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

### **4.1 HA, inflammation, and the role of inflammatory cells in tumourigenesis 4.1.1 Macrophages**

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,

Hyaluronan Associated Inflammation and Microenvironment

dendritic cells which help to eradicate neoplasms.

DC maturation in tumour bearing animals (Kuang *et al*., 2008).

**4.1.4 Dendritic cells and mast cells** 

Remodelling Influences Breast Cancer Progression 219

2002). The majority of stromal B cells localize to perivascular regions within tumours and chronic B cell activation promotes tumours by recruiting macrophages and activating an innate immune response. However, the role of B cells in BCA progression is complicated since, for example, B cells may also recruit antigen presenting cells, such as CD8+ T cells and

Dendritic cells (DC) can also exhibit HA dependent characteristics that either promote or inhibit tumourigenesis. HA or chondroitin sulphate, in conjunction with CSF-1, activate DC from an immature to differentiated state via an NFκB regulated process, illustrating the importance of HA in eliciting an immune response (Yang *et al*., 2002). Pedroza-Gonzalez *et al* (2011) recently showed that human BCA produces thymic stromal lymphopoietin (TSLP) which induces expression of OX40L on DCs, polarizing them towards a Th2 inflammatory response. *In vitro* this drives the production of IL-13 and TNF by Th2 polarized T cells (Pedroza-Gonzalez *et al*., 2011). DC also become tumour insensitive and, as a result, do not mature and differentiate into cytotoxic cells. Furthermore, HA fragment build ups are at least partly responsible for preventing

In BCA, c-kit expression by mast cells, a protein which is usually only present in specific tissue types, such as germ cells, predicts primary tumour recurrence (Khazaie *et al*., 2011). However, an abundance of stromal mast cells in invasive BCA is associated with good prognosis (Rajput *et al*., 2008). The mast cell line HMC-1 expresses high levels of CD44s and, through an interaction with HA, adheres to stromal tissue (Fukui *et al*., 2000). Therefore, in

both mast cells and DC, a CD44-HA interaction may result in anti-tumour responses.

**4.2.1 Breast cancer cells and their contribution to a pro-inflammatory environment**  BCA cells secrete a variety of cytokines and chemokines which promote tumour progression. Studies by Tafani *et al*. (2010), showed that MCF-7 cells upregulate proinflammatory gene transcription and translation *in vitro*, and a pro-inflammatory gene expression profile can be seen in human BCA tumours even in the absence of an immune infiltrate. This illustrates that BCA cells themselves contribute to the pro-inflammatory/protumourigenic TME. One or both of HER2 and ERα, which are often expressed on BCA cells, promote the expression and secretion of CXCL8 (IL-8) through the PI3K and ERK pathways. CXCL8 is a pro-angiogenic chemokine and secretion of CXCL8 by the MCF7 BCA line (which express both HER2 and ERα) is additive upon stimulation of both of these receptors (Haim *et al*., 2008). The pro-inflammatory chemokines CCL2 and CCL5 are also secreted by BCA cells (Ben-Baruch, 2003) and expression and secretion of all three chemokines requires HA fragment/CD44 interactions on TAMs, tumour associated fibroblasts (TAFs) and BCA tumour cells. Both CCL2 and CCL5 are monocyte-recruiting chemokines and their expression in BCA tumours is correlated with poor prognosis, and in the case of CCL2, proangiogenesis factors and vascular invasion (Soria and Ben-Baruch, 2008). TNF-α secretion by TAMs activates a positive feedback loop in BCA tumour cells, stimulating further secretion of growth promoting chemokines (Ben-Baruch *et al*., 2003). Eck *et al* (2009) also showed that conditioned media from BCA cells stimulates the expression of pro-inflammatory genes in normal mammary fibroblasts, polarizing them towards a TAF phenotype. Furthermore, TAF migration is increased, along with the secretion of MMP-1 and CXCR4 (IL-1/SDF-1

**4.2 HA regulation of a pro-inflammatory environment by non-immune cells** 

receptor), both of which are important factors in BCA progression (Eck *et al*., 2009).

c-Myc, MMP, and VEGF, which are all involved in promoting tumourigenesis. In BCA, NOS2 expression within tumour cells themselves is correlated with increased tumour grade and angiogenesis (Ambs and Glynn, 2011).
