**3.3 TLR2 and TLR4**

Toll like receptors (TLR) are part of a cellular defence mechanism that is based on pattern recognition. TLRs recognize and bind bacterial lipopolysaccharides, DNA, and, in the case of TLR2,4, small HA fragments. In general, HA-TLR2,4 interactions control innate immunity through several mechanisms. For example, TLR 2,4 activation results in cytokine and chemokine release and leads to expression of metalloproteinases (MMPs) in immune cells (Voelcker *et al.*, 2008). Versican, which is associated with poor prognosis and relapse in BCA, interacts with HA polymers to form cord-like structures that link TLR2 on endothelial cells and fibroblasts. This, in turn, causes the secretion of pro-inflammatory cytokines (Theocharis *et al.*, 2010). HA-TLR2,4 interactions also stimulate NFκB signalling and activate TNFα. In BCA cells, TLR 2,4 interact with CD44 and act as co-receptors to stimulate signalling through HA and CD44 regulated pathways which may play a role in breast

Hyaluronan Associated Inflammation and Microenvironment

(Matsumoto *et al*., 2010).

**4.1.1 Macrophages** 

**BCA** 

Remodelling Influences Breast Cancer Progression 217

suggests that LYVE-1 plays a role in tumour cell adhesion which is dependent on HA-LYVE-1 interaction. Apart from its effect on tumour cell adhesion, LYVE-1 has also been proven to be a prognostic factor in tongue squamous cell carcinoma and decreased levels of LYVE-1 in the invasive front of tumours predicts cervical lymph node metastasis

**4. HA expression and signalling in different cell types and its relationship to** 

HA has a major role in macrophage biology during inflammation, wound repair, and tumourigenesis and at least part of the detrimental effects of HA accumulation during tumourigenesis is due to the activation of tumour associated macrophages (TAMs). For instance, TAMs preferentially traffic to stromal compartments formed within HA producing tumours (Kobayashi *et al*., 2010). Macrophages are classed into type 1 and 2 according to the adaptive immune polarization with which they associate. Type 1 macrophages are antigenpresenting cells which promote the cytotoxic response, resulting in tumour cell killing. Type 2 macrophages, however, are classically associated with tissue remodelling, angiogenesis, and scavenging/phagocytosis. TAMs are similar to type 2 polarized macrophages which have decreased or inhibited cytotoxic activity (Mytar *et al*., 2003). Kuang *et al*. (2007) found that overexpression of HAS2 was able to polarize macrophages towards a malignant TAM phenotype. Additionally, exposure to solid tumour cell culture supernatant elicits a proinflammatory response in monocytes and their subsequent TAM-like polarization, showing that the tumour cells themselves are responsible for the immunosuppressive macrophage phenotype observed in solid tumours (Kuang *et al*., 2007). The importance of TAM recruitment in BCA dissemination was additionally illustrated by CSF-1 null mice crossed with the MMTV transgenic mouse model of BCA. In these mice, a failure to recruit macrophages into the primary tumour results in delayed primary tumour invasion and metastasis to the lungs compared to wildtype MMTV mice. The addition of exogenous CSF-1 rescues macrophage recruitment and restores tumour and metastasis development to baseline levels (Lin *et al*., 2001). After injury, or during tissue inflammation, small fragments of HA associate with TLR4 and control macrophage cytokines and chemokines (Termeer *et al*., 2000). For example, BCA cell associated HA promotes the production of proinflammatory cytokines and chemokines, such as TNF-α and IL-12, as well as ROS, by TAMs, an effect which can be alleviated by either blocking CD44 receptors on monocytes, or by the addition of non-BCA cell associated HA (Mytar *et al*., 2001). HA regulation of proinflammatory cytokine production also occurs in monocytes pre-exposed to a variety of solid tumour cell types and culture supernatants, including the BCA line MCF-7 (Mytar *et al*., 2003, del Fresno *et al*., 2005), modulating the IRAK family of NFκB regulatory molecules, this further downregulating TNF-α and IL-12 production. HA-mediated CD44 cross-linking induces this activity and is prevented by the addition of exogenous HYAL (Mytar *et al*., 2003). TAMs are recruited and regulated in response to NFκB, whose activation is often HAmediated through TLR4 (del Fresno *et al*., 2005) and NFκB overexpression results in tumour metastasis (Mantovani *et al*., 2007). Nitric oxide, which is the product of nitric oxide synthase 2 (NOS), is stimulated by hypoxia and CSF-1, among others, and is a signalling molecule integrated within the NFκB inflammatory pathway. NOS2 signals the upregulation of CD44,

**4.1 HA, inflammation, and the role of inflammatory cells in tumourigenesis** 

tumour cell migration/infiltration. The human BCA cell line MDA-MB-231 expresses mainly TLR4, and siRNA mediated knock-down of TLR4 significantly reduces cell survival and expression of the cytokines Il-6 and Il-8, suggesting that TLR4 is a promising target for BCA therapy (Yang *et al.*, 2010).

Fig. 3. HA initiates the signalling of RHAMM and CD44 regulated pathways, resulting in a variety of pro-tumourigenic outcomes.
