**3.3 Immune evasion in PCa**

The most common immunosuppressive factors in the tumour tissue include vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β), interleukin (IL)- 10 and adenosine which inhibit proliferation, differentiation and activation of T lymphocytes and DC.

VEGF supports tumour growth and metastasis by initiating endothelial cell proliferation and the formation of new blood vessels, thus providing a continuous blood supply to the tumour (Carmeliet & Jain, 2000). Increased angiogenesis has been observed in PCa tissue compared to benign prostatic hyperplasia (BPH) (Jackson et al., 1997). VEGF production by PCa cells in vitro is enhanced by addition of IL-1 and tumour necrosis factor alpha (TNF-α), both of which are found in the tumour microenvironment in PCa (Ferrer et al., 1997). VEGF is also important in immune suppression not only by inhibiting DC maturation and T cell development but also by acting as a chemoattractant for MDSC (Gabrilovich et al., 1996; Ohm et al., 2003; Oyama et al., 1998).

TGF-β is a pleiotropic cytokine. During PCa development it first acts as a tumour suppressor and later switches roles to become a tumour promoter and immunosuppressor in the tumour environment. TGF-β regulates immune cells by inhibiting cytotoxic T cell function, supporting the development of Treg cells and interfering with DC differentiation (Wan & Flavell, 2007; Wrzesinski et al., 2007). PCa-derived TGF-β has been shown to convert CD4+CD25- T cells into CD4+ CD25+ Foxp3+ Treg cells (Liu et al., 2007).

The anti-inflammatory cytokine IL-10 secreted by tumour and stromal cells can inhibit proliferation, differentiation and activation of T lymphocytes via impaired DC (Sato et al., 2002). In the presence of IL-10, alternatively activated DC maintain an immature phenotype in the tumour microenvironment and induce tolerance rather than immune activation and support Treg cell development (H Huang et al., 2010).

Extracellular adenosine is generated from ATP or ADP via the combined action of CD39 and CD73. These are ecto-nucleoside triphosphate diphosphohydrolases which, in the tumour tissue, are predominantly expressed on Treg cells while CD73 is also expressed on CD8+ T cells and tumour cells (CD73) (Stagg & Smyth, 2010). CD39 and CD73 are also expressed on

Combination of Immunotherapy & Radiotherapy for the Treatment of Prostate Cancer 201

Tumour cells, not killed by IR, respond to radiation by increasing the production of proinflammatory cytokines that include TNFα, IL-1α/β, IL-6 and IL-8 (Shiao & Coussens, 2010; Formenti & Demaria, 2009; Van Der Meeren et al., 1999; Matsumura & Demaria, 2010). The main effect of these cytokines is an inflammatory response and the recruitment of activated T cells and macrophages to irradiated tumours (Formenti & Demaria, 2009). The widely used PCa cell line, LNCaP, is extremely sensitive to TNFα (Chopra et al., 2004). TNFα-treatment results in growth-arrest and apoptosis of LNCaP cells but not of normal prostate epithelial cells, suggesting that IR-induced TNFα expression may selectively induce apoptosis of tumour cells without affecting normal prostate epithelial cells. Immune cell recruitment is further enhanced by the production of the chemokine CXCL16 which is induced by IL-1β and TNFα, both upregulated by IR (Lu et al., 2008). PCa cell lines (LNCaP, DU145, C4-2B and PC3) produce CXCL16 in culture without IR

In tumour cells, not killed by IR, surface molecules such as MHC, the death receptor Fas and heat-shock proteins become upregulated (Shiao & Coussens, 2010; Garnett et al., 2004; Lugade et al., 2008). IR-induced upregulation of MHC Class I molecules, on both tumour cells and APC, improves antigen presentation and may enhance tumour cell recognition by activated CD8+ cells that infiltrate the tumour at an enhanced rate following radiation (Reits

Adhesion molecules, such as intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and platelet endothelial cell adhesion molecule (PECAM)-1, along with integrins, selectins and cadherins are also upregulated in the tumour tissue by IR (Baluna et al., 2006; Lugade et al., 2008). ICAM-1 is known to be upregulated by inflammatory cytokines such as TNFα, IL-1α/β and IL-6 thus resulting in lymphocyte and macrophage accumulation in inflamed tissues. As previously discussed, these cytokines are upregulated in the tumour tissue as a response to IR. ICAM-1 has an important role in enhancing T cell killing via cell-cell adhesion to lymphocyte function-associated antigen (LFA)-1 and by directly co-stimulating activated T cells (Garnett et al., 2004; Baluna et al., 2006). PCa cells express ICAM-1 and VCAM-1 and in tissue areas of high lymphocyte and neutrophil accumulation the expression of ICAM-1 is significantly elevated (Fujita et al., 2008; Rokhlin & Cohen, 1995). These data suggest that ICAM-1 upregulation by IR may facilitate immune responses by recruiting lymphocytes and macrophages to the tumour site. Increased adhesion between tumour cells with upregulated ICAM-1 and activated CD8+ T

**4.3 Irradiated tumour cells become sensitive to immune cell attack** 

cells expressing LFA-1+ may result in more powerful cytotoxic T cell activity.

Immune cells are highly susceptible to IR-induced damage and readily undergo apoptosis. Therapeutic doses of RT often result in lymphopenia. One of the potential immunologically positive effects of direct IR on tumour-infiltrating immune cells is the depletion of Treg cells. However, there is some controversy regarding this question. Cao et al. observed that the proportion of Foxp3+ cells within purified CD4+CD25+ T cell population decreased significantly (48.2% to 23.3%) following irradiation with 1.8 Gy in vitro and was abolished (1.2%) by 30 Gy. The suppressive function of these Treg cells was also impaired by IR

**4.2 Chemokine and cytokine induction by IR** 

treatment (Lu et al., 2008).

et al., 2006; Lugade et al., 2005).

**4.4 Direct effect of IR on immune cells** 

tumour-derived exosomes, thus potentially extending the immunosuppressive tumour microenvironment (Clayton et al., 2010). Adenosine, generated by CD39 (ATP to ADP) and CD73 (ADP and AMP to adenosine) engage androgen receptors on effector T cells, monocytes and DC. As a consequence, pro-inflammatory cytokine production (IL-2, IL-4, IFNγ, TNFα, IL-12) and co-stimulatory molecule expression (e.g. CD86) decreases and cyclic AMP (cAMP) accumulates in these cells. cAMP further amplifies anti-inflammatory responses (Ernst et al., 2010).

Cytokines, such as monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor-1 alpha (SDF-1α) secreted by tumour cells and tumour-associated stromal cells have been associated with enhanced prostate epithelial cell proliferation and migration (Begley et al., 2005; Lu et al., 2006). They also induce the recruitment of myeloid cells at the tumour site and suppress immune responses (Allavena et al., 2008; Loberg et al., 2007). TAM also produce MCP-1 resulting in an amplification loop for further monocyte recruitment (Allavena et al., 2008).

### **4. Immunological aspects of radiation therapy**

The effects of IR on human tissue have been studied for decades. The key therapeutic effect is thought to be via direct killing of tumour cells by initiating irreparable double-stranded DNA breaks. However, the consequences of RT are much more complex than that, as radiation also affects tumour stroma, including tumour-resident immune cells, and results in the re-modelling of the tumour microenvironment. One of the consequences is the reduction of immunosuppression in the tumour tissue.

#### **4.1 IR-mediated release of cellular tumour antigens**

The details of the multiple cellular events downstream of radiation-induced DNA damage are beyond the scope of this chapter. The damage results in activation of damage recognition pathways and proliferative arrest, which can ultimately be repaired (fully or partially), or lead to cell death. RT-induced apoptotic and necrotic tumour cell death provide a cellular source of tumour antigens. The tissue damage attracts phagocytic cells to the site of radiation. Monocytes, macrophages and dendritic cells (DC) phagocytose and process dead tumour cells and carry TAA into draining lymph nodes where antigen presentation and T cell stimulation occur. Contrary to natural cell death which occurs without generating an inflammatory response, IR-treated tumour cells express heat shock proteins, translocate antigens such as calreticulin (CR) from the endoplasmic reticulum to the cell surface and passively release high mobility group protein B1 (HMGB1). DNA, RNA and ATP release are also observed at the site of radiation damage. Phagocytosis and simultaneous signalling in DC by HMGB1 via Toll-like receptors (TLR) such as TLR4 and TLR2, or via RAGE (receptor for advanced glycan end products) trigger DC to release IL-1β and present TAA in an immunogenic manner to T cells and B cells (Ma et al., 2011). ATP, released by damaged cells, also contributes to DC maturation via stimulating purinergic P2RX7 receptors and driving IL-1β secretion (Aymeric et al., 2010). Local radiation of mouse B16 tumour has generated DC efficiently cross-present tumour antigens in a Type I interferon (IFN)-dependent manner (Burnette et al., 2011). Single nucleotide polymorphisms (SNP) of TLR4 (Asp299Gly and Thr399Ilr) or P2RX7 (Glu496Ala) (Sluyter et al., 2004; Arbour et al., 2000), which affect the function of these molecules, may be associated with worse prognosis, as shown in breast cancer patients undergoing chemotherapy (Apetoh et al., 2007).

#### **4.2 Chemokine and cytokine induction by IR**

200 Prostate Cancer – Diagnostic and Therapeutic Advances

tumour-derived exosomes, thus potentially extending the immunosuppressive tumour microenvironment (Clayton et al., 2010). Adenosine, generated by CD39 (ATP to ADP) and CD73 (ADP and AMP to adenosine) engage androgen receptors on effector T cells, monocytes and DC. As a consequence, pro-inflammatory cytokine production (IL-2, IL-4, IFNγ, TNFα, IL-12) and co-stimulatory molecule expression (e.g. CD86) decreases and cyclic AMP (cAMP) accumulates in these cells. cAMP further amplifies anti-inflammatory

Cytokines, such as monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor-1 alpha (SDF-1α) secreted by tumour cells and tumour-associated stromal cells have been associated with enhanced prostate epithelial cell proliferation and migration (Begley et al., 2005; Lu et al., 2006). They also induce the recruitment of myeloid cells at the tumour site and suppress immune responses (Allavena et al., 2008; Loberg et al., 2007). TAM also produce MCP-1 resulting in an amplification loop for further monocyte recruitment

The effects of IR on human tissue have been studied for decades. The key therapeutic effect is thought to be via direct killing of tumour cells by initiating irreparable double-stranded DNA breaks. However, the consequences of RT are much more complex than that, as radiation also affects tumour stroma, including tumour-resident immune cells, and results in the re-modelling of the tumour microenvironment. One of the consequences is the

The details of the multiple cellular events downstream of radiation-induced DNA damage are beyond the scope of this chapter. The damage results in activation of damage recognition pathways and proliferative arrest, which can ultimately be repaired (fully or partially), or lead to cell death. RT-induced apoptotic and necrotic tumour cell death provide a cellular source of tumour antigens. The tissue damage attracts phagocytic cells to the site of radiation. Monocytes, macrophages and dendritic cells (DC) phagocytose and process dead tumour cells and carry TAA into draining lymph nodes where antigen presentation and T cell stimulation occur. Contrary to natural cell death which occurs without generating an inflammatory response, IR-treated tumour cells express heat shock proteins, translocate antigens such as calreticulin (CR) from the endoplasmic reticulum to the cell surface and passively release high mobility group protein B1 (HMGB1). DNA, RNA and ATP release are also observed at the site of radiation damage. Phagocytosis and simultaneous signalling in DC by HMGB1 via Toll-like receptors (TLR) such as TLR4 and TLR2, or via RAGE (receptor for advanced glycan end products) trigger DC to release IL-1β and present TAA in an immunogenic manner to T cells and B cells (Ma et al., 2011). ATP, released by damaged cells, also contributes to DC maturation via stimulating purinergic P2RX7 receptors and driving IL-1β secretion (Aymeric et al., 2010). Local radiation of mouse B16 tumour has generated DC efficiently cross-present tumour antigens in a Type I interferon (IFN)-dependent manner (Burnette et al., 2011). Single nucleotide polymorphisms (SNP) of TLR4 (Asp299Gly and Thr399Ilr) or P2RX7 (Glu496Ala) (Sluyter et al., 2004; Arbour et al., 2000), which affect the function of these molecules, may be associated with worse prognosis, as shown in breast cancer patients undergoing

responses (Ernst et al., 2010).

(Allavena et al., 2008).

**4. Immunological aspects of radiation therapy** 

reduction of immunosuppression in the tumour tissue.

**4.1 IR-mediated release of cellular tumour antigens** 

chemotherapy (Apetoh et al., 2007).

Tumour cells, not killed by IR, respond to radiation by increasing the production of proinflammatory cytokines that include TNFα, IL-1α/β, IL-6 and IL-8 (Shiao & Coussens, 2010; Formenti & Demaria, 2009; Van Der Meeren et al., 1999; Matsumura & Demaria, 2010). The main effect of these cytokines is an inflammatory response and the recruitment of activated T cells and macrophages to irradiated tumours (Formenti & Demaria, 2009). The widely used PCa cell line, LNCaP, is extremely sensitive to TNFα (Chopra et al., 2004). TNFα-treatment results in growth-arrest and apoptosis of LNCaP cells but not of normal prostate epithelial cells, suggesting that IR-induced TNFα expression may selectively induce apoptosis of tumour cells without affecting normal prostate epithelial cells. Immune cell recruitment is further enhanced by the production of the chemokine CXCL16 which is induced by IL-1β and TNFα, both upregulated by IR (Lu et al., 2008). PCa cell lines (LNCaP, DU145, C4-2B and PC3) produce CXCL16 in culture without IR treatment (Lu et al., 2008).
