with a high incidence in Bernese mountain dogs, Rottweilers, Retrievers and Pembroke Welsh Corgis breeds (KRAJE et al., 2001; AFFOLTER and MOORE, 2002; IDE et al., 2011). Canine HS belongs to the category of canine histiocytic proliferative disorders and is characterized by infiltration of neoplastic cells arising from interstitial dendritic cells (FULMER and MAULDIN, 2007; ABADIE et al., 2009; MOORE, 2014). Canine HS can occur in two forms; localized which involves predominantly the skin and subcutis of the extremities, and disseminated, which typically occurs in multiple internal organs such as spleen, lung and lymph nodes (MOORE, 2014).
Disseminated HS show a rapidly progressive clinical course with poor prognosis and affected dogs are often euthanized early in the disease course due to limitations in therapeutic options (AFFOLTER and MOORE, 2002). Although surgical resection is beneficial in some cases of localized HS, the efficacy of surgery and chemotherapy is still limited in cases of disseminated HS (TAKAHASHI et al., 2014). The main reason for the poor prognosis of disseminated HS are distant metastases into internal organs (FULMER and MAULDIN, 2007). A better understanding of biological mechanisms involved in cancer progression and metastasis may be beneficial for exploration of new effective therapies to prevent metastasis and to improve the survival time of patients (WAN et al., 2013).
Besides that, canine HS are considered to occur more frequently in some canine breeds compared to histiocytic malignancies in humans where they represent rare but very aggressive diseases (FAVARA et al., 1997; MOORE, 2014). Both diseases share many pathological features and are histologically comparable (AFFOLTER and MOORE, 2002). Thus canine HS may represent a good translational animal model and may provide a unique opportunity to understand some aspects in the pathogenesis of the human disease (HEDAN et al., 2011).
Role of MMPs and their inhibitors in canine histiocytic sarcomas and xenotransplanted histiocytic sarcomas
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases which are mainly known for their major role in extracellular matrix (ECM) turnover and degradation of ECM components (WOESSNER, 1991). In many previous in vitro and in vivo studies, MMPs
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have been strongly implicated in cancer progression (JACOB and PREKERIS, 2015). In addition, up-regulation of MMPs was found to be associated with increased tumor cell invasion and metastatic rate (EGEBLAD and WERB, 2002). Moreover, increased MMP expression is observed in almost every type of human cancer and this correlates with advanced tumor stage, invasion and metastatic rate (COUSSENS and WERB, 1996;
EGEBLAD and WERB, 2002).
Canine HS especially the disseminated from of the disease are very aggressive tumors with a high invasiveness and metastatic rate (AFFOLTER and MOORE, 2002). Therefore, MMPs and their inhibitors might play an important role in the progression of this disease. In the present study the expression of selected MMPs and their inhibitors, namely MMP-2, -9, -14 and TIMP-1 was studied in spontaneous canine HS and compared to a mouse model of subcutaneously xenotransplanted canine HS. Both spontaneous neoplasms and subcutaneously xenotransplanted tumors displayed a high expression of MMP-2, -9 and -14 at the invasive front of the tumor compared to the tumor center. Similar results were found for the expression of MMP-2 and MMP-9 in human head and neck squamous cell carcinomas (ONDRUSCHKA et al., 2002).
In contrast to MMPs, conflicting data were reported for the role of TIMP-1 in tumor progression as TIMP-1 showed to possess both, MMP inhibiting activity as well as tumor promoting activity independent of its MMP-regulatory function (HORNEBECK et al., 2005).
Therefore it is difficult to predict the importance of a TIMP-1 expression on tumor progression and invasion. However, in the present study, TIMP-1 expression was increased at the invasive front of both spontaneous neoplasms and subcutaneously xenotransplanted canine HS. Similar to these results, an increased expression of TIMP-1 was reported in colorectal cancer and was associated with poor prognosis in laryngeal squamous cell carcinoma patients (KAHLERT et al., 2008; MA et al., 2014). Hence, further studies are needed to address this paradox in the role of TIMP-1 in tumor progression.
MMPs promote the process of invasion and metastasis through cleavage of extracellular matrix proteins thus triggering tumor cell migration in the early phases of tumor invasion and also in the late phases through facilitating the extravasation of neoplastic cells and establishment of secondary growth sites (CHAMBERS and MATRISIAN, 1997;
KOSHIKAWA et al., 2000; EGEBLAD and WERB, 2002). A high expression of MMP-2, -9 and -14 was found in both spontaneous canine HS and subcutaneously xenotransplanted neoplasms in the present study. Therefore MMPs might represent one main factor contributing to the aggressive behavior of this tumor type. Unfortunately, due to lacking
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clinical data and unknown survival time of dogs with spontaneous HS it was not possible to determine the prognostic significance of MMPs in the present study.
An additional mechanism through which MMPs might promote tumor progression and metastasis in HS is regulating of angiogenesis.
Supporting this idea, MMPs were able to regulate angiogenesis, either through increasing the bioavailability of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) or through degradation of the ECM to facilitate endothelial cell migration and invasion (BERGERS et al., 2000; RODRIGUEZ-MANZANEQUE et al., 2001). To test the possible role of MMPs in the regulation of angiogenesis in HS, micovessel density in subcutaneously xenotransplanted HS was correlated to MMP expression. Interestingly a positive correlation between MMP-14 expression and micovessel density was found. Therefore, MMP-14 might have a direct impact in promoting angiogenesis and consequently might be one essential factor determining the aggressive behavior of canine HS.
Role of inflammation in canine histiocytic sarcomas and xenotransplanted histiocytic sarcomas
Similar to MMPs, inflammation has been shown to play a critical role in tumor progression and development and inflammatory cells within the tumor microenvironment contribute to nearly every aspect of tumor evolution (COUSSENS and WERB, 2002; GRIVENNIKOV et al., 2010). Tumor infiltrating inflammatory cells include a wide range of cell types, such as macrophages, dendritic cells, neutrophils, eosinophils and lymphocytes (MACARTHUR et al., 2004). Such inflammatory cells can either promote or suppress tumor progression through production of cytokines, MMPs and other mediators (COUSSENS and WERB, 2002). In addition, tumor associated macrophages (TAM) often represent a large part of the tumor mass and are considered as the major link between tumors and an inflammatory response (SIVEEN and KUTTAN, 2009). These cells can produce a variety of growth factors which can stimulate tumor cell growth or promote angiogenesis (POLLARD, 2004; SIVEEN and KUTTAN, 2009; SOLINAS et al., 2009). Therefore, a detailed inflammatory cell profile may provide important information about tumor progression. Previous studies have reported marked inflammatory infiltrates within canine HS tumors (CONSTANTINO-CASAS et al., 2011; PAZDZIOR-CZAPULA et al., 2015). However, a detailed inflammatory cell profile is lacking so far. The present study demonstrated a predominance of macrophages and T lymphocytes infiltrating spontaneous HS whereas B lymphocytes were only present in low numbers. A similar predominance of infiltrating macrophages was found in xenotransplanted
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HS. In addition, few B lymphocytes and some T lymphocytes were observed as single scattered cells or small aggregates, which presumably are inactive.
Interestingly, the present study demonstrated a high expression of MMP-9, -14 and TIMP-1 in tumor associated macrophages in xenotransplanted HS. Supporting this finding, a high expression of MMP-9 in tumor associated macrophages in hepatocellular carcinoma and lungs tumors (RODERFELD et al., 2010; WANG et al., 2011). Therefore, TAM are an important source of MMPs and TIMP-1 and might therefore promote tumor cell invasion and tumor progression through the influence on tumor microenvironment (HAO et al., 2012).
However TAM represent a large heterogeneous cell population which renders precise predictions about their function difficult in individual tumor types and developmental stages.
One possibility to predict the impact of TAM on tumor progression is determination of their polarity since M1 TAM are known to suppress and M2 TAM to favor tumor growth in many cancer types (MANTOVANI et al., 2002; SOLINAS et al., 2009). This phenotype change occurs in response to cytokines and other mediators (MANTOVANI et al., 2002; VERRECK et al., 2004). It is now generally accepted that TAMs mostly resemble the M2 phenotype (SOLINAS et al., 2009). This macrophage polarization during tumor progression may represent an attractive candidate for therapeutic intervention. Therefore, it seems valuable to investigate the polarity of TAMs in canine HS using xenotransplanted HS as an animal model under standardized conditions.
Summarized, the present study demonstrates a pivotal role of MMPs in HS progression.
Therefore, the use of MMP inhibitors is assumed to be an effective potential therapy to control tumor progression and metastases. However, the use of MMP inhibitors apart from the immune system may lead to disappointing results as matrix degradation is necessary for immune surveillance against tumor cells. In consequence, further studies are needed to clarify the involvement of MMPs and their inhibitors as well as the immune system at different stages of tumor progression.
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