Breast Cancer Metastasis and Tumour Microenvironment

Breast cancer is the second major cause of mortality among women in the United States. Approximately one in eight women (12%) will develop invasive breast cancer over the course of their lifetimes (Siegel et al. 2014). Furthermore, 3,327,552 American women were estimated to be living with breast cancer in 2014, and older women, within 55-64 age, were found to have an increased incidence rate of the disease (American Cancer Society. Breast Cancer Facts & Figures 2015-2016). Although several risk factors for breast cancer have been identified for the majority of women, it is not possible to develop causal relationships that inform efficient clinical treatment options (Lacey, Devesa, and Brinton 2002). In general, a family history of breast cancer and mutations in the genes BRCA1, BRCA2 and P53 are well established risk factors (Kelsey and Bernstein 1996). Furthermore, reproductive factors associated with prolonged exposure to endogenous estrogens, such as early menstruation, late menopause and late age at first childbirth are among the most important risk factors for breast cancer. Such exogenous hormones exert a higher risk for breast cancer, so users of oral contraceptives and hormone replacement therapy are at higher risk of developing cancer than non-users (Lacey, Devesa, and Brinton 2002). Early cases of breast cancer were first reported by Egyptians; from then breast cancer cases have been reported for thousands of years and remain a menacing disease (Rayter 2003). One primary treatment developed to cure breast cancer is mastectomy, which is removal of the affected breast tissue using surgical procedures. However, despite effectiveness of this procedure, in the mid-1800s surgeons noted that the disease had a high recurrence rate post operations because the cancer spread to nearby regions (Rayter 2003). LeDran was one of the pioneers who suggested that breast cancer begins as a local disease and spreads to other organs, so local treatment options are not completely effective for treatment. It was later established that metastasis of cancer to secondary sites results in a high recurrence rate and poor prognosis (Rayter 2003). To better understand metastasis, there have been numerous in vivo animal and in vitro cell-based models developed to study cancer metastasis (Wang, Eddy, and Condeelis 2007). Further improvements in the fields of microfabrication, tissue engineering, high resolution cell-imaging, and genetic/molecular pathways analysis have led to developing better diagnostic and therapeutic approaches (DeVita Jr and Rosenberg 2012).

1. METASTASIS

Metastasis is the primary cause of mortality in individuals with breast cancer (Nguyen, Bos, and Massagué 2009). It is a process by which tumor cells migrate from the primary tumor to one or multiple distant organs (Spano et al. 2012). Metastasis progresses through a complex multistep process termed as the “Metastatic Cascade”, which involves several stages as listed below (Valastyan and Weinberg 2011) (Figure 1.1):

1. Invasion of cancer cells through surrounding extracellular matrix and stroma
2. Intravasation into the lumen and blood vessels
3. Surviving and transport through vasculature
4. Lodging at distant sites
5. Extravasation to form micro metastatic structure
6. Surviving harsh foreign micro environment
7. Re-initiation of proliferation at distant site

It is well respected that invasion and metastasis does not just depend on cancer cells but various biochemical and biophysical factors within the local tumor microenvironment (Hu et al. 2008). During metastasis, the surrounding environment becomes activated due to cancer cells secreting several growth factors such as Transforming growth factor beta (TGF-), Hepatocyte growth factor (HGF), Vascular Endothelial Growth Factor (VEGF), Platelet-derived growth factor (PDGF), etc. This activated tumor microenvironment will in turn influence tumor cell polarity, circulation, and migration (Wolf and Friedl 2003). Thus, before tumor cells undergoes metastasis, a conducive microenvironment assembles, which eventually promotes the formation of the secondary tumor (Kucia et al. 2005, Orimo et al. 2005).

Cancer cell invasion involves the cells disseminating from the well-defined primary tumor, entering the surrounding stroma, and then moving into adjacent tissues (Wolf and Friedl 2003). The ability of cancer cells to undergo migration and invasion allows neoplastic cancer cells to enter blood vessels and metastasize to distant organs (Chambers, Groom, and MacDonald 2002). To invade, carcinoma cells must first overcome the basement membrane (BM), a specialized membrane surrounding epithelial tissues. Active proteolysis of BM occurs by secretion of matrix metalloproteinases (MMPs) by cancer cells (Bissell and Hines 2011). Once the carcinoma cells dissolve the BM, they reach the stroma where they encounter the stromal cells. With the association of tumor cells, stromal cells transition to a phenotype where they are commonly known as “Tumor-Associated stromal cells” (Kessenbrock, Plaks, and Werb 2010). To overcome barriers they face during invasion, cancer cells opt to a cell biological program called the Epithelial–Mesenchymal Transition (EMT), which is one of the crucial steps during invasion (Thiery et al. 2009). During the EMT, tight junctions and adherents between the cells are dissolved and cell polarity is lost. The individual cells dissociate from epithelial cell sheets and exhibit mesenchymal cell traits which are associated with high cell motility and invasion (Wolf and Friedl 2003). A prerequisite for the EMT is that cells lose their cell-cell junctions and express migration promoting factors like integrin, followed by loss of cadherin or catenin and increase of proteases. Also, there will be downregulation of epithelial markers and upregulation of mesenchymal markers. To migrate, a cell modifies its shape and interacts with the surrounding microenvironment. Specifically, the cell becomes polarized and forms a pseudopod by extending its leading edge, and the entire cell body contracts, generating traction forces that will lead to gradual forward movement of the cell (Friedl and Bröcker 2000). In vitro and in vivo studies have shown that tumor cells have diverse movement patterns, such as, they can migrate as individual cells (individual cell migration), as cell sheets, strands, or clusters (collective cell migration) (Wolf and Friedl 2003). From previous studies, it was shown that motile tumor cells originate from multicellular components, but they lose their cell-cell contacts, detaching and migrating as individual cells (van Zijl, Krupitza, and Mikulits 2011). In collective migration, the cell aggregates move as one functional unit. In contrast to individual movement, cell-cell adhesions in cell groups leads to cell assembly, forming large sized multicellular bodies (Wolf and Friedl 2003). Many tumor cells lack stop signaling in migratory events, which causes a large imbalance and drives them to invade surrounding tissue and migrate to distant organs (Wolf and Friedl 2003) (Figure 1.1 b).

After invading, cancer cells migrate and enter blood or lymphatic vessels, a process known as ‘intravasation’(Gupta and Massagué 2006). Once, the carcinoma cells successfully enter blood vessels, they can translocate to different parts of the body through venous and arterial circulation. Cancer cells spreading through lymphatic vessels (i.e. lymphogenous spread) is observed in humans at significant levels, but the major mechanism of cancer cell dissemination is through blood circulation (i.e. hematogenous spread) (Gupta and Massagué 2006). Furthermore, cancer intravasation can be either active or passive. Active intravasation occurs when tumor cells migrate towards the blood or lymphatic vessels following gradients of secreted chemokines. On the other hand, passive intravasation happens when blood vessels are in close proximity to the tumor and cancer cells are able to enter nearby luminar structures (van Zijl, Krupitza, and Mikulits 2011). Another important distinction is whether tumor cells intravasate into the vessels through the endothelial cell body or between endothelial cell-cell junctions (Reymond, d’Água, and Ridley 2013). Recent studies have demonstrated a large involvement of endothelial junctional in both paracellular and transcellular migration, suggesting that intravasation is facilitated by the ability of cancer cells to modify and cross the endothelial cell junction and pericyte barrier. Matrix metalloproteases (MMP-1) secreted by cancer cells modify Protease-activated receptors (PAR1) on the endothelial cells and remodel the endothelial junctions (Bergers et al. 2000). Also, Disintegrin and Metalloproteinase domain-containing protein 12 (ADAM12) induce cleavage of VE-cadherin and ang-1 receptor TIE2 which can disrupt endothelial junctions (Reymond, d’Água, and Ridley 2013). Macrophages that are present in the stroma promote intravasation by secreting Epidermal Growth Factor (EGF) and Tumor Necrosis Factor alpha (TNF-), which also induce retraction of endothelial junctions. Cancer cells also use NOTCH receptors to transmigrate through endothelial junctions (Gupta and Massagué 2006).

The mechanism of intravasation is strongly influenced by tumor-associated angiogenesis, where tumor cells stimulate the formation of new blood vessels. The alterations to the tumor microenvironment (e.g. inadequate oxygen supply (hypoxia), insufficient nutrients) result in tumor cells secreting growth factors, which lead to the formation of vascular networks (Partridge, Deryugina, and Quigley 2008). There are many mechanisms through which tumor cells induce angiogenesis(Vaupel 2004). However, VEGF is known to be one of the major factor secreted; VEGF stimulates the formation of new blood vessels, a process termed as ‘Neoangiogenesis’. In contrast to normal vascular networks, the newly formed vasculatures are leakier and permeable due to weak inter cellular junctions and absence of pericyte lining. Permeability of endothelial cells also facilitates intravasation (Gupta and Massagué 2006). Only about 0.01% of circulating tumor cells (CTC) will reach and form tumors at secondary sites, but it is still important to study the process of intravasation to understand the mechanism in-depth and decipher the efficiency of metastasis. There are several in vivo and in vitro systems developed to study intravasation, and they have clarified the role of different cells, signaling pathways, and molecules that contribute to intravasation (Woodfin et al. 2011, Stoletov et al. 2010). Despite progress in identifying tumor-cell autonomous intravasation mechanisms, recent studies suggest that the tumor microenvironment also plays a major role in regulating tumor cell dissemination (Joyce and Pollard 2009). Hence, a comprehensive understanding of the underlying biological mechanisms of cancer cell intravasation, both at the intracellular and tumor microenvironment levels, is critical for identifying and developing novel targeted therapies (Figure 1.1 b).

Once the cancer cells successfully intravasate, they become circulating tumor cells within the blood stream. These cells are in between the primary tumor and metastatic sites, so they are called ‘Metastatic Intermediates’ (Meng et al. 2004). After surviving circulation and reaching the foreign site, carcinoma cells must cross the endothelial cell lumina to reach the tissue parenchyma of the host tissue, this process is known as ‘extravasation’ (Valastyan and Weinberg 2011). To overcome barriers during extravasation, the carcinoma cells make the vessels more permeable and perturb the microenvironment. They achieve this by secreting different factors such as Angiopoietin-like 4 (ANGPT14), Epiregulin (EREG), Cyclooxygenase (COX-2), MMP-1, MMP-2 and VEGF, which renders the nearby vessels more permeable and assist the tumor cells during extravasation (Gupta et al. 2007, Padua et al. 2008).

Figure 1.1: Metastatic cascade (a) Schematic of the metastatic cascade. Tumor growth and development (b) Angiogenesis, cancer cell invasion, and intravasation. (c) Finally, surviving cancer cells and cancer circulate through the body, attach to blood vessels, and extravasate to form secondary metastases. Adapted from Peela et al. with permission from Elsevier [Biomaterials], copyright (2017) (Peela et al. 2017)

The extravasated cancer cells must survive the foreign environment to further proliferate and form secondary cancer. Until the foreign environment is hospitable, the cancer cells are dormant and will be in the hibernating phase. Some models suggest that cancer cells adapt to the foreign environment by releasing signals that upregulate fibronectin, leading to mobilization of ‘VEGF receptor-positive hematopoietic progenitor cells’ towards metastatic sites. These cells change the distant microenvironment to more hospitable sites, which are called pre-metastatic niches (Psaila and Lyden 2009). Initiation and development of this pre-metastatic niche is observed to originate from many different factors. Apart from factors involving the presence of chemokines such as stromal-derived factor 1 (SDF-1), the pre-metastatic niche contains microenvironmental components such as fibroblasts and endothelial cells, which secrete growth factors and chemokines that influence tumor cell polarity, circulation, and migration (Psaila and Lyden 2009).  The ability of disseminated tumor cells to adapt to the host site and proliferate depends on their proficiency to change the foreign microenvironment to a hospitable environment (Chambers, Groom, and MacDonald 2002) (Figure 1.1 c).

The successful completion of each of these stages would result in metastasis and the development of a secondary tumor. Although great advances have been made in early diagnostics and curing cancer, metastasis remains as one of the major fatal challenges. Numerous processes, signaling pathways and growth factors are involved in metastasis. However, very few molecules have been translated effectively for metastasis prevention and treatment.

2. TUMOR MICROENVIRONMENT

Cancer cells develop a complex surrounding microenvironment and depend on it for their survival, growth, invasion, and metastasis called ‘Tumor Micro Environment’(TME) (Quail and Joyce 2013). The microenvironment is composed of both stromal cells and Extra Cellular Matrix (ECM) proteins. The Basement membrane in the ECM principally interacts with the epithelium and is primarily composed of collagen type IV, laminin (LM) (LM-111 and LM-332), glycoproteins (epiligrin and entactin) and proteoglycans (Oskarsson 2013). The ECM helps to maintain tissue structure and architecture as well as homeostasis. The surrounding stroma includes fibroblasts, adipocytes, endothelial cells as well as immune cells (Place, Huh, and Polyak 2011), and there is a bi-directional communication between the tumor cells and their surrounding stroma. It is shown that the interactions between the tumor and tumor microenvironment are critical for the disease initiation, progression, and metastasis (Quail and Joyce 2013). Several studies and observations in human patients have led to the postulation that accumulation of genomic instability in the stroma might lead to genomically unstable epithelium and consequently, neoplastic transformation (Weber et al. 2006).

Rudolf Virchow in 1863 first proposed that there is a link between tumorigenesis and chronic inflammation. Furthermore, the infiltration of leukocytes was identified to be a key hallmark of tumors (Balkwill and Mantovani 2001). Tumor-associated macrophages (TAM) play an important role in cancer progression; although macrophages are regarded as effector cells in the immune system, studies have shown that the TAM play a supporting role during cancer progression where they drive invasive phenotypes in cancer cells (Biswas and Mantovani 2010).

Fibroblasts are one of the predominant cells in the connective tissues. In the TME, Cancer Associated Fibroblasts (CAFs) are present in high numbers and they support the tumor by depositing ECM and maintaining homeostasis (Olumi et al. 1999). It is unclear where CAFs arise during disease progression, but studies suggest that they may transition EndMT (Endothelial-Mesenchymal Transition) of cells. CAFs in the TME are activated by growth factors and cytokines in TME like Fibroblast Growth Factor (FGF), PDGF, TGF-, monocyte chemotactic protein 1(MCP1) and many secreted proteases. (Kalluri and Zeisberg 2006).

Tumor angiogenesis is now one of the accepted hallmarks of cancer, which is essential for nutrient and oxygen supply without which the tumor would become dormant (Hanahan and Weinberg 2011). Judah Folkman proposed that all tumors are angiogenesis dependent (Folkman 1971). Tumor angiogenesis requires multiple cells in TME like endothelial cells, pericytes, precursor cells, etc. These stromal cells are hypoxia driven (LaGory and Giaccia 2016). Angiogenesis is activated with the help of factors such as epidermal growth factor (EGF), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor (FGF) which are secreted by cancer cells. In addition to this, many other stromal cells and Mesenchymal Stem cells (MSCs) contribute to activation of endothelial cells (Weis and Cheresh 2011). In vivo and in vitro assays developed so far have been enormously helpful in elucidating the diverse mechanisms involved in the interactions of tumor cells with the surrounding microenvironment. Better relevant models that can capture the complexities of the microenvironment will give a better understanding and may be key to combat cancer.