**3.3.1 Tumor-associated macrophages in cholangiocarcinoma**

As mentioned previously, cholangiocarcinoma shares a long-standing relationship with chronic inflammation (Gores 2003). Indeed, cholangiocarcinoma cells are known to overproduce many inflammatory cytokines, with IL-6 being the most studied (Isomoto et al. 2007). The role of TAMs in the development and progression of cholangiocarcinoma is poorly understood. However, recent studies have demonstrated that the density of infiltrating macrophages (as demonstrated using the MAC387 antibody to specifically stain macrophages) was high in over half of the tumor samples studied and that a high density of MAC387-positive cells correlated to a poor survival rate although conclusive proof that these cells were of the M2-phenotype is lacking (Subimerb et al. 2010a). Similarly, a subset of monocytes (CD14+CD16+) thought to be the precursors of tissue-resident macrophages are increased in the blood from cholangiocarcinoma patients, the levels of which were correlated with the density of MAC387-positive infiltrating macrophages (Subimerb et al. 2010b). The circulating CD14+CD16+ monocytes also expressed higher levels of angiogenic factors such as VEGF and the chemokine CXCL3 (Subimerb et al. 2010b). Lastly, Hasita et al. demonstrated that the macrophages infiltrating intrahepatic cholangiocarcinoma are mainly of the M2 phenotype (using CD163 as a marker of M2-type macrophages); their number correlates closely with neovascularization and infiltration of FOXP3+ regulatory T cells (Hasita et al. 2010). Furthermore, treatment of macrophages in culture with the supernatant from a number of CCA cells induced macrophage polarization toward the M2 phenotype and induced the macrophage-derived expression and secretion of VEGF-A, IL-10 and TGF (Hasita et al. 2010). Taken together, these data suggest that TAMs may play a role in cholangiocarcinoma progression. However, the molecules regulating the crosstalk between M2-type TAMs and cholangiocarcinoma cells needs to be further clarified.

### **3.4 Lymphangiogenesis in cancer**

Tumor metastasis is the most lethal aspect of cancer. The spread of tumor cells is often via the lymphatic vasculature and the presence of tumor foci in lymph nodes is considered an adverse prognostic factor in most carcinomas (Achen and Stacker 2008). Metastatic spread of tumor cells via the lymphatic system was previously thought to be via a passive process by

Logically, most TAMs have an M2-like phenotype (Mantovani and Sica 2010) thought to be brought about by various signals expressed within the tumor microenvironment, including interleukin-10, transforming growth factor- and colony stimulating factor-1 (Sica 2010). These signals responsible for the M2-activation of macrophages have been reported to come from myeloid-derived suppressor cells, IL-10+ B lymphocytes, Th2 subtype of T helper cells and the tumor cells themselves (Sica 2010). Once alternatively activated, TAMs exhibit reduced antitumor activities, while increasing the production of mediators of angiogenesis such as VEGF and IL-10 (Mantovani and Sica 2010), as well as M2-specific genes that are known to be involved in promoting cell proliferation (Mantovani and Sica 2010). These events are

It has been proposed that strategies to inhibit the M2- and activation of the M1-inducing signals may lead to the restoration of the anti-tumor functions of TAM and help to remove the protective signals originating from the M2 TAM (Sica and Bronte 2007), which may

As mentioned previously, cholangiocarcinoma shares a long-standing relationship with chronic inflammation (Gores 2003). Indeed, cholangiocarcinoma cells are known to overproduce many inflammatory cytokines, with IL-6 being the most studied (Isomoto et al. 2007). The role of TAMs in the development and progression of cholangiocarcinoma is poorly understood. However, recent studies have demonstrated that the density of infiltrating macrophages (as demonstrated using the MAC387 antibody to specifically stain macrophages) was high in over half of the tumor samples studied and that a high density of MAC387-positive cells correlated to a poor survival rate although conclusive proof that these cells were of the M2-phenotype is lacking (Subimerb et al. 2010a). Similarly, a subset of monocytes (CD14+CD16+) thought to be the precursors of tissue-resident macrophages are increased in the blood from cholangiocarcinoma patients, the levels of which were correlated with the density of MAC387-positive infiltrating macrophages (Subimerb et al. 2010b). The circulating CD14+CD16+ monocytes also expressed higher levels of angiogenic factors such as VEGF and the chemokine CXCL3 (Subimerb et al. 2010b). Lastly, Hasita et al. demonstrated that the macrophages infiltrating intrahepatic cholangiocarcinoma are mainly of the M2 phenotype (using CD163 as a marker of M2-type macrophages); their number correlates closely with neovascularization and infiltration of FOXP3+ regulatory T cells (Hasita et al. 2010). Furthermore, treatment of macrophages in culture with the supernatant from a number of CCA cells induced macrophage polarization toward the M2 phenotype and induced the macrophage-derived expression and secretion of VEGF-A, IL-10 and TGF (Hasita et al. 2010). Taken together, these data suggest that TAMs may play a role in cholangiocarcinoma progression. However, the molecules regulating the crosstalk between

trigger an innate immune response, thereby reducing tumor size (Sica 2010).

M2-type TAMs and cholangiocarcinoma cells needs to be further clarified.

Tumor metastasis is the most lethal aspect of cancer. The spread of tumor cells is often via the lymphatic vasculature and the presence of tumor foci in lymph nodes is considered an adverse prognostic factor in most carcinomas (Achen and Stacker 2008). Metastatic spread of tumor cells via the lymphatic system was previously thought to be via a passive process by

**3.4 Lymphangiogenesis in cancer** 

**3.3.1 Tumor-associated macrophages in cholangiocarcinoma** 

summarized in Figure 4.

which detached tumor cells enter pre-existing lymphatic vessels in the vicinity of the tumor (Achen and Stacker 2008). However, recent studies suggest that the formation of new lymphatic vessels in the tumor microenvironment correlates with lymphatic metastasis (Achen et al. 2005).

To date, the growth factors recognized to be associated with the control of lymphangiogenesis are similar to those that control angiogenesis. That is, the most characterized factors are VEGF-C and VEGF-D, which are secreted from the tumors, and then activate VEGFR-3 expressed on lymphatic endothelium (Lymboussaki et al. 1998). Activation of VEGFR-3 induces the proliferation of lymphatic endothelial cells *in vitro* (Makinen et al. 2001) and the formation of new lymphatic vessels *in vivo* (Veikkola et al. 2001). Other identified lymphangiogenic factors include VEGF-A (Nagy et al. 2002), fibroblast growth factor-2 (Kubo et al. 2002), angiopoietin-2 (Gale et al. 2002) and plateletderived growth factor-BB (Cao et al. 2004).

Because of the overlap in angiogenic and lymphangiogenic activity of the above-mentioned factors, agents designed to block angiogenesis may also be effective in blocking lymphangiogenesis. For example, inhibitors that block the VEGF-C/VEGF-D/VEGFR3 signalling mechanism might have the potential to not only block angiogenesis, but to also block lymphangiogenesis and hence to block lymphogenous metastatic spread (Baldwin et al. 2002; Stacker et al. 2002a; Stacker et al. 2002b). Indeed, a neutralizing VEGF-D monoclonal antibody designed to block the interaction between VEGF-D and its receptors, inhibited angiogenesis, lymphangiogenesis and metastatic spread via the lymphatics in a mouse tumor model (Stacker et al. 2001). Further studies into therapeutic strategies designed to block lymphangiogenesis are required in an attempt to stop the metastatic spread of tumors.
