The DDR is orchestrated by two conserved protein kinases, ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3 related), which in concert control the cellular response to DNA double strand breaks (DSBs) and single-stranded (ss) DNA (Smith et al., 2010). ATM is recruited to DSBs by the Mre11–Rad50–Nbs1 (MRN) complex and phosphorylates the histone H2 variant H2AX, thus generating an interaction platform for other DDR constituents required for DSB repair (Shiloh, 2006). Concomitantly, ATM activates the DNA damage checkpoint by initiating the resection of the broken strand(s) leading to the generation of ssDNA, a DNA damage repair intermediate which, in turn, activates ATR kinase (Hurley and Bunz, 2007). ATR and ATM phosphorylate and activate two respective effector kinases termed Chk1 and Chk2. Collectively, these four protein kinases instigate multiple cellular pathways culminating in transient or permanent cell cycle arrest, DNA damage repair, and/or cell death (Jackson and Bartek, 2009). A crucial target of the ATM/ATR cascade is the tumor suppressor protein p53, a transcription factor, whose function is lost or compromised in more than 50% of all cancers (Levine, 1997). Under steady-state conditions, p53 protein is sustained at very low levels, since it is continuously being ubiquitinylated by the Mdm2 ubiquitin ligase and thus targeted for subsequent proteolytic degradation via the 26S proteasome. Yet, phosphorylation of p53 by kinases of the ATM/ATR pathway induces the dissociation of p53 from MDM2, and the subsequent reduction in p53 ubiquitinylation leads to a deceleration of proteasome-mediated degradation and hence a stabilization of p53 (Meek, 2009). Depending on the cell cycle phase and the type as well as the extent of DNA damage, p53 can function as a modulator of the DNA damage repair process, or as a transcriptional activator of genes, which are involved in transient or permanent cell cycle arrest, and/or cell death, respectively (Sengupta and Harris, 2005).

Apoptosis is one type of programed cell death. It is commonly considered to be the prevalent form of cell death underlying daily tissue regeneration and renewal. Morphologically, it is characterized by cellular shrinkage, chromatin condensation, nuclear fragmentation, and membrane blebbing (Figure 1). In response to radiotherapy, apoptosis is predominantly observed in cells of the hematopoietic system, and it is critically regulated by the mitochondrial, intrinsic death pathway (Rudner et al., 2001; Eriksson and Stigbrand, 2010). The pivotal events in this context involve the permeabilization of the mitochondrial outer membrane (MOMP) and the release of various proteins, including cytochrome c, from the mitochondrial intermembrane space into the cytosol, thus stimulating the formation of the apoptosome and the activation of procaspase-9. Activated caspase-9, in turn, triggers the activation of downstream effector caspases, which execute the final stages of apoptosis and the disintegration of the cell (Taylor et al., 2008). Crucial regulators of mitochondrial permeabilization and cytochrome c release are proteins of the B cell lymphoma-2 (Bcl-2) family, including the pro-apoptotic BH3-only (e.g., Puma) and the anti-apoptotic (e.g., Bcl-2) family members, which control MOMP via their impact on the oligomerization of the effector members Bax and Bak (Youle and Strasser, 2008). p53 links this signaling pathway to radiation-induced DNA damage by transactivating the expression of pro-apoptotic Bcl-2 family members, such as Puma and Noxa (Sengupta and Harris, 2005). Apart from the intrinsic pathway, apoptosis can be induced extrinsically via the ligation of death receptors, such as CD95 or the TRAIL receptors 1 and 2, by their corresponding ligands (Debatin and Krammer, 2004). Receptor clustering leads to recruitment and activation of the pro-caspases-8 and -10, triggering of the caspase cascade, and thus to apoptosis. Various proteins of the death receptor pathway are known to be upregulated in response to ionizing radiation (p53-dependently as well as -independently) and thus might contribute to apoptosis induction (Belka et al., 1998; Haupt et al., 2003). However, the intrinsic death pathway appears to be the major signaling mechanism of irradiation-induced apoptosis (Rudner et al., 2001). Notably, although p53 essentially controls the expression of various key regulators of apoptosis, irradiation-induced apoptosis can be observed in cancer cells with defective p53 function. Here, mechanisms, such as p63-/p73-dependent induction of pro-apoptotic Bcl-2 members and p53-independent stimulation of death receptor signaling have been described to be involved (Afshar et al., 2006; Wakatsuki et al., 2008).

In tumor cells of epithelial origin, which reveal limited apoptosis induction in response to radiotherapy, radiation-induced DNA damage – especially when combined with hyperthermia - has been reported to stimulate necroptosis (Mantel et al., 2010; Schildkopf et al., 2010; Figure 1). The crucial events in this context include the hyperactivation of the DNA repair enzyme poly-ADP-ribose-polymerase (PARP) and the subsequent and substantial depletion of intracellular ATP levels (Vandenabeele et al., 2010; Vanlangenakker et al., 2012). This – in a so far poorly understood way – couples to the activation of receptor interacting protein (RIP), the formation of the high-molecular weight necrosome, and finally the execution of necroptosis as characterized by the production of reactive oxygen species (ROS), lipid peroxidation, swelling of organelles, rupture of the plasma membrane, and release of intracellular contents (Vandenabeele et al., 2010). Apart from necroptosis, ionizing radiation – particularly when applied in high single doses during ablative radiotherapy – can trigger necrosis, an accidental, uncontrolled form of cell death as a consequence of excessive physico-chemical stress (Vandenabeele et al., 2010). Moreover, secondary necrosis can occur when apoptotically dying cells are not properly and timely engulfed by neighboring cells or professional phagocytes, respectively (Munoz et al., 2010a; Silva, 2010). This is of specific relevance when the local phagocytic compartment is overwhelmed due to massive apoptosis induction in the context of tumor radiotherapy. In both cases the integrity of the plasma membrane is lost and cellular contents, often in an oxidatively modified and partially degraded form, leak into the surrounding tissue.

Radiation-induced senescence is a condition of permanent cell cycle arrest, which can be observed in cells, where DNA damage is excessive and cell cycle checkpoints are still intact (Figure 1). The hallmarks of cellular senescence include an enlarged and flattened cellular morphology, increased granularity, upregulation of cyclin-dependent kinase inhibitors, such as p16^INK4a, p21^Waf1, and p27^Kip1, and positive staining for the senescence-associated β-galactosidase (SA-β-Gal). The key players in this scenario are p53 and pRB, yet senescent phenotypes have also been reported in the absence of functional p53 (Nardella et al., 2011). Senescent cells exit the cell cycle and do not further undergo cell division, but may remain metabolically active. Interestingly, they have been shown to release factors, which can be tumor suppressing as well as tumor promoting, and which can alter the immune response (Kuilman and Peeper, 2009; Coppe et al., 2010).

Higher organisms have developed impressively efficient mechanisms of dying cell clearance as can be appreciated from the fact that dying cells are rarely to be observed in normal tissues, although – according to careful estimations – approximately one million cells per second undergo apoptosis in the course of everyday tissue turnover and regeneration (Reed, 2006). These cells are swiftly phagocytosed and degraded, and the immune system had to acquire the capability to distinguish them from cells, which are dying in the context of an infection. Whereas several types of amateur phagocytes, including fibroblasts, endothelial cells, and mesothelial cells, have been described, professional phagocytes, such as macrophages and dendritic cells (DCs), apparently play a crucial role in this scenario, particularly when the local amateur phagocytic compartment is overwhelmed (Lauber et al., 2004; Ravichandran, 2011; Wagner et al., 2011). Macrophages and DCs serve as professional dying cell scavengers with discrepant tasks. While tissue resident macrophages can proficiently engulf and degrade huge amounts of dying prey cells, DCs act as sentinels, which are highly potent in presenting and cross-presenting antigens, thus stimulating, skewing, or inhibiting adaptive immune responses, respectively (Steinman, 2007; Biswas and Mantovani, 2010).

The sophisticated process of dying cell removal involves distinct phases: phagocyte recruitment, prey cell engulfment, and the post-phagocytic response (Ravichandran, 2010; Figure 2).

It is well acknowledged that the induction of tumor cell death and the abrogation of clonogenic survival by ionizing irradiation are key determinants of its therapeutic success. However, there is accumulating experimental evidence that particularly in the context of ablative radiotherapy, during which radiation is applied in high single doses of 10 Gy or more, complex immune mechanisms contribute to tumor regression. One of the initial reports on this issue showed that local high dose radiation therapy of transplanted mouse B16 melanoma stimulates the generation of tumor antigen-specific, IFN-γ producing T cells (Lugade et al., 2005). In the same mouse model, ablative, but not fractionated radiotherapy was observed to drastically enhance T cell priming in tumor draining lymph nodes paralleled by a reduction/eradication of the primary tumor as well as distant metastases in a CD8⁺ T cell-dependent manner (Lee et al., 2009). A mechanistic explanation of how these T cells are primed was provided by a recent study showing that the intra-tumoral production of IFN-α/β in response to high dose radiotherapy enhances the cross-presenting capacity of tumor infiltrating DCs (Burnette et al., 2011). Hence, it appears that ablative radiotherapy triggers a temporal cascade of IFNs, which is well-known from the field of tumor immunoediting, where IFN-α/β produced by CD11c⁺ cells (presumably DCs and macrophages) enhances the cross-priming activity of CD8α⁺ DCs, thus stimulating the generation of IFN-γ producing CD8⁺ T cells, and finally tumor rejection (Figure 5; Diamond et al., 2011; Fuertes et al., 2011). Notably, there is a clear difference in how IFN-α/β and IFN-γ contribute to a reduction in tumor burden. IFN-α/β predominantly affects macrophages, DCs, and NK cells leading to their activation and/or maturation, the upregulation of chemokine expression, the enhancement of antigen-presentation and cross-presentation by DCs and a robustly augmented induction of adaptive immune responses (Dunn et al., 2006). Consequently, in the context of cancer immunoediting, IFN-α/β-responsiveness is specifically required in DCs and macrophages, whereas it appears to be dispensable in tumor cells (Diamond et al., 2011; Fuertes et al., 2011). Nevertheless, tumor rejection in response to radiotherapy might also be improved – at least in part – by a direct effect of IFN-α/β on the tumor via its reported capacity of radiosensitization (Morak et al., 2011). In strong contrast to IFN-α/β, tumor cell responsiveness to IFN-γ obviously is an essential prerequisite for the development of anti-tumor immune responses. Various anti-tumor mechanisms have been reported to be exerted by IFN-γ, including inhibition of tumor cell proliferation, apoptosis induction, inhibition of angiogenesis, and an overall enhancement of tumor immunogenicity as characterized by an upregulation of the MHC-I pathway, modulation/extension of the MHC-I ligandome, and downregulation of NKG2D ligands (Dunn et al., 2006; Reits et al., 2006; Lugade et al., 2008). Notwithstanding these direct effects on the tumor, IFN-γ is key for the stimulation of an anti-tumor immune response. As such, IFN-γ is vitally involved in T_H1/T_C1 cell differentiation/activation, and it exerts similar effects as IFN-α/β in terms of innate immune cell activation and the promotion of DC-mediated antigen cross-presentation (Dunn et al., 2006).

Once more it should be emphasized that this IFN-controlled cascade of innate and adaptive immune responses was only observed in case of ablative but not conventional, fractionated radiotherapy (Lee et al., 2009). A feasible explanation for this might be that ablative and fractionated radiotherapy trigger different modalities of tumor cell death with only high dose irradiation stimulating primary and/or secondary necro(pto)sis. The corresponding liberation of danger signals, including HMGB-1 and ATP, in turn stimulates the TLR4-dependent production of IFN-α/β and other pro-inflammatory cytokines, initiating the above described IFN cascade and the DC-mediated instigation of anti-tumor T cell responses (Apetoh et al., 2007).

A key question that arises from an immuno-radiotherapeutic point of view at this point is: How can the process of dying cell clearance be instrumentalized or manipulated in order to enhance the efficacy of fractionated radiotherapy? Various putative approaches can be envisioned in this regard (Figure 6). The observations made with high dose ablative radiotherapy suggest that the temporary induction of primary/secondary tumor cell necro(pto)sis might be beneficial. Initial studies on this issue in fact provide evidence that the combination of radiotherapy with hyperthermia results in the induction of an immunogenic type of cell death as characterized by the release of danger signals, including HMGB-1 and HSP70, which foster the maturation of DCs in vitro (Schildkopf et al., 2009, 2010, 2011; Mantel et al., 2010). However, it remains to be elucidated, whether this translates into the productive stimulation of anti-tumor T cell responses in vivo. Alternatively, radiotherapy might be combined with photodynamic therapy (PDT), since PDT has been reported to induce the expression of HSPs and the release of danger signals (Castano et al., 2006). Alone or in combination with chemotherapy PDT leads to the stimulation of anti-tumor T cell responses in vivo (Castano et al., 2008; Kammerer et al., 2011). Yet, if this holds also true for its combination with radiotherapy awaits further clarification. For tumors, which predominantly undergo apoptosis in response to fractionated radiotherapy, caspase inhibition might represent an approach to overcome the tolerogenic nature of this form of cell death, since caspase inhibition in apoptosing cells has been reported to directly trigger necroptosis (He et al., 2009b). However, caspase inhibition might also confer radioresistance due to an overall inhibition of cell death. Therefore, the applicability of this approach has to be very carefully evaluated.

Directly interfering with apoptotic cell clearance in order to promote the accumulation of secondary necrotic tumor cell material might be an alternative approach to instigate the IFN cascade described above and a concomitant anti-tumor immune response. In this regard, annexin A5 and a mutant form of MFG-E8, which no longer is able to bind to the vitronectin receptor, might be valuable tools (Hanayama et al., 2002; Bondanza et al., 2004; Gaipl et al., 2007b; Munoz et al., 2007a,b; Frey et al., 2009). In a mouse model of tumor vaccination, annexin A5 has been shown to impair the uptake of irradiated apoptotic lymphoma cells by macrophages and to specifically target them to CD8α⁺ DCs, thus inducing the release of proinflammatory cytokines and tumor-specific immune memory, which contributed to the regression of growing tumors and conferred resistance against tumor re-challenge (Bondanza et al., 2004). Redirecting dying cell clearance toward Fcγ receptor-mediated phagocytosis also appears a promising strategy for the induction of anti-tumor immune responses. For example, opsonization with autoantibodies has been reported to potently trigger FcyR-dependent phagocytosis of dying cells and subsequent pro-inflammatory cytokine production (Manfredi et al., 1998; Sarmiento et al., 2007; Grossmayer et al., 2008). Hence, “eat-me” signal specific antibodies, including anti-PS antibodies, or tumor antigen specific antibodies, like anti-HSP70 antibodies, might help to utilize pro-inflammatory dying cell phagocytosis for the induction of anti-tumor immunity (Ran et al., 2005; Gehrmann et al., 2008; He et al., 2009a; Stangl et al., 2011a,b). Engineering these antibodies into bi-specific antibodies coupling to Clec9A, is of special interest in this regard, since thereby dying tumor cell material could be specifically targeted toward the recycling endosomal compartment, thus favoring cross-presentation in BDCA3⁺ DCs, the alleged human equivalent to mouse CD8α⁺ DCs (Schreibelt et al., 2012). Seminal data obtained with different mouse models of viral vaccination point to the direction that this represents a successful approach of inducing vaccine-specific immune responses (Idoyaga et al., 2011; Iborra et al., 2012; Zelenay et al., 2012). It should be noted that Clec9A ligation alone is not sufficient to induce DC maturation and a concomitant adaptive immune response against dying cells (Ahrens et al., 2012). Coligation of additional receptors for DAMPs instead appears to be required. Research in the field of autoimmunity has convincingly shown that dying cell-derived nucleosomal material is very potent in this regard (Frisoni et al., 2007; Urbonaviciute et al., 2008). Hence, interfering with DNase II activity, which in phagocytes mediates the proper degradation of engulfed prey cell DNA, could represent a promising strategy to stimulate the described IFN cascade required for the induction of productive immune responses and well-known for its crucial role in the etiopathogenesis of autoimmune diseases, such as SLE (Kawane et al., 2006; Marshak-Rothstein, 2006; Deng and Tsao, 2010). To this end, cell-permeable, small molecule inhibitors of DNase II would be helpful, and it remains to be elucidated whether such compounds can technically be developed. Finally, further studies are required to improve and expand our knowledge on the process of senescent cell clearance with special focus on its applicability and potential utilization for the enhancement of tumor immunogenicity in the context of fractionated radiotherapy.

Undoubtedly, the induction of tumor cell death and the abrogation of clonogenic survival by ionizing irradiation are key to its therapeutic success. However, pioneering immuno-radiotherapeutic studies have convincingly shown that a contribution of complex immune mechanisms – particularly in the context of ablative radiotherapy – can no longer be neglected. The clearance of dying tumor cells by phagocytic cells of the innate immune system represents a crucial initiating step in this scenario. Valuable lessons can be learned from research in the field of autoimmunity on how the form of cell death skews the subsequent immune response, which cell types and molecules are involved in this context, and how these might be utilized for the induction of productive anti-tumor immune responses in combination with radiotherapy. Notably, these immune responses would not only target the local tumor but could also reach distant, out-of-field metastases. To this end, combined, multi-modal treatment regimes have to be developed with the capacity to induce immunogenic forms of tumor cell death and concomitantly activate the immune system. The correct timing will be an essential parameter affecting the therapeutic success of these approaches, since the priming of adaptive immune responses takes place in the tumor draining lymph nodes, which due to the risk of tumor cell spread and metastasis formation are commonly removed by surgery. Hence, it might be worth considering – carefully and on an individualized basis – to postpone lymph node surgery until the initial anti-tumor immune priming has successfully been accomplished. Not only in this regard, the close collaboration of clinical radiation oncologist, surgeons, radiobiologists, molecular oncologists, and immunologists is indispensable in order to develop and optimize the personalized therapeutic regime with the highest benefit for each individual patient.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.