**4.2 CXCL-8/IL-8**

A recent paper examined the presence of cytokines, chemokines and their receptors in colon cancer using a Taqman Low Density Array with probes for 24 ligands and 17 receptors. This revealed CCL3, CCL4 and CXCL8 levels to be significantly increased in colon cancer samples compared to normal tissue (Erreni, 2009). Further analysis of the levels of CCL3, CCL4 and CXCL8 mRNA expression showed that CCL4 and CCL3 levels were higher in normal and tumour tissue. However, CXCL8 expression was significantly increased in tumour tissue (p<0.0001). There was no correlation with tumour stage for all three inflammatory markers. SW620 and HT29 cell lines showed low but detectable levels of CXCL8 which increased with stimulation with TNFα and TGFβ. Interestingly there was high levels of two anti-angiogenic chemokines found in these samples also, CXCL9 and CXCL10 but their receptor was not significantly expressed.

The main function of CXCL8 is the recruitment of neutrophils. These leucocytes have a short life span and have no presence or role in the tumour micro-environment. CXCL8 has however been reported to have a role in the tumour micro-environment by stimulating tumour advancement and invasion by stimulating the process of neoangiogenesis and by activating tumour matrix proteases (Zhu, 2004; Bates, 2004). However, CCL3 and CCL4 are not products of TLR3 and TLR4 signalling, but CXCL8 is produced as a result of signalling by TLR3 and TLR4. Elevated levels of CXCL8 or Interleukin-8 has been associated with increased angiogenesis in normal and transformed tissue adjacent to colon cancer tumours (Fox, 1998; Kuniyasi, 2000). Also, increased CXCL8 protein expression in primary colo-rectal tumours increases the risk of metastasis (Haraguchi, 2002). To date CXCL8 has two known receptors, CXCR1 and CXCR2. These receptors are known broadly as G-protein coupled receptors (GPCR). Recently GPCR antagonists have been developed which can block the biological effects of GPCR signalling. [D-Arg1, D-Trp5,7,9,Leu11]SP or substance P antagonist is a potent GPCR antagonist and has been shown to inhibit small cell lung cancer cell proliferation both in vitro and in vivo (Seckl, 1997) and in pancreatic cancer cell proliferation in vitro and in vivo (Guha, 2005). The GPCR ligand/receptor interaction has been shown to result in neuropeptide-induced Ca2+ mobilisation which increases intra-cellular Ca2+ and this proves useful as a marker of GPCR function (Ryder, 2001). A GPCR antagonist has previously been shown to prevent GPCR agonist induced increase in Ca2+, DNA synthesis and anchorage independent growth in pancreatic cancer cells (Guha, 2005). The role of a GPCR antagonist in colon cancer has yet to be established. IL-8 is produced and has been shown to be produced by tumour cells and the level of its production correlates directly with the metastatic potential of the tumour (Abdollahi, 2003; Bruserud, 2004).

#### **5. Immuno-surveillance**

398 Advances in Cancer Therapy

Both TLR3 and TLR4 signalling produce a pro-inflammatory response including chemokines and it has previously been shown that the pro-inflammatory response may contribute to tumorigenesis (Kelly, 2006). Chemokines and cytokines are markers of inflammation which normally recruit leucocytes and aid in blood vessel remodelling. Pathophysiological roles for chemokines include acting as autocrine growth factors, disruption of basement membranes, increasing motility and tumour invasion (Rollins, 2006;

A recent paper examined the presence of cytokines, chemokines and their receptors in colon cancer using a Taqman Low Density Array with probes for 24 ligands and 17 receptors. This revealed CCL3, CCL4 and CXCL8 levels to be significantly increased in colon cancer samples compared to normal tissue (Erreni, 2009). Further analysis of the levels of CCL3, CCL4 and CXCL8 mRNA expression showed that CCL4 and CCL3 levels were higher in normal and tumour tissue. However, CXCL8 expression was significantly increased in tumour tissue (p<0.0001). There was no correlation with tumour stage for all three inflammatory markers. SW620 and HT29 cell lines showed low but detectable levels of CXCL8 which increased with stimulation with TNFα and TGFβ. Interestingly there was high levels of two anti-angiogenic chemokines found in these samples also, CXCL9 and

The main function of CXCL8 is the recruitment of neutrophils. These leucocytes have a short life span and have no presence or role in the tumour micro-environment. CXCL8 has however been reported to have a role in the tumour micro-environment by stimulating tumour advancement and invasion by stimulating the process of neoangiogenesis and by activating tumour matrix proteases (Zhu, 2004; Bates, 2004). However, CCL3 and CCL4 are not products of TLR3 and TLR4 signalling, but CXCL8 is produced as a result of signalling by TLR3 and TLR4. Elevated levels of CXCL8 or Interleukin-8 has been associated with increased angiogenesis in normal and transformed tissue adjacent to colon cancer tumours (Fox, 1998; Kuniyasi, 2000). Also, increased CXCL8 protein expression in primary colo-rectal tumours increases the risk of metastasis (Haraguchi, 2002). To date CXCL8 has two known receptors, CXCR1 and CXCR2. These receptors are known broadly as G-protein coupled receptors (GPCR). Recently GPCR antagonists have been developed which can block the biological effects of GPCR signalling. [D-Arg1, D-Trp5,7,9,Leu11]SP or substance P antagonist is a potent GPCR antagonist and has been shown to inhibit small cell lung cancer cell proliferation both in vitro and in vivo (Seckl, 1997) and in pancreatic cancer cell proliferation in vitro and in vivo (Guha, 2005). The GPCR ligand/receptor interaction has been shown to result in neuropeptide-induced Ca2+ mobilisation which increases intra-cellular Ca2+ and this proves useful as a marker of GPCR function (Ryder, 2001). A GPCR antagonist has previously been shown to prevent GPCR agonist induced increase in Ca2+, DNA synthesis and anchorage independent growth in pancreatic cancer cells (Guha, 2005). The role of a GPCR antagonist in colon cancer has yet to be established. IL-8 is produced and has been shown to be produced by tumour cells and the level of its production correlates directly

with the metastatic potential of the tumour (Abdollahi, 2003; Bruserud, 2004).

**4. Role of chemokines and cytokines** 

CXCL10 but their receptor was not significantly expressed.

**4.1 Overview** 

Balkwill 2004).

**4.2 CXCL-8/IL-8** 

The interaction between cancer cells and the innate and adaptive immune system is complex. As described previously, pro-inflammatory cytokines promote tumour growth; however the immune system has a means of restricting cancer development through immuno-surveillance and immuno-editing. The pro-tumorigenic effect of the inflammatory immune response appears generally to be a consequence of the innate immune system, whereas the adaptive immune system exerts anti-tumorigenic effects (Karin, 2005).

Cancer immunosurveillance is a theory formulated in 1957 by Burnet and Thomas, who proposed that lymphocytes act as sentinels in recognising and eliminating continuously arising transformed cells (Dunn, 2002; Burnet, 1957). Cancer immunosurveillance appears to be an important host protection process that inhibits carcinogenesis and maintains regular cellular homeostasis (Kim 2007). It has been shown that when mice are deficient in genes that encode for cells integral to the adaptive immune response such as T cells, B cells and natural killer T (NKT) cells, tumour incidence and tumour growth are increased (Shankaran, 2001). With the aid of additional studies, natural killer cells and T cells now appear to be the dominant cells involved in tumour immuno-surveillance. Interestingly, interferon-γ (IFN-γ) is produced by both NKT and T cells. Furthermore, IFN-γ is the most influential cytokine released by these cells. Recent studies have now shown that mice deficient in IFN-γ are more susceptible to spontaneous carcinogenesis (Shankaran, 2001).

The mechanism involved in the anticancer protective effect of the immune system is complex. Generally, it is theorised that cells of the immune system recognise the presence of a growing tumour which has undergone stromal remodelling, causing local tissue damage. This is followed by the induction of inflammatory signals which recruits natural killer cells, NKT cells and macrophages to the tumour site. During this phase, the infiltrating lymphocytes such as the natural killer cells and NKT cells are stimulated to produce IFN-γ. Newly synthesised IFN-γ then induces tumour death as well as promoting the production of chemokines which play an important role in promoting tumour death by blocking the formation of new blood vessels.

Similarly, IL-4 is released in large quantities by NKT cells upon activation and is a key regulator in the adaptive immunity. Therapeutic attempts to recruit the immune system to curtail cancer growth have now become a keen area of interest. Although, attempts have met with limited success thus far, this is the basis of vaccine guided anti-cancer therapy which is a rapidly progressing area of research (Dranoff, 2004).

It is clear that elements of the immune system can restrict tumour growth. However, as discussed previously pro-inflammatory cytokine release initiated by the immune system can also promote tumour growth. Therefore, the immune system appears to have the effect of a double edged sword on cancer growth. If we had full understanding and control of these pathways, this could potentially lead to successful cures for cancer.

#### **5.1 Vaccines**

Recently vaccines have been developed to take advantage of the role played by the immune system in cancer. The discovery of tumour associated antigens (TAA) expressed by colorectal carcinoma as well as recent advances in tumour immunology are providing new focus to develop biologically targeted immunotherapeutic strategies. It has been reported that immuno-deficient animals as well as humans, e.g. transplant patients, are at greater risk of developing malignancy (Penn, 2000). It has also been reported that cytotoxic T

The Role of Inflammation in Cancer 401

intervention in these patients is thought not only to accelerate their progression but also to be responsible for activating dormant micrometastases that may have remained inactive in the absence of surgery. In addition, the extent of the surgery is proportional to the postoperative recurrence rate. This is evidenced by the meta-analyses that show that minimally invasive surgery is more effective than open surgery in improving tumour recurrence, and cancer-related survival (Hensler, 1997), although equally studies exist that do not support this idea (Jayne, 2010). Considering surgery is the only curative treatment of solid tumours, this knowledge has prompted efforts to understand this undesired effect of surgery. It is hoped that this understanding will eventually help develop therapeutic treatments that may

Considering what is already known concerning the role of inflammation in tumourigenesis, it is not surprising that surgery is tumorigenic. Surgery confers a traumatic insult to the body which like all traumas induce a potent pro-inflammatory response. Surgery is followed by a biologic period of repair to help restore homeostasis. It induces an early hyper-inflammatory response, which is characterised by pro-inflammatory TNF-α, IL-1 and IL-6 cytokine release (Walker, 1999) and neutrophil activation (Hensler, 1997). Several of these cytokines have been shown to potentiate tumour growth (Coussens, 2004). The massive and continuous IL-6 release subsequently accounts for the up-regulation of major anti-inflammatory mediators, such as PGE2, IL-10, and TGF-ß (Walker 1999). The magnitude and duration of this hyper-inflammatory response is proportional to the severity of the trauma which may explain how laparoscopic versus open surgery may result in lower tumour recurrence (Colacchio, 1994; Baigrie, 1992). Following surgery, angiogenesis is also stimulated as the body initiates a period of healing and biological repair. Pro-angiogenic substances such as VEGF become elevated post-operatively. Moreover, anti-angiogenic substances such as endostatin and angiostatin are not detectable in the serum shortly after tumour excision (Li, 2001; Holmgren 1995; O'Reilly, 1994). This incites the formation of capillaries and new blood vessels not only to the areas of tissue insult but also to all parts of the body including areas of residual metastatic disease which promotes tumour growth. Furthermore, surgery also influences immune system function. Immuno-suppression is a feature of the post-operative stress response and is also associated with anaesthesia, blood transfusion and the release of acute-phase proteins (Lee, 1977). The immune system appears to be an important regulator in identifying and eliminating any abnormal cells with cancerous potential. Hence, any disruption of immune function as a result of surgery can also lead to potentiating tumour growth. Interestingly, this immuno-suppression is greater dependent on the extent of surgery (Da Costa, 1998; Da Costa, 1999). The immunosuppressive effects of surgery can last anywhere from between 4 to 14 days depending on the size of surgical trauma induced but peaks day three post-operatively in most cases. Likewise, this may explain why laparoscopic versus open surgery in colon cancer patients confers a greater survival advantage, although the evidence supporting this

Endotoxaemia also occurs following surgery involving bowel manipulation. Endotoxin, a potent inflammatory mediator, is a component on the wall of Gram negative bacteria often found in the lumen of the gastrointestinal system. Upon manipulation, endotoxin or LPS translocates across the intestinal lumen and enters the circulation. Endotoxaemia further

attenuate or eliminate this unwelcome consequence of surgery.

**6.1 Mechanisms of surgery induced tumourigenesis** 

is not equivocal.

lymphocytes (CTL) and antibodies specific for TAA have been demonstrated in patients with cancers including colorectal cancer (Nagorsen, 2000). Therefore there is an ideal niche in cancer treatment for an active specific immunotherapy. Previously identified or undefined TAAs can be administered to cancer patients in order to cause a systemic immune response which will lead to malignant cell destruction (Rosenberg, 2001).

Despite the huge amount of evidence and theoretical potential of this treatment strategy, the clinical results are limited to date. There is evidence to support the tumouricidal effects of active specific immunotherapy however it appears cancer cells are able to survive the tumouricidal effects as the disease progresses. This phenomenon has become known as 'tumour immune escape'. Several mechanisms have been proposed as to how this phenomenon occurs. One body of evidence shows evidence that cancer genetic instability leads to TAA/HLA downregulation as well as to disruption of the TAA processing/presenting machinery which then allows malignant cells to evade the surveillance of immune sentinels (Seliger, 2001; Yang, 2003). Another proposed mechanism lies in the ability of cancer cells to produce immunosuppressive cytokines e.g. IL-10, and thus counteract the immune system response (Mocellin, 2001; Walker, 1997). The challenge for tumour immunologists currently is to overcome this 'tumour immune escape' and increase the proportion of patients mounting an immune response and increase the rate of responses from the targeted tumour.

The role of macrophages in the development of colorectal cancer is controversial. One study demonstrated that increased numbers of macrophages in all areas of the tumour correlated with an advanced tumour stage (Bailey, 2007). On the other hand Forssell et al showed that the presence of macrophages positively influenced prognosis in colorectal cancer (Forssell, 2007). Another group demonstrated that macrophages have the ability to inhibit or stimulate tumour growth according to their polarisation and state of activation (Mantovani, 2005). This group showed that M1-polarised macrophages activated by IFNγ and bacterial products like LPS display tumouricidal effects whereas M2 macrophages differentiated in the presence of Th2 cytokines, IL-4, IL-13 or IL-10, have the opposite effect. These macrophages favour tumour cell proliferation and stimulate tumour progression and tumour invasion. These studies are crucial in demonstrating the important role that inflammatory mediators such as chemokines and cytokines play in the establishment of the tumour microenvironment. The tumorgenicity of proinflammatory mediators such as interleukin-6 and tumour necrosis factor-α (TNFα) was shown by Coussens et al (Coussens, 2002).
