Anti-cancer therapies can also result in non-apoptotic cell death , including necrosis , autophagy and mitotic catastrophe.

Necrosis was historically considered a ‘messy’ form of cell destruction, morphologically characterized by random degradation of nuclear DNA, organelle degeneration and swelling and rupture of the cell membrane with release of intracellular components [4]. Recent work, however, suggests that necrosis can occur in a regulated manner, which is dependent on specific signalling modules such as RIP1 activation and can be triggered by a number of processes including alkylating DNA damage and death receptor ligation [35–38]. Zong et al. [39] first demonstrated that in vitro cell death caused by the alkylating agents nitrogen mustard and MNNG (N-methyl-N-nitro-N-nitrosoguanidine) occurred independently of apoptotic factors, but required activation of the DNA repair protein poly (ADP-ribose) polymerase 1 (PARP1), suggesting a necrotic form of programmed cell death. More recently, the alkylating agent cyclophosphamide was shown to cause tumor regression in a xenograft mouse tumor system in both apoptosis-competent and apoptosis-deficient tumor cells, with the observation of sporadic necrosis in both groups, as identified using cell morphology, high mobility box group 1 (HMBG1) extracellular release and activation of innate immune cells [31]. In an effort to define the molecular signalling network that regulates necrotic pathways, Hitomi et al. [36] carried out a genome-wide siRNA screen and identified 432 genes that regulate necroptosis of which 32 acted downstream of regulators of RIP, 32 genes were required for death-receptor-mediated apoptosis and 7 genes that were involved in both pathways. Together, these data suggests that necrosis can occur in a regulated manner that is independent of apoptosis and may play a role in several physiological and pathological settings, but its role in tumorigenesis, in chemotherapy-induced cell death , or in the antitumor immune response is yet to be fully elucidated.

Autophagy , or ‘autophagic cell death’, is a process that leads to cytoplasmic vacuolization without features of apoptosis . It often constitutes a cytoprotective response activated by dying cells as a defense against acute stress, and inhibiting autophagy can actually mediate death rather than prevent it [40–42]. It is therefore unclear whether autophagy can be responsible for cell death , or rather is a process that can accompany cell death, without participating in the lethal catabolic cascade [11].

A third death mechanism, ‘mitotic catastrophe’, refers to cell death triggered by aberrant mitosis and occurring during mitosis or in subsequent interphase [43]. A number of chemotherapeutic agents, namely spindle poisons or microtubule inhibitors (taxanes and vinca alkaloids) interfere with the function of microtubules during mitosis, resulting in activation of the spindle assembly checkpoint (SAC) and causing mitotic arrest. The result is caspase-mediated cell death, but the molecular mechanism connecting initial SAC activation to caspase activation has not been clearly defined [44]. Interestingly, recent work has indicated that these mitotic aberrations can lead to cell senescence or cell death, which can occur either through apoptosis or necrosis [45, 46]. It was therefore proposed that mitotic catastrophe may not be a ‘pure’ cell death pathway but an oncosuppression mechanism that ultimately leads to cell death or senescence. Whether the duration of mitotic arrest truly has an influence on the cell fate remains unclear [11, 47].

Cellular senescence is a form of permanent cell cycle arrest with typical morphological and biochemical changes including the induction of senescence-associated b-galactosidase activity. It can be induced by cellular stresses such as DNA damage and oncogenic stress, which can be regulated by tumor suppressors including p53, p16^INK4a and retinoblastoma protein (RB) [4]. Cellular senescence may play a role in suppressing tumorigenesis by disabling apoptosis , as suggested by the early cancer development in murine cells with senescence defects [48, 49]. Moreover, senescence may contribute to chemotherapy sensitivity in primary murine lymphomas, where cyclophosphamide can lead to p16^INK4a activation, p53-dependent cell-cycle arrest and premature senescence in vivo [50].

Morphological classification of cell death dominated the cell death literature for several decades before the development of many of the biochemical tests available today. However, it is becoming increasingly clear that morphologically similar cell deaths may demonstrate significant functional, biochemical and immunological heterogeneity [43, 51, 52]. In addition, a specific morphological appearance may not necessarily be sufficient to establish a link between a causative process and subsequent cell death . Conversely, biochemical classifications also have drawbacks. For example, a cell death pathway frequently associated with a process may still occur in the absence of that process. Phosphatidylserine (PS) exposure, an early marker of cell death, was shown not to occur in autophagy-deficient cells undergoing apoptosis [53]. The Nomenclature Committee on Cell Death (NCCD), which has previously published two rounds of recommendations in 2005 and 2009, has formulated a novel systematic classification of cell death based on biochemical features intended for both in vitro and in vivo applications [11].

The immune system has long been thought to play a role in cancer development and surveillance . The multi-step process of carcinogenesis can lead to the accumulation of mutations that may affect the antigenic properties of proteins within tumor cells, which can in turn be presented by MHC class I molecules to CD8 ⁺  T cells . In addition, the oncogenic stress occurring in early tumor development can also lead to activation of immune responses by the innate and/or adaptive immune system that may destroy cells prior to development of overt malignancy. It is now accepted that the ability of tumor cells to evade immune destruction is one of the hallmarks of cancer [54, 55].

The original model of immune function described by Burnet [56] suggested that the immune system functions by differentiating between ‘self’, which is defined early in life, and ‘non self’, which represents anything that comes later. The model evolved over the years to accommodate new data and address incompatible findings such as somatic hypermutation of B cells and transplant rejection, and consequently the ‘danger model’ was proposed [57]. This model suggests that it is the damage or ‘danger’ that is of greater importance to the immune system, rather than ‘foreignness’. As such, established tumors that do not cause damage, i.e. do not elicit endogenous or exogenous danger signals through cellular stress or injury such as ‘immunogenic cell death ’, will not elicit an immune response and conversely ‘self-ness’ of healthy tissues does not guarantee tolerance. Further incarnations of the danger model lead to the current understanding that the immune system would seek to answer two questions when faced with a threat: firstly whether to respond or not—and this is determined by the nature of the threat as determined by ‘danger signals ’; and second, is what kind of immune response is elicited, a decision that will be primarily determined according to the tissue where the response occurs, rather than the nature of the invading pathogen [57, 58].

When we consider the immune response to a tumor that has escaped immune surveillance and destruction, the antitumor immune response can involve components of the innate immune system as well as the humoral (antibody-mediated) and cellular (T cell mediated) arms of the adaptive immune system. However, CD8⁺  cytotoxic lymphocytes are generally considered the most effective of the anti-tumor immune responses for a number of reasons. Firstly, the importance of CD8 ⁺  effector cells is highlighted by the observation that greater proportions and activation of CD8⁺  tumor infiltrating lymphocytes (TILs) correlates with a better prognosis in several cancers, as discussed in Sect. 1.4.2.3. Secondly, both treated and untreated tumors have been shown to grow more rapidly when CD8 ⁺  cells, but not NK cells or B cells are depleted from the tumor microenvironment [59, 60]. Moreover, some chemotherapy can selectively deplete B cells while preserving CD8 ⁺  function without obvious detriment to antitumor immunity [61]. The demonstrated efficacy of adoptive T-cell transfer therapy in some patients with advanced melanoma, and the use of graft versus leukaemia effect with donor leucocyte infusions for relapse after allogeneic bone marrow transplantation also highlight the prominent role for CD8 ⁺  T cells for antitumor immunity [62–64].

An understanding of the steps required to elicit an antitumor immune response, especially a specific CD8⁺  antitumor response, as well as the effect of various anticancer therapies on antitumor immunity is necessary to enable the interpretation as well as the rational design of preclinical and clinical studies. This will enable us to harness the immune response and utilize the expanding armament of immunotherapies that can be combined with chemotherapy to address immune evasion resulting from tumor or chemotherapy i.e. the ‘brakes’ on the immune response; and/or to synergize with the immune-stimulatory effects of treatment discussed below to achieve better outcomes with chemo-immunotherapy .

There are six key steps postulated for an effective antitumor CD8⁺T-cell response:

The induction of immunogenic cell death is an important goal for anticancer chemotherapy. Apoptotic cell death, the most common modality of chemotherapy-induced cell death, has classically been assumed to be non-immunogenic (or tolerogenic), whereas necrotic cell death was considered truly immunogenic. As millions of cells die every second by ‘physiological’ apoptosis, PS residues are exposed on the cell surface, which leads to the rapid and silent clearance of apoptotic bodies by macrophages and stimulates the production of immunosuppressive cytokines [65, 66], thus protecting the host from overwhelming inflammation and autoimmunity.

Other studies, however have challenged the theory that apoptosis is uniformly non-immunogenic, and demonstrated that the pre-apoptotic cellular responses to stress, the surface characteristics of apoptotic bodies and the process of apoptosis itself can be quite heterogeneous biochemically, despite apparently similar morphologies [28, 60, 67–69]. We have shown that in a murine model of malignant mesothelioma, chemotherapy-induced apoptosis increased antigen cross-presentation and cross-priming rather than tolerance, and demonstrated a synergistic effect between immunogenic chemotherapy and immunotherapy [25, 70].

Kroemer and colleagues demonstrated that although the chemotherapeutic drugs mitomycin C and doxorubicin both cause caspase-dependent apoptosis, only cell death caused by doxorubicin is immunogenic and mediated by dendritic cells (DCs) . They found that caspase inhibition by the broad-spectrum caspase inhibitor Z-VAD-fmk, or using cells transfected with the baculovirus caspase inhibitor p35 delayed, but did not inhibit doxorubicin induced death; and inhibited uptake of dead tumor cells by DCs, but had no effect on their ability to elicit DC maturation. Moreover, the selective depletion of dendritic cells (DCs) or CD8 ⁺  T cells , but not natural killer (NK) cells, abolished the antitumor immune response [60].

Although it has been shown that chemotherapy-induced cell death can be immunogenic, it is less clear which anti-cancer agents, and in what settings, can trigger danger signals that would be seen by the immune system and elicit an antitumor immune response. A number of features of dangerous cell death have been well described, and various chemotherapy agents have been analyzed in vitro and in vivo to determine their relative danger and immunogenicity .

Conventional cytotoxic chemotherapy has long been assumed to have either a neutral effect or, more commonly, a negative effect on the immune system. This was based on the inhibitory effect on proliferation of immune constituents, observations of clinical lymphopenia, and the production of apoptotic cell death, which was thought to be immunologically ‘bland’, as discussed above. In line with this, conventional cytotoxic drug development has involved the selection of agents that are active against human tumor cells in vitro and against transplanted xenografts in immunodeficient mice [99].

There is now a significant body of existing and emerging evidence that chemotherapy can have both immune-stimulatory and immune inhibitory effects over and above the distinctive properties of immunogenic vs. non-immunogenic cell death discussed above.