RESUMEN

1.3.4.1 Role of CAFs in tumour progression/metastasis

Metastasis requires ECM degradation, invasion/intravasation into lymph nodes or blood vessels, extravasation and the ability to proliferate and establish a secondary tumour at the new site. CAFs can play an important role in many of these aspects and here I will summarise some key known functions.

1.3.4.2 Effect of CAFs on ECM remodelling

A recent paper investigated the role of CAFs in ECM remodelling of breast tumours (Wang et al. 2017). CAFs were shown to affect the assembly and re-organisation of collagen I and

43 fibronectin, two important components of the ECM. First, CAFs lay down unfolded

fibronectin fibres, that is followed by a denser matrix of collagen I after remodelling, resembling scar tissue after wound healing. Matrix metalloproteins (MMP-2, MMP-9 and MMP-12) secreted by CAFs also play an important role in ECM remodelling (Karagiannis et al. 2012). CAFs aid metastasis also by upregulating palladin expression and secreting proteolytic enzymes to degrade the ECM (Brentnall et al. 2012).

1.3.4.3 Effect of CAFs on cancer cell invasion

The collective invasion of cancer and fibroblasts were observed under 3D in vitro conditions, using organotypic culture model (Gaggioli et al. 2007) (Gaggioli 2008). This model involves the culture of cells on top of a gel matrix, composed of collagen I, as well as laminins and collagen IV. Cancer cells alone do not show any migration within the gel, unlike in the presence of fibroblasts, where invasion of cells down the gel is observed. Furthermore, the degree of invasion was dependent on the number of fibroblasts that were co-seeded with the epithelial cancer cells. A closer look at the invading cells revealed fibroblasts to be at the leading front, followed by cancer cells in tracks of degraded gel matrix.

1.3.4.4 Role of CAFs in EMT

EMT is a process whereby epithelial cells undergo morphological changes and become more mesenchymal-like. Cells undergoing EMT lose expression of epithelial marker E- cadherin and lose apico-basal polarity, whilst increasingly expression of mesenchymal markers such as vimentin, N-cadherin and Zeb1. This results in cells that are more motile, invasive and less polarised.

CAFs can trigger EMT in breast epithelial cells, which can that result in cancer stem cell- like aggressive behaviour, driving cancer metastasis (Soon et al. 2013). Factors such as TGF-β, CCL2 and IL-1β drive this process (Yu et al. 2014) (Tsuyada et al. 2012) (P et al.

44 2016). Moreover, CAFs also deposit their own ECM that has been shown to differ from the ECM laid down by normal fibroblasts. CAF-deposited ECM is more aligned in pattern (Dumont et al. 2013), induces EMT in tumour cells and contains high levels of fibronectin, biglycan and ECM-modulating enzyme lysyl oxidase.

1.3.4.5 Role of CAFs in altering metastatic tropism

Moreover, CAFs has shown to also pay a role in influencing metastatic tropism. For

example, TNBC, that have a high tendency to metastasise to visceral organs, can alter their tropism to form bone metastases through exposure to CAF-secreted CXCL12 and IGF (Zhang et al. 2013). Another way in which CAFs may be involved in influencing the metastatic site, is by migrating together with the cancer cells to the metastatic site. This has been demonstrated, by Duda et al, in lung metastases (Duda et al. 2010). The presence of stromal cells is thought to support the initiation and initial growth of secondary tumours in the lung.

1.3.4.6 Effect of CAFs on tumour drug response

A number of studies provide evidence to suggest the involvement of CAFs in drug

resistance. Teicher et al observed different susceptibility of cancer cells to chemotherapy between in vitro and in vivo conditions (Teicher et al. 1990). Resistance demonstrated in

vivo was absent when tested in vitro, indicating that factors present in vivo are required

for developing resistance. In breast and colon cancer, the tumour-stroma ratio is predictive of the outcome of patients receiving adjuvant chemotherapy (Dekker et al. 2013) (Park et al. 2014). The gene expression profiles of the stroma of breast tumours have also shown to be able to predict outcome of patients undergoing neoadjuvant chemotherapy (Farmer et al. 2009).

The TME can affect response to therapy via intrinsic and acquired mechanisms (Klemm and Joyce 2015). Intrinsic mechanisms include reduced drug delivery, pro-survival

45 signalling and paracrine signalling loops. Acquired resistance can arise after exposure to therapy.

The ECM provides structural support to the TME, where collagen is a key player and contributes to mechanical properties that has implications for intrinsic drug resistance (Barocas and Tranquillo 1997). Studies have demonstrated that the amount and

organisation of collagen affects interstitial diffusion and thereby delivery of drugs from the bloodstream. Eliminating collagen in tumours, using collagen-degrading enzyme

collagenase, resulted in improved diffusion (Netti et al. 2000).

Tamoxifen resistance is an example that has been attributed to CAFs, mediated by upregulation of PI3K/AKT and MAPK/ERK pathways and phosphorylation of ER (Pontiggia et al. 2012). Metabolic changes in CAFs driven by cancer cells results in CAFs undergoing aerobic glycolysis, inducing oxidative stress and autophagy. This provides adjacent cancer cells with nutrients and mediates tamoxifen resistance; mimicked in co-culturing MCF7 with fibroblasts or supplementing with metabolic substrates such as ketone bodies. This mechanism of tamoxifen resistance was overcome by inhibiting the mitochondria in MCF7 cells. (Martinez-Outschoorn et al. 2011). Factors secreted by CAFs also contribute to drug resistance. For example, HGF that activates AKT and MAPK signalling pathways and chemokine CCL2 disable drug-induced apoptosis (Tsuyada et al. 2012).

Furthermore, a recent study identified two different sub-populations of CAFs in ER- positive breast tumours, that have varying effects on sensitivity to oestrogen (Brechbuhl et al. 2017). CD146neg _{CAFs decrease oestrogen sensitivity and thus induce resistance to} tamoxifen, by diminishing the expression of ER in tumours. On the other hand, CD146pos population of CAFs maintain sensitivity to tamoxifen, due to unaffected levels of ER expression.

46 Non-malignant cells within the TME are genetically stable, unlike cancer cells, and are therefore stable therapeutic targets. The intrinsic roles of CAFs and their interaction with cancer and non-cancerous cells within the TME, makes them potential therapeutic targets.

Several studies have shown the potential of targeting CAFs directly and indirectly in cancer (Togo et al. 2013). Work by Loeffler et al (Loeffler et al. 2006) demonstrated that DNA vaccines generated for FAP (specifically overexpressed in fibroblasts within colon, breast, and lung carcinomas (Scanlan et al. 1994) can reduce tumour growth in combination with doxorubicin.