Ejecución instrumental Compromiso directo e indirecto con la música

Carcinogenesis is driven by a multitude of factors with an increasing recognition of the critical role of inflammation on numerous processes in aiding cancer progression (86). Studies have reported the use of markers of systemic inflammatory response such as CRP, albumin, GPS, and NLR to predict patient clinical outcomes and response to chemotherapy. Patients with elevated markers of systemic inflammation were found to have poor chemotherapy response and reduced OS (104, 113). It is still not completely understood why patients with elevated systemic inflammation have poor clinical outcomes.

There is increasing evidence which supports the relationship between local immune response within the tumour microenvironment and systemic inflammation (68). The crosstalk between these two systems is mediated by a complex plethora of soluble mediators produced by the tumour microenvironment and secreted into the systemic circulation such as cytokines, chemokines, growth factors and matrix-degrading enzymes and a host of immune and stromal cells. Inflammatory mediators, such as IL-6, initiate the systemic inflammatory response and ECM remodelling to orchestrate tumour growth, progression, invasion, angiogenesis and metastasis and educate recruited immune cells for immune evasion within the tumour microenvironment (64-67, 77). Thus, understanding the relationships of soluble mediators released during advanced stages of carcinogenesis may potentially lead to greater knowledge of the fate of a malignant cell and assist in predicting the clinical outcomes of patients with mCRC.

Numerous studies support the importance of the ECM in the tumour microenvironment, not only for regulating cellular and structural functions directly, but acting as inflammatory stimuli and chemo-attractants for immune cells via degraded fragments of ECM proteins (72, 76). In cancer, deregulation of ECM dynamics is a vicious cycle controlled by a tight-network of protein-protein interactions, proteolytic enzymes such as MMPs and their endogenous

Chapter 2 Investigation of plasma fibulin-1 as a prognostic marker in mCRC patients

Page | 54 inhibitors, TIMPs, and cell surface receptors called integrins (71, 74). ECM remodelling involves two actions: 1) synthesis and deposition of components of the ECM and 2) degradation of the ECM. Increased activity in either action is indicative of highly invasive tumours and poorer patient survival (72). There is also evidence that highlights the role of ECM organisation and composition within the tumour microenvironment in influencing drug concentration and sensitivity of chemotherapy agents on tumour cells via the interaction of cell adhesion

molecules and integrins with components of the ECM (85, 181). Despite the role the ECM plays in carcinogenesis and chemotherapy resistance, there are no ECM proteins that are used clinically to predict prognosis and chemotherapy response in patients with CRC.

Fibulin-1 is a critical ECM component located within the stromal compartment of tissues and secreted as a plasma glycoprotein (135). Due to its ability to interact with various ECM

components, such as fibronectin, elastic fibers, angiogenin and laminin-1, fibulin-1 has a range of functional roles that influence cellular communication and behaviour and regulate ECM structural dynamics within the tumour microenvironment. Changes in the cellular expression of fibulin-1 in tumour cells has been shown to increase apoptosis and inhibit cell proliferation, adhesion, migration, invasion and angiogenesis (142, 151, 154, 161, 179).

In CRC, a reduction of tumour fibulin-1 expression has been observed in comparison to adjacent normal mucosal tissue in patients (153). Fibulin-1 expression was significantly reduced in more advanced stages of CRC and associated with increased tumour cell

proliferation (153). Cellular expression of fibulin-1 in CRC tumour cells was also found to be regulated by the inflammatory chemokine, CXCL1 (153). Repression of CXCL1 using siRNA increased the expression of fibulin-1 and inhibited the growth of CRC cell line HCT-15 in vitro. Based on these finding, it may be hypothesized that in CRC patients with inflammation, fibulin- 1 is reduced leading to enhanced tumour proliferation and poorer clinical outcomes. Other

Page | 55 studies investigating hepatocellular, cutaneous melanoma and bladder cancer have shown that reduced fibulin-1 expression is associated with poorer survival (147-149).

Whilst the use of immunohistochemistry (IHC) has been the recommended standard method for examining clinical pathological biological specimens, they only provide a small glimpse into the local tumour microenvironment at one static time point, i.e. at diagnosis, and little

information about the temporal changes or secreted versions involved during carcinogenesis. Plasma is easily accessible, can provide a snap shot of a disease state in the body at any given point in time and accessed serially for longitudinal monitoring of proteins (182, 183).

Interestingly, fibulin-1 is one of the few ECM proteins found to be secreted at relatively high levels within the blood (132, 133); although, very little is known about the origin and clinical significance of fibulin-1 in the circulation. Increased fibulin-1 levels in the blood have been found in patients with respiratory diseases such as asthma and idiopathic pulmonary fibrosis, chronic kidney disease and type 2 diabetes (132, 133, 184-186). Recently, plasma fibulin-1 has been implicated in dysfunction of smooth muscle in asthma, and identified as a potential biomarker of renal impairment and arterial ECM remodelling in patients with type 2 diabetes related to arterial stiffness (132, 133, 185, 186). An increase in plasma fibulin-1 in diabetes patients was also predictive of higher overall mortality and cardiovascular mortality (132). These studies provide evidence of the role of fibulin-1 in ECM remodelling and as a prognostic marker.

There are limited studies which have investigated the expression and biological function of secreted fibulin-1 in carcinogenesis. Secreted fibulin-1 expression has only been investigated in one study which found secreted fibulin-1 expression was increased in ovarian cancer cells in the presence of oestradiol (156). Overexpression of fibulin-1 in CM of ACHN renal cancer cells was found to reduce tube formation in vitro, only to be restored by pre-treatment with anti- fibulin-1 antibody (151).

Chapter 2 Investigation of plasma fibulin-1 as a prognostic marker in mCRC patients

Page | 56 The prognostic value of plasma fibulin-1 in predicting chemotherapy response and clinical outcomes in CRC patients is still not known. Therefore, the development of a highly sensitive plasma-based assay is required to allow for the detection of fibulin-1 and determine its prognostic application in the clinic.

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