Targeting immunogenic cell stress and death for cancer therapy

Abstract

Immunogenic cell death (ICD), which results from insufficient cellular adaptation to specific stressors, occupies a central position in the development of novel anticancer treatments. Several therapeutic strategies to elicit ICD - either as standalone approaches or as means to convert immunologically cold tumours that are insensitive to immunotherapy into hot and immunotherapy-sensitive lesions - are being actively pursued. However, the development of ICD-inducing treatments is hindered by various obstacles. Some of these relate to the intrinsic complexity of cancer cell biology, whereas others arise from the use of conventional therapeutic strategies that were developed according to immune-agnostic principles. Moreover, current discovery platforms for the development of novel ICD inducers suffer from limitations that must be addressed to improve bench-to-bedside translational efforts. An improved appreciation of the conceptual difference between key factors that discriminate distinct forms of cell death will assist the design of clinically viable ICD inducers.

Figure 1.. Conceptual differences between immunogenic and non-immunogenic cell death.

Depending on a number of parameters, dying cells can: (1) be largely ignored by the innate and adaptive immune system, a physiological scenario generally reflecting the rapid uptake of dying cells or their corpses by macrophages; (2) be actively tolerogenic, i.e., promote the establishment of antigen-specific peripheral tolerance upon the activation of regulatory T (T_REG) cells that generally results from antigenicity in the absence of proper adjuvant signals; (3) activate innate (but not adaptive) immune mechanisms, notably inflammatory responses, as a result of abundant adjuvanticity in the absence of antigens that can be recognized by the mature T cell repertoire; or (4) overtly immunogenic, when cell death occurs in the context of sufficient adjuvanticity, failing adaptation to stress coupled with the emission of damage-associated molecular patterns (DAMPs) and pro-inflammatory cytokines, and under microenvironmental conditions that are permissive for dendritic cell recruitment, activation and consequent T cell priming (at cell death sites) as well as for T cell infiltration and effector functions (where the cellular targets of adaptive immunity reside). CTL, cytotoxic T lymphocyte; DC, dendritic cell.

Figure 2.. Core mechanisms of immunogenic cell stress and death.

The perception of cell stress and death as overtly immunogenic (i.e., resulting in the activation of adaptive immune responses specific for dead cell-associated antigens) mechanistically relies on the following core components: (1) the site of cell death must contain or be permissive for the recruitment dendritic cells (DCs) or their precursors, and must not be dominated by immunosuppressive signals that may prevent DC activation (microenvironmental conditions for priming); (2) cell death must occur in the context of unsuccessful responses to stress (cytotoxicity) that are associated with the spatiotemporally regulated emission of damage-associated molecular patterns (DAMPs) and cytokines that: (a) unless the site of cell death is abundantly infiltrated by immune cells a priori, recruit DC precursors, (b) attract DC precursors to the close proximity of dying cells, (c) promote the phagocytic uptake of dying cells or material thereof by DC precursors, (d) provide robust immunostimulatory signals to DC precursors for their maturation and functional licensing, and (e) enable mature DCs to reach secondary or tertiary lymphoid structures and to cross-prime antigen-specific T lymphocytes (adjuvanticity); (3) dying cells must express antigenic determinants that can be recognized by the mature T cell repertoire (antigenicity); and (4) the microenvironment of cells targeted by such an adaptive immune responses is permissive for infiltration by antigen-specific T lymphocytes and the activation of their effector functions (microenvironmental conditions for effecting). ANXA1, annexin A1; CALR, calreticulin; CD91 (official name: LRP1), LDL receptor related protein 1; C-X-C motif chemokine ligand, CXCL; DNGR-1 (official name: CLEC9A), C-type lectin domain containing 9A; FPR1, formyl peptide receptor 1; GZMB, granzyme B; HMGB1, high-mobility group box 1; IFN, interferon; IFNAR, IFN interferon alpha and beta receptor; IFNG, interferon gamma; P2RX7, purinergic receptor P2X 7; P2RY2, purinergic receptor P2Y2; TLR, Toll-like receptor.

Figure 3.. Potential pipeline for preclinical development of novel ICD-relevant drugs.

Both immunogenic cell death (ICD)-inducing (a) and ICD-enhancing (b) drug candidates can be identified in vitro by medium-to-high screening efforts based on: (a) human cancer cell lines expressing biosensors or treated with chemical dyes that enable the assessment of autophagy activation, calreticulin (CALR) exposure on the plasma membrane, ATP secretion and high mobility group box 1 (HMGB1) loss; or (b) a conditionally immortalized mouse dendritic cell (DC) precursor cell line that can be de-immortalized ad hoc and exposed to mouse cancer cells undergoing ICD in the presence of potential ICD enhancers, followed by the assessment of ICD-related parameters including phagocytic capacity, expression of DC maturation markers, and T cell cross-priming proficiency. In the case of ICD inducers, low throughput in vitro validation with alternative technologies should be followed by in vivo assays based on prophylactic vaccination, therapeutic tests in immunocompetent vs immunodeficient hosts, and/or therapeutic assays in bilateral tumor models (when ICD induction can be achieved by local interventions). In the case of ICD enhancers, low throughput in vitro validation can involve human DCs or mouse bone-marrow derived DCs, followed by in vivo validation based on therapeutic DCs vaccination assays upon ex vivo DC exposure to candidate drugs, or ICD inducer plus enhancer treatment of mouse tumors established in syngeneic immunocompetent mice or mice lacking conventional type 1 DCs (cDC1s), such as Batf3^−/− mice or mice administered with recombinant cytochrome c, somatic (CYCS). The pipeline for the discovery of agents restoring deficient ICD signaling in cancer cells, so-called ICD correctors, strictly resembles the one for ICD inducer discovery, except for the fact that combinatorial strategies are tested (i.e., suboptimal ICD inducer plus potential ICD corrector). Along similar lines, candidate ICD boosters be identified in vitro by high-throughput strategies testing regulatory T (T_REG) cell or myeloid-derived suppressor cell (MDSC) viability and immunosuppressive functions, but can only be validated by low throughput in vivo assays. IFN, interferon.