Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines

Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

ipilimumab (Yervoy) in 2011 for the treatment of metastatic melanoma patients (1), two mAbs targeting programed death (PD)-1 receptor signaling (nivolumab and pembrolizumab) have very recently joined the list of FDA-approved checkpoint blockers (respectively, for the treatment of metastatic squamous non-small cell lung cancer and relapsed/refractory melanoma patients) (2,3).
However, the primary goal of cancer immunotherapy is to activate the immune system in cancer patients. This requires the induction of tumor-specific T-cell-mediated antitumor immunity. Checkpoint blockers are only able to abrogate the brakes of a functioning antitumoral immune response, implying that only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade. This is evidenced by the observation that ipilimumab may be more effective in patients who have pre-existing, albeit ineffective, antitumor immune responses (4). Hence, combining immune checkpoint blockade with immunotherapeutic strategies that prime tumor-specific T cell responses might be an attractive and even synergistic approach. This relatively new paradigm has lead to the revival of existing, and to date disappointing (as monotherapies), active immunotherapeutic treatment modalities. One promising strategy to induce priming of tumor-specific T cells is dendritic cell (DC)-based immunotherapy.
Dendritic cells are positioned at the crucial interface between the innate and adaptive immune system as powerful antigen-presenting cells capable of inducing antigen-specific T cell responses (5). Therefore, they are the most frequently used cellular adjuvant in clinical trials. Since the publication of the first DC vaccination trial in melanoma patients in 1995, the promise of DC immunotherapy is underlined by numerous clinical trials, frequently showing survival benefit in comparison to non-DC control groups (6)(7)(8). Despite the fact that most DC vaccination trials differ in several vaccine parameters (i.e., site and frequency of injection, nature of the DCs, choice of antigen), DC vaccination as a monotherapy is considered safe and rarely associates with immune-related toxicity. This is in sharp contrast with the use of mAbs or cytokine therapies. Ipilumumab has, for instance, been shown to induce immune-related serious adverse events in up to one-third of treated melanoma patients (1). The FDA approval of Sipuleucel-T (Provenge), an autologous DC-enriched vaccine for hormone-resistant metastatic prostate cancer, in 2010 is really considered as a milestone in the vaccination community (9). After 15 years of extensive clinical research, Sipileucel-T became the first cellular immunotherapy ever that received FDA approval, providing compelling evidence for the substantial socio-economic impact of DC-based immunotherapy. DC vaccinations have most often been applied in patients with melanoma, prostate cancer, high-grade glioma, and renal cell cancer. Although promising objective responses and tumor-specific T cell responses have been observed in all these cancer-types (providing proof-of-principle for DC-based immunotherapy), the clinical success of this treatment is still considered suboptimal (6). This poor clinical efficacy can in part be attributed to the severe tumor-induced immune suppression and the selection of patients with advanced disease status and poor survival prognostics (6,(10)(11)(12).
There is a consensus in the field that step-by-step optimization and standardization of the production process of DC vaccines, to obtain a Th1-driven immune response, might enhance their clinical efficacy (13). In this review, we address some recent DC vaccine adaptations that impact DC biology. Combining these novel insights might bring us closer to an ideal DC vaccine product that can trigger potent CTL-and Th1-driven antitumor immunity.
One factor requiring more attention in this production process is the immunogenicity of the dying or dead cancer cells used to load the DCs. It has been shown in multiple preclinical cancer models that the methodology used to prepare the tumor cell cargo can influence the in vivo immunogenic potential of loaded DC vaccines (14)(15)(16)(17)(18)(19). Different treatment modalities have been described to enhance the immunogenicity of cancer cells in the context of DC vaccines. These treatments can potentiate antitumor immunity by inducing immune responses against tumor neo-antigens and/or by selectively increasing the exposure/release of particular damage-associated molecular patterns (DAMPs) that can trigger the innate immune system (14,(17)(18)(19). The emergence of the concept of immunogenic cell death (ICD) might even further improve the immunogenic potential of DC vaccines. Cancer cells undergoing ICD have been shown to exhibit excellent immunostimulatory capacity owing to the spatiotemporally defined emission of a series of critical DAMPs acting as potent danger signals (20,21). Thus far, three DAMPs have been attributed a crucial role in the immunogenic potential of nearly all ICD inducers: the surface-exposed "eat me" signal calreticulin (ecto-CRT), the "find me" signal ATP and passively released high-mobility group box 1 (HMGB1) (21). Moreover, ICD-experiencing cancer cells have been shown in various mouse models to act as very potent Th1-driving anticancer vaccines, already in the absence of any adjuvants (21,22). The ability to reject tumors in syngeneic mice after vaccination with cancer cells (of the same type) undergoing ICD is a crucial hallmark of ICD, in addition to the molecular DAMP signature (21).
Here, we review the effects of three frequently used immunogenic modalities and four potent ICD inducers on DC biology and their application in DC vaccines in preclinical as well as clinical settings (Tables 1 and 2). Moreover, we discuss the rationale for combining different cell death-inducing regimens to enhance the immunogenic potential of DC vaccines and to ensure the clinical relevance of the vaccine product.  (19,24,26,27) Oxidation-inducing modalities (HOCl/H2O2 treatment or freeze-thaw cycles followed by X-ray irradiation)
Targeting cancer stem cells is another promising development, particularly in the setting of glioma (87). Glioma stem cells can foster tumor growth, radio-and chemotherapy-resistance, and local immunosuppression in the tumor microenvironment (87,88). Furthermore, glioma stem cells may express higher levels of tumor-associated antigens and MHC complex molecules as compared to non-stem cells (89,90). A preclinical study in a rodent orthotopic glioblastoma model has shown that DC vaccines loaded with neuropsheres enriched in cancer stem cells could induce more immunoreactivity and survival benefit as compared  (14) Only in combination with γ-irradiation and heat shock in B-cell lymphoma patients (57) Oxidation-inducing modalities (HOCl/H2O2 treatment or freeze-thaw cycles followed by X-ray irradiation) ID8-ova ovarian carcinoma model in C57BL/6 mice -weekly curative immunizations (14); orthotopic GL261 high-grade glioma model in C57BL/6 mice -both prophylactic and curative vaccination settings induced a pro-inflammatory shift in the brain-infiltrating immune cells and the protein carbonyl content in the tumor lysate positively correlated with tumor rejection (18) Freeze-thaw cycles in combination with high-dose irradiation: often reported in clinical trials involving high-grade glioma and melanoma patients (8,(58)(59)(60)(61)(62)(63)(64)(65)(66) HOCl: pilot study in five recurrent ovarian cancer patients demonstrated potent T cell responses against tumor antigens, decreased circulating Treg levels, and serum IL-10 levels and two patients experienced durable PFS responses of ≥24 months (14) Heat shock PANCO2 pancreatic cancer model in C57BL/6 mice -curative vaccinations (17); in combination with 30 Gy irradiation in B16-ova model in C57BL/6 mice -prophylactic vaccinations (16) Non-randomized trial in newly diagnosed glioblastoma patients (67): significantly improved tumor control rates and survival rates in DC vaccine group than in control group; increased proportions of peripheral CD4 + and CD8 + T cells post vaccination compared to control group; in combination with other cell killing modalities in B-cell lymphoma and melanoma patients (57,68) inducers of immunogenic cell death Radiotherapy B16 melanoma in C57BL/6 -prophylactic immunization model with critical involvement of CD4 + and CD8 + T cells (15,37); E.G7 (SCCVII) in C57BL/6 -curative vaccination model (40) Radiotherapy as a single intervention: multiple clinical trials in melanoma patients (8) and two clinical trials in high-grade glioma patients (69,70). This study by Cho and colleagues reported a survival advantage of more than 15 months in the vaccinated glioblastoma patients in comparison to the control group (receiving conventional treatment) Radiotherapy as part of an ICD-inducing cell death protocol in B-cell lymphoma patients (57 order to generate a strong T cell response. In view of this finding, the route of administration is another vaccine parameter that can influence the homing of the injected DCs to the lymph nodes. In the context of prostate cancer and renal cell carcinoma it has been shown that vaccination routes with access to the draining lymph nodes (intradermal/intranodal/intralymphatic/subcutaneous) resulted in better clinical response rates as compared to intravenous injection (93). In melanoma patients, a direct comparison between intradermal vaccination and intranodal vaccination concluded that, although more DCs reached the lymph nodes after intranodal vaccination, the melanoma-specific T cells induced by intradermal vaccination were more functional to DCs loaded with GL261 cells grown under standard conditions (91). Currently there are four clinical trials ongoing in highgrade glioma patients evaluating this approach (NCT00890032, NCT00846456, NCT01171469, and NCT01567202). With regard to the DC maturation status of the vaccine product, a phase I/II clinical trial in metastatic melanoma patients has confirmed the superiority of mature antigen-loaded DCs to elicit immunological responses as compared to their immature counterparts (92). This finding was further substantiated in patients diagnosed with prostate cancer and recurrent high-grade glioma (93,94). Hence, DCs need to express potent costimulatory molecules and lymph node homing receptors in FiGURe 1 | A schematic representation of immunogenic DC vaccines. Cancer cells show enhanced immunogenicity upon treatment with UV irradiation, oxidizing treaments, and heat shock, characterized by the release of particular danger signals and the (increased) production of tumor (neo-)antigens. Upon loading onto DCs, DCs undergo enhanced phagocytosis and antigen uptake and show phenotypic and partial functional maturation. Upon in vivo immunization, these DC vaccines elicit Th1-and cytotoxic T lymphocyte (CTL)-driven tumor rejection. (95). Furthermore, the frequency of vaccination can also influence the vaccine's immunogenicity. Our group has shown in a cohort-comparison trial involving relapsed high-grade glioma patients that shortening the interval between the four inducer DC vaccines improved the progression-free survival curves (58,96).
Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE2 (97,98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97,98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99,100). Recently, a novel cytokine cocktail, including TNF-α, IL-1β, poly-I:C, IFN-α, and IFN-γ, was introduced (101, 102). The type 1-polarized DCs obtained with this cocktail produced high levels of IL-12 and could induce strong tumorantigen-specific CTL responses through enhanced induction of CXCL10 (99). In addition, CD40-ligand (CD40L) stimulation of DCs has been used to mature DCs in clinical trials (100,103). Binding of CD40 on DCs to CD40L on CD4 + helper T cells licenses DCs and enables them to prime CD8 + effector T cells.
A final major determinant of the vaccine immunogenicity is the choice of antigen to load the DCs. Two main approaches can be applied: loading with selected tumor antigens (tumor-associated antigens or tumor-specific antigens) and loading with whole tumor cell preparations (13). The former strategy enables easier immune monitoring, has a lower risk of inducing auto-immunity, and can provide "off-the-shelf " availability of the antigenic cargo. Whole tumor cell-based DC vaccines, on the other hand, are not HLA-type dependent, have a reduced risk of inducing immuneescape variants, and can elicit immunity against multiple tumor antigens. Meta-analytical data provided by Neller et al. have demonstrated enhanced clinical efficacy in several tumor types of DCs loaded with whole tumor lysate as compared to DCs pulsed with defined tumor antigens (104). This finding was recently also substantiated in high-grade glioma patients, although this study was not set-up to compare survival parameters (105).

TOwARD A MORe iMMUNOGeNiC TUMOR CeLL CARGO
The majority of clinical trials that apply autologous whole tumor lysate to load DC vaccines report the straightforward use of multiple freeze-thaw cycles to induce primary necrosis of cancer cells (8,93). Freeze-thaw induced necrosis is, however, considered non-immunogenic and has even been shown to inhibit toll-like receptor (TLR)-induced maturation and function of DCs (16). To this end, many research groups have focused on tackling this roadblock by applying immunogenic modalities to induce cell death.
immunogenic Treatment Modalities Tables 1 and 2 list some frequently applied treatment methods to enhance the immunogenic potential of the tumor cell cargo that is used to load DC vaccines in an ICD-independent manner (i.e., these treatments do not meet the molecular and/or cellular determinants of ICD). Immunogenic treatment modalities can positively impact DC biology by inducing particular DAMPs in the dying cancer cells ( Table 1). Table 2 lists the preclinical and clinical studies that investigated their in vivo potential. Figure 1 schematically represents the application and the putative modes of action of these immunogenic enhancers in the setting of DC vaccines.

Ultraviolet Irradiation
Ultraviolet (UV) light is considered an electromagnetic nonionizing radiation with a wavelength between 100 and 400 nm. Its immunogenic potential was discovered in 1991 when Begovic et al. demonstrated that vaccination of immunocompetent mice (but not immunodeficient nude mice) with UV-irradiated cancer cells could induce resistance to subsequent rechallenge with live tumor cells (23,106,107). This antitumor effect was crucially mediated by NK cells and CD8 + T cells. UV-treated cancer cells are efficiently engulfed by DCs, leading to phenotypic maturation and increased IL-12 production (19, 24, 26) ( Table 1). Moreover, these matured DCs in turn stimulated the polarization of T cells toward IFN-γ producing CD8 + T cells (24,26). Of note, human DCs that had ingested UV-irradiated apoptotic tumor cells were shown to be more effective in generating CD8 + CTLs than DCs pulsed with freeze-thaw lysates (27). In addition, immunization with DCs loaded with UV-treated tumor cells could elicit effective antitumor therapeutic efficacy in a B16 mouse melanoma model, albeit nonsuperior to DCs loaded with necrotic freeze-thaw lysate (19) ( Table 2). The induction of specific DAMPs, such as ecto-CRT, and the release of heat shock protein 70 (HSP70) and HMGB1 determines the immunogenicity of UV irradiation (23, 24, 33) ( Table 1). Moreover, as UV light is known to affect mainly DNA, mutation-induced tumor neo-antigens might also contribute to increasing the host antitumor immune response (108). T cells reactive against mutated neo-antigens are theoretically less susceptible to central and peripheral tolerance. Vaccination with UV-induced tumor neo-antigens might be particularly useful in UV-induced tumors (e.g., cutaneous and uveal melanoma) that might share the ex vivo UV-induced tumor neo-antigens. Besides, it has previously been shown that immunization of tumor-bearing mice with mutated melanoma-derived selfantigens can elicit efficient cross-reactive CD8 + T cell responses against multiple non-mutated epitopes of the tumor protein and against the melanoma cells (109). This led to the rejection of established poorly immunogenic B16 melanoma tumors (109). To the best of our knowledge, there are no reports of clinical trials that used UV irradiation as a single treatment for obtaining an antigen source to pulse DC vaccines ( Table 2). This is probably related to the fact that UV light as a single treatment is not able to induce high levels of cancer cell death in the vaccine, an absolute requirement for clinical translation.

Oxidation-Inducing Modalities
In recent years, an increasing number of data were published concerning the ability of oxidative stress to induce oxidationassociate molecular patterns (OAMPs), such as reactive protein carbonyls and peroxidized phospholipids, which can act as DAMPs (28,29) (Table 1). Protein carbonylation, a surrogate indicator of irreversible protein oxidation, has for instance been shown to improve cancer cell immunogenicity and to facilitate the formation of immunogenic neo-antigens (30,31).
One prototypical enhancer of oxidation-based immunogenicity is radiotherapy (21,23). In certain tumor types, such as highgrade glioma and melanoma, clinical trials that apply autologous whole tumor lysate to load DC vaccines report the random use of freeze-thaw cycles (to induce necrosis of cancer cells) or a combination of freeze-thaw cycles and subsequent high-dose γ-irradiation (8,18) (Table 2). However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. In light of the oxidation-based immunogenicity that is associated with radiotherapy, we recently demonstrated the superiority of DC vaccines loaded with irradiated freeze-thaw lysate (in comparison to freeze-thaw lysate) in terms of survival advantage in a preclinical high-grade glioma model (18) ( Table 2). This survival advantage was associated with an increased tumor infiltration of Th1 cells and CTLs and accompanied by a reduced invasion of regulatory cells (Tregs), macrophages, and myeloid-derived suppressor cells. Moreover, this study revealed a significant positive correlation between the level of protein carbonylation -as a measure of the total oxidative content -in the tumor lysates used to load the DCs and the percentage of mice able to reject the aggressive intracranial tumors. Treatment of the tumor lysate with hydrogen peroxide (H2O2, a strong oxidant) even induced higher tumor protection than irradiated freeze-thaw lysate, warranting the preclinical investigation of other strong oxidizing modalities to further potentiate the immunogenicity of whole tumor antigen-pulsed DC vaccinations.
In line with these results and through a series of elegant ex vivo an in vivo mouse experiments, Chiang et al. recently selected hypochlorous acid (HOCl)-based oxidation (to induce primary necrosis of tumor cells) as the method of choice (as compared to UVB irradiation and freeze-thaw cycles) for preparing whole tumor lysate-loaded DC vaccines in the preclinical ID8 ovarian cancer model (14) ( Table 2). Interestingly, T cells stimulated by DCs loaded with HOCl-induced oxidatively modified tumor cells were still able to recognize non-modified tumor cells, an essential requirement if the cells are to exert antitumor activity (30). In a pilot study containing five recurrent ovarian cancer patients, these autologous DCs loaded with HOCl-oxidized autologous tumor lysate could produce high levels of IL-12, elicited strong antigen-specific T cell responses and reduced the levels of circulating Tregs and serum IL-10 (14). Moreover, two patients experienced durable progression-free survival intervals of more than 24 months after vaccination ( Table 2).

Heat Shock Treatment
Heat shock is a term that is applied when a cell is subjected to a temperature that is higher than that of the ideal body temperature of the organisms of which the cell is derived. Heat shock can induce apoptosis (41-43°C) or necrosis (>43°C) depending on the temperature that is applied (110). The immunogenicity of heat shock treated cancer cells largely resides within their ability to produce HSPs, such as HSP60, HSP70, and HSP90 (17, 32) ( Table 1). These HSPs can function as chaperones for tumor antigens, facilitating their cross-presentation (17). Moreover, after recognition by their receptors (CD91, TLR2/4), these HSPs can instigate the attraction of neutrophils and monocytes and the activation of NK cells and DCs (111). These events are crucial for the initiation of tumor-specific immune responses. Independent of the induction of HSPs, heat shock treatment has also been shown to upregulate the transcription of specific tumor-associated antigens (34).
Co-incubation of heat-stressed apoptotic cancer cells with immature DCs resulted in the upregulation of DC maturation markers (CD40, CD80, and CD86) and higher IL-12 levels (32) ( Table 1). Interestingly, splenocytes from mice immunized with heat-stressed apoptotic cancer cells got polarized toward a Th1 cytokine profile. Furthermore, DCs loaded with heat shock stressed melanoma cells can efficiently cross-prime tumor-antigen-specific CTLs both in vitro and in vivo (34). Of note, direct comparison of heat shock treated tumor lysate with freeze-thaw tumor lysate in a DC vaccine setup demonstrated a stronger tumor regression in favor of heat shock lysate in a mouse model for pancreatic cancer ( Table 2). Again, this was associated with a stronger priming of tumor-specific CTL responses (17).
Dendritic cells loaded with heat shocked cancer cells have already been successfully applied in clinical practice in highgrade glioma patients (  (67). The vaccinated patients had a significantly improved overall survival and progression-free survival. Interestingly, the proportions of peripheral CD4 + T cells, CD8 + T cells, and NK cells were significantly higher after DC vaccination in comparison to the control group. Moreover, increased levels of IFN-γ, IL-2, and IL-12 were measured in the sera of DC vaccinated patients.
All together, these data suggest that an immunogenic treatment of cancer cells can positively impact the potency of DCs interacting with them (Figure 1). In light of this finding, the relatively new concept of ICD of cancer cells can be considered a promising strategy for loading DC-based anticancer vaccines, potentially giving rise to a next generation of potent Th1-driving DC vaccines (111,112) (Figure 2).

inducers of immunogenic Cell Death
Immunogenic cell death is a cell death regimen that is associated with the spatiotemporally defined emission of immunogenic DAMPs that can trigger the immune system (20,21,113). ICD has been found to depend on the concomitant induction of reactive oxygen species (ROS) and activation of endoplasmatic reticulum (ER) stress (111). Besides the three DAMPs that are most crucial for ICD (ecto-CRT, ATP, and HMGB1), other DAMPs such as surface-exposed or released HSPs (notably HSP70 and HSP90) have also been shown to contribute to the immunogenic capacity of ICD inducers (20,21). The binding of these DAMPs to their respective immune receptors (CD91 for HSPs/CRT, P2RX7/P2RY2 for ATP, and TLR2/4 for HMGB1/HSP70) leads to the recruitment and/or activation of innate immune cells and facilitates the uptake of tumor antigens by antigen-presenting cells and their cross-presentation to T cells eventually leading to IL-1β-, IL-17-, and IFN-γ-dependent tumor eradiation (22). This in vivo tumor rejecting capacity induced by dying cancer cells in the absence of any adjuvant, is considered as a prerequisite for an agent to be termed an ICD inducer. Recently, a classification system for ICD inducers was proposed based on whether an ICD inducer triggers apoptotic cell death as a consequence of direct action at the ER (Type II ICD inducer), or whether it initiates both ER stress-dependent danger signaling and apoptosis through divergent mechanisms (Type I ICD inducer) (111).
Although the list of ICD inducers is constantly growing (113), only few of these immunogenic modalities have been tested in order to generate an immunogenic tumor cell cargo to load DC vaccines (Tables 1 and 2). Figure 2 schematically represents the preparation of ICD-based DC vaccines and their putative modes of action.

Radiotherapy
Ionizing X-ray or γ-ray irradiation exerts its anticancer effect predominantly via its capacity to induce DNA double-strand breaks leading to intrinsic cancer cell apoptosis (114). The idea that radiotherapy could also impact the immune system was derived from the observation that radiotherapy could induce T-cell-mediated delay of tumor growth in a non-irradiated lesion (115). This abscopal (ab-scopus, away from the target) effect of radiotherapy was later explained by the ICD-inducing capacity (116). Together with anthracyclines, γ-irradiation was one of the first treatment modalities identified to induce ICD. Although this type I ICD inducer is known to induce ROS, its ER stress-inducing capability remains largely unexplored (111). The DAMPs that are induced following radiotherapy treatment of cancer cells include the exposure of ecto-CRT (23,24,35) and ecto-HSP70 (36), and the release of HMGB1 (33, 35) ( Table 1). Irradiated B16 melanoma cells have been shown to be efficiently phagocytosed by DCs and to induce phenotypic DC maturation (15,37). In addition, human DCs pulsed with irradiated tumor cells could efficiently stimulate antigen-specific CTL responses (40) ( Table 1). Furthermore, mice immunized with DCs loaded with irradiated cancer cells could efficiently suppress tumor growth following inoculation with live syngeneic tumor cells in multiple preclinical cancer models (15,40). In this setting, splenocytes from vaccinated animals could efficiently prime CD4 + and CD8 + T cells and exerted antigen-specific cytolytic activity (15) ( Table 2).
Dendritic cell vaccines exposed to irradiated cancer cells have also been successfully implemented in clinical practice in melanoma and HGG patients (8,69,70) (Table 2). Cho et al. have shown that the implementation of DC vaccines loaded with irradiated autologous tumor cells in the conventional treatment regimen of newly diagnosed glioblastoma patients could significantly prolong the median overall survival (by more than 15 months) as compared to a control group receiving solely conventional treatment (69). Interestingly, the group of Di Nicola reported that vaccination with DCs loaded with dying autologous tumor cells after exposure to a cell death protocol consisting of heat shock, γ-ray, and UV ray could elicit clinical responses in 6 out of 18 relapsed B-cell lymphoma patients (117). Later, they showed the impaired ability of the neoplastic cells used to vaccinate non-responders to undergo ICD upon exposure to the cell death protocol (57). Importantly, they revealed a positive association between the extent of CRT and HSP90 surface expression in the DC antigenic cargo and the clinical and immunological responses achieved (57).

Shikonin
The phytochemical shikonin, a major component of Chinese herbal medicine, is known to inhibit proteasome activity. It serves multiple biological roles and can be applied as an antibacterial, antiviral, anti-inflammatory, and anticancer treatment. The latter application has been shown to yield responsiveness in late-stage lung cancer patients (118). Apoptotic cell death elicited by this type I ICD inducer can be inhibited by anti-oxidants, suggesting a role of shikonin-induced ROS (119,120). The link between shikonin treatment and ER stress is not evidenced yet. The ICD that is induced in shikonin-treated cancer cells is characterized by the early induction of HSP70, HSP90, GRP78, and HMGB1 (41) ( Table 1). Importantly, shikonin treatment could significantly improve the survival of mice bearing P388 leukemia and this antitumor effect of shikonin was less pronounced in immunodeficient mice (120). Moreover, the tumor lysate from shikonin-treated B16 cells could enhance phenotypic and functional DC maturation and differentiation of Th1 and Th17 cells, two important features of ICD-associated antitumor immunity (41) ( Table 1). Additionally, curative vaccination of B16 melanoma-inoculated mice with shikonin-lysate-loaded DCs could delay tumor growth (41). This was associated with increased cytolytic activity of splenocytes on target tumor cells ( Table 2). Although shikonin is administered to breast cancer patients for observational application (NCT01287468), clinical experience evaluating shikonin-lysate-loaded DC vaccines is unfortunately still lacking ( Table 2).

High-Hydrostatic Pressure
High-hydrostatic pressure (HHP) is an established method to sterilize pharmaceuticals, human transplants, and food. HHP between 100 and 250 megapascal (MPa) has been shown to induce apoptosis of murine and human (cancer) cells (121)(122)(123). While DNA damage does not seem to be induced by HHP <1000 MPa, HHP can inhibit enzymatic functions and the synthesis of cellular proteins (122). Increased ROS production was detected in HHPtreated cancer cell lines and ER stress was evidenced by the rapid phosphorylation of eIF2α (42).
The anticancer activity of HHP was already demonstrated more than four decades ago in bladder cancer patients (124). Later, preclinical experiments demonstrated in vivo immunogenicity of HHP-treated cancer cells in the B16 melanoma model and the 3LL-D122 lung metastasis model (125,126). Subsequently, it was shown that HHP-treated mammalian cancer cell lines undergoing apoptosis can release HSP70 and HMGB1, while retaining their immunogenicity in vivo (127). Very recently, Fucikova and colleagues have shown the ability of HHP to induce prototypical ICD in human prostate and ovarian cancer cell lines and in acute leukemia cells (42). HHP treatment induced the rapid expression of ecto-HSP70, ecto-HSP90, and ecto-CRT and the release of HMGB1 and ATP (Table 1). Interestingly, HHP-treated cancer cells were rapidly phagocytosed by DCs and induced the upregulation of CD83, CD86, and HLA-DR, and the release of pro-inflammatory cytokines ( Table 1). This led to the stimulation of high numbers of tumor-specific T cells without inducing Tregs. Hence, all ICD-associated molecular criteria are fulfilled for HHP. This group is currently testing the in vivo immunogenicity of HHP killed tumor cells in prophylactic and curative murine vaccination settings ( Table 2). Moreover, they have initiated multiple clinical trials to evaluate the potential of DC vaccines loaded with HHP-treated cancer cells in ovarian and prostate cancer patients (71).

Oncolytic Viruses
Oncolytic viruses are self-replicating, tumor selective virus strains that can directly lyse tumor cells. Over the past few years, a new oncolytic paradigm has risen; entailing that, rather than utilizing oncolytic viruses solely for direct tumor eradication, the cell death they induce should be accompanied by the elicitation of antitumor immune responses to maximize their therapeutic efficacy (128). One way in which these oncolytic viruses can fulfill this oncolytic paradigm is by inducing ICD (128).
Thus far, three oncolytic virus strains can meet the molecular requirements of ICD; coxsackievirus B3 (CVB3), oncolytic adenovirus and Newcastle disease virus (NDV) ( Table 1) (113). Infection of tumor cells with these viruses causes the production of viral envelop proteins that induce ER stress by overloading the ER. Hence, all three virus strains can be considered type II ICD inducers (113). While CVB3 and oncolytic adenoviruses induce the surface expression of CRT, followed by the release of ATP and the passive release of HMGB1 in apoptotic tumor cells (in nonsmall cell lung carcinoma and adenocarcinoma cells, respectively) (43,44), NDV induces necroptosis accompanied by the surface exposure of ATP and the post-necroptotic release of HMGB1 in GL261 glioma cells, with no contribution of ATP ( Table 1) (45). In addition, NDV-infected GL261 cells upregulated the expression of the PMEL17 tumor antigen (45).
Intratumoral administration of CVB3 in nude mice resulted in the marked infiltration of NK cells, macrophages, granulocytes, and mature DCs into the tumor tissue ( Table 1) (44). Tumorinfiltrating DCs expressed significantly higher levels of costimulatory molecules CD80 and CD86, as well as the lymph node homing receptor CCR7 (44). CD40-ligand encoding oncolytic adenoviruses have also been shown to facilitate the recruitment of DCs to the tumor tissue, this way entailing efficient Th1 and CD8 + CTL responses ( Table 1) (43). Measles virus is another oncolytic virus that requires further investigation. Although extensive analysis of in vitro ICD determinants is lacking for this virus (only the release of HMGB1 has been documented), DCs exposed in vitro to measles-virus treated melanoma cells showed increased CD80 and CD86 expression levels ( Table 1) (46). This resulted in the efficient priming of melanoma-specific cell killing by IFN-γ producing CD8 + T cells. Moreover, in terms of priming these melanoma-specific CTL responses, measles virusinfected melanoma cells constituted more effective tumor lysates (also termed oncolysates) for loading of DCs than uninfected melanoma cell lysates (46). The DC stimulatory capacity of NDVderived oncolysates has already been demonstrated more than a decade ago by Schirrmacher et al. (47). DCs derived from breast cancer patients pulsed with NDV-oncolysates showed increased expression of costimulatory molecules in comparison to DCs loaded with tumor lysate from non-infected breast carcinoma cells ( Table 1) (47). In addition, NDV-oncolysate-loaded DCs were more effective in stimulating bone-marrow-derived reactive memory T cells in vitro (47).
Oncolytic viruses hold great potential for application in ICD-based DC vaccines given their potential to elicit several ICD-related DAMPs. Furthermore, these viruses might directly affect DC maturation and activation through interaction with pathogen recognition receptors on the tumor cells. This way, biological oncolysates may render the use of an artificial maturation cocktail otiose. Unfortunately, there are no preclinical in vivo data available yet to evince the efficacy of DC vaccines loaded with immunogenic oncolysates ( Table 2). Nevertheless, several studies have documented the beneficial effect of intratumoral application of oncolytic viruses in combination with tumor-directed systemic DC vaccinations (72,73). Very recently, Schirrmacher et al. disclosed a case report of a breast cancer patient with liver metastasis that was treated with local hyperthermia, intravenously administered NDV, and subcutaneous vaccination with DCs loaded with NDV-infected breast cancer cells (oncolysate) (74). This combination therapy led to long-lasting tumorspecific memory T cell responses and stable disease for more than 66 months in this particular patient. The use of autologous DCs loaded with NDV-mediated oncolysate is licensed by the Paul Ehrlich Institute to the Immunologic-Oncologic Centre Cologne (IOZK) since May 2015.
Of note, in October 2015, the FDA approved the first oncolytic virus, Imlygic (a genetically modified live oncolytic herpes virus) for the treatment of melanoma lesions in the skin and lymph nodes. This FDA approval should facilitate the approval of other oncolytic viruses as well as the application of oncolysates in DC vaccine settings.

Photodynamic Therapy
Photodynamic therapy (PDT) is an established, minimally invasive anticancer treatment modality. It has a two-step mode of action involving the selective uptake of a photosensitizer by the tumor tissue, followed by its activation by light of a specific wavelength. This activation results in the photochemical production of ROS in the presence of oxygen (129)(130)(131). One attractive feature of PDT is that the ROS-based oxidative stress originates in the particular subcellular location where the photosensitizer tends to accumulate, ultimately leading to the destruction of the tumor cell (132). PDT-based antitumor effects are multifactorial and depend on its abilities to damage the tumor vasculature, directly kill tumor cells, exert cytotoxic effects toward tumor-infiltrating immune cells, and recruit and activate immune cells that can initiate adaptive antitumor immune responses (131).
Increasing preclinical information is available regarding the impact of PDT on the immune system. Recent studies have demonstrated that PDT can effectively generate several DAMPs. HSP70, the best studied DAMP associated with PDT, is exposed on the surface of cancer cells treated with photofrin-PDT, 5-Aminolevulinic acid (5-ALA)-PDT, and Foscan-PDT (51,133,134). Of note, the uptake of tumor antigens and DC maturation induced by 5-ALA-PDT treated GBM spheroids were inhibited when HSP70 was blocked (133). Later, it was reported that photofrin-PDT also promotes the early/mid-apoptotic surface expression of CRT and the post-apoptotic release of HMGB1 (52) ( Table 1). Very recently, the DAMPs profile induced by Rose Bengal Acetate (RBAc)-based PDT was unraveled. RBAcphotosensitized apoptotic/autophagic Hela cells were found to expose and/or release ATP, HSP70/90, HMGB1, and CRT (135). In terms of its immunogenicity, hypericin can be considered the best studied photosensitizer. Recently, hypericin-PDT became the first PDT modality capable of inducing prototypical ICD in cancer cells (20,48,49,111). Hypericin localizes predominantly in the ER and upon irradiation it causes photo-oxidative ER stress, making hypericin-PDT the only known modality able to induce ICD through focused ROS-based ER stress (Type II ICD inducer), eventually culminating in mitochondrial apoptosis (49,136). In the pre-apoptotic stage, it induces the active emission of three crucial ICD-associated DAMPs, i.e., ecto-CRT, ecto-HSP, and secreted ATP (at a faster rate than what was previously published for these DAMPs), followed by the passive release of Improved Immunogenicity of DC Vaccines Frontiers in Immunology | www.frontiersin.org HSP70 and HMGB1 (48,49) (Table 1). Interestingly, this ICDsubroutine was more effective in comparison to chemotherapyor radiotherapy-induced ICD (48,49).
The immunogenic features of Hyp-PDT-treated cancer cells have also been confirmed by ex vivo and in vivo experiments (Tables 1 and 2). Hyp-PDT-treated cancer cells form a productive interface with DCs in terms of phagocytosis (CRTdependent) and maturation (49) ( Table 1). More specifically, the interacting DCs exhibit functional stimulation (NO high , IL-10 absent , IL-6 high , IL-1β high , and IL-12p70 median ) and phenotypic maturation (CD80 high , CD83 high , CD86 high , and MHC-II high ) (49,53). Moreover, these immunogenic and fully mature DCs induce the clonal expansion of human IFN-γ producing CD4 + and CD8 + T cells (53,54). Consequently, this in vitro antitumor immunity induced by Hyp-PDT-induced ICD led to the efficient rejection of murine tumors in vivo in the absence of any adjuvants (both in prophylactic and curative vaccination models) (49,137). Besides hypericin-based PDT, photofrin-based PDT is to date the only PDT modality that is capable to fulfill this critical in vivo requirement for ICD characterization. Here, curative immunization with benzoporphyrin-based PDT-treated squamous cell carcinoma cells constituted a potent anticancer vaccine in this poorly immunogenic model (56).
Importantly, inoculation of mature DCs in PDT-treated tumors resulted in the cytolytic activation of T cells and NK cells, leading to effective tumor eradication (75). Moreover, DC vaccines loaded with PDT-induced tumor lysates have been shown to cure fully established solid non-orthotopic tumors. This was associated with enhanced CTL responses and Th1 immunity (138) ( Table 2)

Combinatorial Regimens
In DC vaccine settings, cancer cells are often not killed by a single treatment strategy but rather by a combination of treatments. In some cases, the underlying rationale lies within the additive or even synergistic value of combining several moderately immunogenic modalities. The combination of radiotherapy and heat shock has, for instance, been shown to induce higher levels of HSP70 in B16 melanoma cells than either therapy alone (16). In addition, a combination therapy consisting of heat shock, γ-irradiation, and UV irradiation has been shown to induce higher levels of ecto-CRT, ecto-HSP90, HMGB1, and ATP in comparison to either therapy alone or doxorubicin, a well-recognized inducer of ICD (57). Besides, the sequence of the applied methodologies seems to matter. The application of radiotherapy prior to freeze-thaw cycles was recently shown to negatively impacted the survival of high-grade glioma-bearing mice (in comparison to freeze-thaw cycles followed by X-ray irradiation) in the context of DC-based immunotherapy (18). A second rationale for combining several cell killing methodologies is to meet the clinical requirement of reaching 100% cancer cell death (14). Subcutaneous injection of irradiated tumor cells has, for instance, induced subcutaneous tumor growth in one glioblastoma patient (139). In general, most single treatment modalities discussed in this review cannot meet this requirement, postulating their combination with other (potentially less immunogenic) cell death modalities. In view of this, preclinical testing should always consider the most clinically relevant version of the vaccine.

CONCLUDiNG ReMARKS
Triggering antitumor immune responses is an absolute requirement to tackle metastatic and diffusely infiltrating cancer cells that are resistant to standard-of-care therapeutic regimens. ICD-inducing modalities, such as PDT and radiotherapy, have been shown to be able to act as in situ vaccines capable of inducing immune responses that caused regression of distal untreated tumors. Exploiting these ICD inducers and other immunogenic modalities to obtain a highly immunogenic antigenic tumor cell cargo for loading DC vaccines is a highly promising application. In case of the two prominent ICD inducers, Hyp-PDT and HHP, preclinical studies evaluating this relatively new approach are underway and HHP-based DC vaccines are already undergoing clinical testing. In the preclinical testing phase, more attention should be paid to some clinically driven considerations. First, one should consider the requirement of 100% mortality of the tumor cells before in vivo application. A second consideration from clinical practice (especially in multi-center clinical trials) is the fact that most tumor specimens arrive in the lab in a frozen state. This implies that a significant number of cells have already undergone non-immunogenic necrosis before the experimental cell killing strategies are applied. In case of ICD inducers, this could potentially hamper the immunogenicity of the tumor cells as these modalities mainly rely on active danger signaling pathways. Finally, for a more clinically relevant evaluation of the effect of immunogenic DC vaccines on tumor cell stromal interactions, orthotopic tumor inoculation should be applied. As tumor cells are implanted in the anatomically appropriate location, orthotopic tumors reflect the clinical situation (e.g., the tumor microenvironment) much better than conventional subcutaneous non-orthotopic models.
Even the most potent active immunotherapeutic strategies such as (ICD-based) DC vaccines will, however, be hampered by the presence of immunomodulatory immune checkpoint molecules (such as PD-1 and CTLA-4) that inhibit cytotoxic immune responses or even induce immune tolerance. The development of drugs that can unleash these inhibitory molecules has become one of the most active areas in oncology. This creates the opportunity to combine checkpoint inhibitors with DC-based immunotherapy. The synergistic action of a CTLA-4 blocking Ab (tremelimumab) in combination with DC therapy has already been demonstrated in advanced melanoma patients and several other trials evaluating this approach are on the horizon (6,140,141).
We believe that the specialty of DC-based immunotherapy is considerably moving forward by focusing on developing more immunogenic Th1-driving vaccines, such as ICD-based DC vaccines. Moreover, the combination of ICD-based DC vaccines with checkpoint inhibitors or other drugs that can inhibit the severe tumor-induced immune suppression might be able to reveal the full efficacy of DC-based immunotherapy for cancer.

AUTHOR CONTRiBUTiONS
LV did the literature study, data collection, and wrote the manuscript. SV provided senior supervision, helped in writing, and critically revised the manuscript. All the other co-authors have substantially contributed to the design of the work. All coauthors critically revised the manuscript and approved the final manuscript. All co-authors can be considered accountable for all aspects of the work.