**2.1.1 Vascular cells**

Tumor blood vessels, like normal vessels, are composed of endothelial cells, pericytes/smooth muscle cells and basement membrane. However, all of these components are morphologically and/or functionally different from the normal counterparts (Baluk et al., 2005).

Tumor-associated endothelial cells (TECs) are the major player in the formation of tumor vasculature through sprouting from pre-existing blood vessels (a process called 'angiogenesis'). During blood vessel formation, endothelial cells proliferate, migrate and form the inner layer of a lumen, followed by basement membrane formation and pericyte attachment. Angiogenesis is stimulated by excessive pro-angiogenic factors secreted by tumor cells or stromal cells in an oxygen-depleted microenvironment. Moreover, bone marrow-derived endothelial progenitor cells recruited to tumor stroma can contribute to blood vessel construction by incorporating into vessels (Lyden et al., 2001). New blood vessel formation is critical for tumor development and progression, as it delivers nutrients and oxygen to growing tumor and removes metabolic wastes. In addition, vascular endothelial cells form a barrier between circulating blood cells, tumor cells and the extracellular matrix (ECM), thus playing a central role in regulating the trafficking of leukocytes and tumor cells (Chouaib et al., 2010). In this regard, endothelial cells are critical for boosting a host immune defense against cancer cells and for controlling tumor metastasis. However, the 'gate-keeping' function of endothelial cells in tumors is heavily compromised. TECs are not tightly associated with each other, resulting in wider interendothelial junctions that cause plasma leakage and hemorrhage (Hashizume et al., 2000). Consequently, tumor vasculature is often leaky and less efficient in blood perfusion, leading to high interstitial fluid pressure, hypoxia and acidic extracellular pH that significantly affect the delivery and efficacy of chemotherapeutic drugs. The leaky blood vessels also facilitate the intravastion of tumor cells and promote tumor metastasis.

TECs are different from endothelial cells in normal tissues at several aspects. It has been reported that human hepatocellular carcinoma-derived endothelial cells, when compared to the ones from adjacent normal liver tissue, show increased apoptosis resistance, enhanced angiogenic activity and acquire more resistance to the combination of angiogenesis inhibitor with chemotherapeutic drugs (Xiong et al., 2009). Studies have also revealed distinct gene expression profiles of TECs and identified cell-surface markers distinguishing tumor versus normal endothelial cells (Seaman et al., 2007).

In blood vessels, pericytes are smooth muscle cell-like cells that cover the vascular tube. They are intimately associated with endothelial cells and embedded within the vascular basement membrane, and play an important role in the maintenance of blood vessel integrity. Pericytes in tumors are different from normal ones: in tumors, pericytes are often less abundant and more loosely attached to the endothelial layer (Abramsson et al., 2002; Morikawa et al., 2002). The abnormality in pericytes weakens the vessel wall and increases vessel leakiness. Pericytes express several markers, though none is pericyte-exclusive, including α-smooth muscle actin (αSMA), platelet-derived growth factor receptor-β (PDGFR-β) and NG2 (Gerhardt and Betsholtz, 2003; McDonald and Choyke, 2003). PDGF-B signaling is important for pericyte recruitment and attachment to endothelial cells during vascular development (Abramsson et al., 2002; Abramsson et al., 2003).

#### **2.1.2 Inflammatory/immune cells**

4 Cancer Prevention – From Mechanisms to Translational Benefits

This condition arises mainly due to an increase in the production of lactic acid by glycolysis along with other proton sources (Gatenby and Gillies, 2004; Helmlinger et al., 2002; Yamagata et al., 1998). Acidosis is a selection force for cancer cell somatic evolution, modulates cancer cell invasion and metastasis, and affects the efficacy of some chemotherapeutic drugs (Cairns et al., 2006; Gatenby et al., 2006; Gatenby and Gillies, 2004). Here we will describe cellular heterogeneity, hypoxia, and acidosis in the tumor microenvironment, and discuss some recent progresses in targeting tumor angiogenesis, inflammation, hypoxia and acidosis-related pathways for cancer prevention and therapy.

Tumor is an aberrantly proliferating tissue that contains cancerous cells and host stromal cells such as vascular cells, inflammatory cells, and fibroblasts. These cells are crucial for cancer initiation, progression and metastasis and have been exploited as targets for cancer therapy and prevention (Ferrara and Kerbel, 2005; Fukumura and Jain, 2007; Hanahan and

Tumor blood vessels, like normal vessels, are composed of endothelial cells, pericytes/smooth muscle cells and basement membrane. However, all of these components are morphologically and/or functionally different from the normal counterparts (Baluk et

Tumor-associated endothelial cells (TECs) are the major player in the formation of tumor vasculature through sprouting from pre-existing blood vessels (a process called 'angiogenesis'). During blood vessel formation, endothelial cells proliferate, migrate and form the inner layer of a lumen, followed by basement membrane formation and pericyte attachment. Angiogenesis is stimulated by excessive pro-angiogenic factors secreted by tumor cells or stromal cells in an oxygen-depleted microenvironment. Moreover, bone marrow-derived endothelial progenitor cells recruited to tumor stroma can contribute to blood vessel construction by incorporating into vessels (Lyden et al., 2001). New blood vessel formation is critical for tumor development and progression, as it delivers nutrients and oxygen to growing tumor and removes metabolic wastes. In addition, vascular endothelial cells form a barrier between circulating blood cells, tumor cells and the extracellular matrix (ECM), thus playing a central role in regulating the trafficking of leukocytes and tumor cells (Chouaib et al., 2010). In this regard, endothelial cells are critical for boosting a host immune defense against cancer cells and for controlling tumor metastasis. However, the 'gate-keeping' function of endothelial cells in tumors is heavily compromised. TECs are not tightly associated with each other, resulting in wider interendothelial junctions that cause plasma leakage and hemorrhage (Hashizume et al., 2000). Consequently, tumor vasculature is often leaky and less efficient in blood perfusion, leading to high interstitial fluid pressure, hypoxia and acidic extracellular pH that significantly affect the delivery and efficacy of chemotherapeutic drugs. The leaky blood vessels also

facilitate the intravastion of tumor cells and promote tumor metastasis.

**2. Tumor microenvironments and cancer progression** 

**2.1 Complex cellular components in solid tumors** 

Weinberg, 2011).

al., 2005).

**2.1.1 Vascular cells** 

Tumors are often infiltrated by inflammatory cells, such as macrophages, neutrophils, lymphocytes, mast cells, and myeloid progenitors. This phenomenon was initially observed by Rudolf Virchow more than a century ago and thought as an immunological response attempting to eliminate cancer cells. Whereas immune cells play a role in recognizing and eradicating early cancer cells (Kim et al., 2007), mounting evidence has also shown that inflammatory cells within tumors can enhance tumor initiation and progression by helping cancer cells acquire hallmark capabilities (Grivennikov et al., 2010; Hanahan and Weinberg, 2011). Inflammation is considered as an 'enabling characteristic' of tumor biology (Hanahan and Weinberg, 2011).

Pathological studies show that the abundance of certain types of infiltrating inflammatory cells, such as macrophages, neutrophils and mast cells, is correlated with poor prognosis of cancer patients (Murdoch et al., 2008). Tumor associated macrophages (TAMs), along with mast cells, neutrophils and other immune cells, produce cytokines (e.g. TNFα and IL-1), chemokines (e.g. CCL2 and CXCL12), angiogenic factors (e.g. VEGF, PDGF, FGF and IL-8), and matrix-degrading enzymes (e.g. MMPs, cathepsin proteases and heparanase) (Grivennikov et al., 2010; Karnoub and Weinberg, 2006). Some inflammatory cells, particularly neutrophils, also generate reactive oxygen and nitrogen species. These bioactive factors promote cancer cell proliferation, invasion and resistance to apoptosis through, for instance, the interleukin-JAK/STAT pathway (Ara and Declerck, 2010), and induce new blood vessel formation in the tumor. Extracelluar matrix-degrading enzymes promote cancer cell invasion and metastasis, whereas accumulation of reactive oxygen and nitrogen species can cause DNA mutagenesis, suppress DNA repair enzymes, increase genomic instability, and aggravate cancer progression.

Targeting Tumor Microenvironments for Cancer Prevention and Therapy 7

CAFs can maintain the myofibroblastic properties even after several passages *in vitro* without further signaling from carcinoma cells. How do CAFs acquire and maintain their activated phenotype? There are some controversial results with regard to the presence of somatic genetic alterations in CAFs. It has been reported that stroma microdissected from various human cancers exhibited some genetic alterations, such as chromosomal loss of heterozygosity (LOH) and somatic mutations (Currie et al., 2007; Kurose et al., 2002; Moinfar et al., 2000; Paterson et al., 2003; Tuhkanen et al., 2004; Wernert et al., 2001). Other reports also demonstrated that in the process of tumor development, fibroblasts that have lost p53 activity were clonally selected, leading to a highly proliferative stroma (Hill et al., 2005; Kiaris et al., 2005). In contrast, several genome-wide genetic analyses, including CGH and SNP arrays, were not able to detect any genetic alterations in the myofibroblasts isolated from various human cancers (Qiu et al., 2008; Walter et al., 2008). Other studies have suggested that epigenetic modifications within the genome of CAFs, such as DNA methylation, might be the reason (Hu et al., 2005; Jiang et al., 2008). Further studies are

Although cancer can originate from a single transformed cell, not all the cancer cells within a tumor are identical; in other words, cancer cells become heterogeneous during the somatic evolution process, reflected by distinct tumor regions with different histopathological characteristics and various degrees of tumor hallmark capacities. Moreover, mounting evidence indicates that tumor cells are also heterogeneous with regard to the capability to generate new tumors (Cho and Clarke, 2008; Lobo et al., 2007). Multiple studies showed that distinct subpopulations of cancer cells could be sorted from primary tumor samples based on their cell-surface antigen profiles. When different subpopulations of cells were injected into immune-deficient mice, only a subset of cells was able to propagate tumor growth, whereas other cells were unable to induce tumor regeneration (Lobo et al., 2007). This population of cancer cells has also been demonstrated to have the ability of self-renewal and differentiation, two hallmark characteristics of stem cells (Clarke et al., 2006). In addition, these cells also expresses some markers of normal stem cells (Al-Hajj et al., 2003); hence, these cells are termed as 'cancer stem cells' (CSCs; also referred as cancer initiating cells or

CSCs were initially identified in leukemia (Bonnet and Dick, 1997; Lapidot et al., 1994) and later in solid tumors that include cancers of breast, brain, pancreas, head and neck, and colon (Al-Hajj et al., 2003; Dalerba et al., 2007; Li et al., 2007; O'Brien et al., 2007; Prince et al., 2007; Ricci-Vitiani et al., 2007; Singh et al., 2004). Studies of leukemia stem cells suggest that, CSCs may arise from normal stem cells that acquire oncogenic mutations and undergo transformation (Fialkow, 1990; Lapidot et al., 1994; Lobo et al., 2007), or progenitor cells that gain the ability to self-renew through oncogenic transformation (Cozzio et al., 2003; Krivtsov et al., 2006; So et al., 2004). However, recent observations suggest that CSCs may also be derived from non-CSCs via the EMT process (Mani et al., 2008; Morel et al., 2008; Singh and Settleman, 2010), which plays an important role in morphogenesis and in promoting tumor cell motility and invasiveness (Hugo et al., 2007; Thiery, 2003). This model indicates that whereas CSCs can differentiate into non-CSCs; non-CSCs may also be reprogrammed and converted to CSCs, suggesting the existence of a dynamic interconversion between CSCs and non-CSCs that is controlled by the tumor microenvironment (Gupta et al., 2009). Such

required to clarify these issues.

tumorigenic cancer cells).

**2.1.4 Cancer stem/initiating cells** 

While the tumor-promoting effects of infiltrating inflammatory cells have been well documented, certain types of immune cells, particularly cytotoxic T cells and natural killer cells, exhibit anti-tumor activities. The high numbers of these cells within a tumor predict a favorable prognosis (de Visser, 2008; Fridman et al., 2011). Immune surveillance is considered as an important mechanism to inhibit carcinogenesis and maintain tumor dormancy (Kim et al., 2007). Evading immune destruction by downregulating tumor antigens, suppressing immune cell function and other means is an emerging hallmark of cancer cells and plays important roles in cancer progression and metastasis (Hanahan and Weinberg, 2011). With regard to cancer therapy, blockade of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4), a negative regulator of T cells, by the monoclonal antibody, ipilimumab, improved overall survival in patients with metastatic melanoma treated in combination with dacarbazine (Robert et al., 2011a). Moreover, expansion of tumor-infiltrating lymphocytes *ex vivo* and adoptive T-cell transfer immunotherapy led to regression of metastatic melanoma and durable responses in patients (Dudley et al., 2002; Rosenberg et al., 2011).

#### **2.1.3 Fibroblasts**

Fibroblasts account for the majority of stromal cells within solid tumors and are the principal source of ECM constituents (Chang et al., 2002). Fibroblasts in tumors are termed as cancer-associated fibroblasts (CAFs).

Tumors have been described as wounds that do not heal (Dvorak, 1986). Indeed, it has been observed that tumor-associated fibroblasts are biologically similar to the ones involved in wound healing or fibrosis (Ryan et al., 1973; Schor et al., 1988). Fibroblasts involved in these processes produce more ECM proteins and proliferate faster than the normal counterparts from healthy tissues (Castor et al., 1979; Muller and Rodemann, 1991). Fibroblasts with these properties are referred as "activated fibroblasts" or "myofibroblasts", due to their characteristic expression of α-smooth muscle actin (α-SMA) (Gabbiani, 2003; Ronnov-Jessen et al., 1996). Fibroblasts can be activated by various stimuli, such as transforming growth factor-β (TGFβ), epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2) (Zeisberg et al., 2000).

CAFs play an important role in promoting tumor initiation and progression by stimulating angiogenesis and tumor cell growth and invasion (Shimoda et al., 2010). The existence of a large number of CAFs in tumors is often associated with poor prognosis (Maeshima et al., 2002; Surowiak et al., 2006). CAFs produce growth factors, cytokines, chemokines and ECM proteases to stimulate angiogenesis and cancer cell proliferation and invasion. For example, CAFs secrete elevated levels of stromal cell-derived factor 1 (SDF-1; also called CXCL12) that facilitates angiogenesis by recruiting endothelial progenitor cells into the tumor (Orimo et al., 2005). SDF-1 can also interact with the CXCR4 receptor expressed on the surface of cancer cells, thus stimulating tumor cell growth and promoting tumor progression *in vivo*  (Orimo and Weinberg, 2006)*.* TGFβ, another factor produced by CAFs, is a critical mediator of the epithelial-to-mesenchymal transition (EMT); therefore, CAFs might contribute to EMT in nearby cancer cells and promote their invasiveness (Shimoda et al., 2010). Moreover, CAFs facilitate cancer cells to invade ECM and metastasize by releasing ECM-degrading proteases, such as matrix metalloproteinases (MMPs) (Boire et al., 2005; Sternlicht et al., 1999).

CAFs can maintain the myofibroblastic properties even after several passages *in vitro* without further signaling from carcinoma cells. How do CAFs acquire and maintain their activated phenotype? There are some controversial results with regard to the presence of somatic genetic alterations in CAFs. It has been reported that stroma microdissected from various human cancers exhibited some genetic alterations, such as chromosomal loss of heterozygosity (LOH) and somatic mutations (Currie et al., 2007; Kurose et al., 2002; Moinfar et al., 2000; Paterson et al., 2003; Tuhkanen et al., 2004; Wernert et al., 2001). Other reports also demonstrated that in the process of tumor development, fibroblasts that have lost p53 activity were clonally selected, leading to a highly proliferative stroma (Hill et al., 2005; Kiaris et al., 2005). In contrast, several genome-wide genetic analyses, including CGH and SNP arrays, were not able to detect any genetic alterations in the myofibroblasts isolated from various human cancers (Qiu et al., 2008; Walter et al., 2008). Other studies have suggested that epigenetic modifications within the genome of CAFs, such as DNA methylation, might be the reason (Hu et al., 2005; Jiang et al., 2008). Further studies are required to clarify these issues.
