**6. ICD and DAMPs in Cancer Therapy**

The literature on ICD in cancer biology has been significantly expanded over the last years, providing new possibilities for cancer treatment. Firstly, the discovery that ICD can be induced by certain classical anticancer agents has made it clear that some treatment regimens should be designed not only to directly eliminate tumor cells, but also to optimize their capacity to induce immune responses against tumor antigens. Secondly, ICD inducers can be used in combination with immunotherapeutics, such as immune checkpoint inhibitors.

Recently, Ganassin et al. (2022) demonstrated that curcumin, a polyphenol obtained from turmeric, causes ER stress and ICD in colorectal adenocarcinoma CT26 cells. The authors observed that, when treated with curcumin, these cells exhibited an initial increase of intracellular Ca2+, which was followed by the activation of XBP1, a protein involved in the UPR response. The ER stress initiated by curcumin was accompanied by the induction of ICD.

Regarding chemotherapeutics, different drugs have been found to induce ICD in preclinical studies [36]. Moreover, chemotherapeutics such as doxorubicin, mitoxantrone, idarubicin, cyclophosphamide, bortezomib and oxaliplatin have already been clinically tested as ICD inducers, mainly in combination with immunotherapeutics [39]. For instance, the combination of doxorubicin, an ICD inducer, with the PD-1-targeting immunecheckpoint blocker, nivolumab, resulted in 35% objective response rates (ORRs) in the treatment of metastatic triple negative breast cancer patients [39,97]. When nivolumab was combined with cisplatin, a non-ICD inducer chemotherapeutic, ORRs were lower (23%) [97]. A number of other clinical studies are already under way to test this immunostimulatory combination using not only immune-checkpoint blockers, but also CAR-T cells, DC-based vaccines, immunostimulatory cytokines, and others [39].

Interesting results of the Phase II study published by Bota et al. (2018) also show that ICD-inducers can improve clinical outcomes in glioblastoma (WHO grade IV astrocytic glioma) immunotherapy [98]. The authors used an allogeneic/autologous therapeutic glioblastoma vaccine (ERC1671, Gliovac), which is a mixture of inactivated tumor cells and lysates of tumor cells derived from the treated patient and three other glioblastoma patients, combined with recombinant colony stimulating factor 2 (CSF2, best known as GM-CSF, used to activate immune responses). The patients received a short regimen of low-dose cyclophosphamide (50 mg/day for 4 days) prior to vaccination. Cyclophosphamide is an ICD-inducer, used in this case, according to the authors, for relaxing the immunosuppressive environment. Indeed, low-dose cyclophosphamide has been reported to suppress Treg cell response, helping to promote DC expansion and antitumor cytotoxic T cell-mediated response [99]. The protocol of treatment described by Bota et al. (2018) also included the treatment of these glioblastoma patients with bevacizumab, a monoclonal antibody specific to VEGF. The results showed increased survival for the patients thus treated (12 months vs. 7.5 months for patients receiving bevacizumab only). Although the authors did not discuss the occasional contribution of ICD to the outcomes, the benefit observed with this protocol makes it worth investigating the possible contribution of ICD in preclinical and clinical models, which could help to improve this combinatory therapy in the future.

Radiotherapy has been shown to induce ICD as well. In clinical practice, radiotherapy and chemoradiotherapy were shown to induce the exposure of the ICD-related DAMPs calreticulin, HSP70 and HMGB1 when applied either as a pre- or post-operative protocol of different cancers [100]. The results, however, are still contradictory. A possible cause for this can be the different experimental settings used and insufficient data regarding ICD hallmarks.

Lämmer et al. (2019) investigated the expression of cytosolic HSP70 in tumor tissue of 60 patients diagnosed with primary glioblastoma. As discussed before in this review, HSP70 is released during ICD, acting as a DAMP. In this study [101], the tumors were surgically resected and patients were then treated with radiotherapy and temozolomide chemotherapy. The progression-free survival and overall survival were significantly longer in patients exhibiting a higher expression of cytosolic HSP70. The authors hypothesized that this result may be due to the induction of ICD by the combination of radiotherapy and chemotherapy given to patients.

Rothammer et al. (2019) analyzed the concentration of HSP70 in the serum of 40 breast cancer patients, who received breast-conserving surgery and adjuvant radiotherapy [102]. The authors observed that patients with higher serum concentrations of HSP70 had an increased probability of developing contralateral recurrence or metastases within two years of receiving radiotherapy.

Protocols based on the combination of radiotherapy and immunotherapeutics have also been tested, and it remains unclear if patients benefit from radiotherapy-induced ICD, as the clinical data are contradictory. For instance, in the treatment of rectal cancer, the increase in HMGB1 was associated both with poorer [103] and with better [104] responses to chemoradiotherapy.

Hongo et al. (2015) studied 75 patients with lower rectal cancer who were treated with preoperative chemoradiotherapy, consisting of radiotherapy (1.8 Gy × 28 fractions) and chemotherapy with a 5-fluorouracil (FU) prodrug (300 mg/m2/day) and leucovorin (75 mg/day). After chemoradiotherapy, tumors were surgically resected and analyzed for their expression of HMGB1. The patients with a higher expression of HMGB1 had a poorer response to chemoradiotherapy [103]. In the study by Huang et al. (2018), the presence of HMGB-1 in the cytosol, resulting from its translocation from the nucleus, was analyzed in the samples of locally advanced rectal cancer from 89 patients. The authors reported that patients whose cancer exhibited cytosolic HMGB-1 before neoadjuvant chemoradiotherapy had a better clinical outcome.

PDT has also been shown to induce ICD. This therapy is based on the pre-treatment of target cells with a photosensitizer, which is next photoactivated to generate reactive species and cause oxidative stress in situ [105]. As a consequence of the photoreactions thus induced, reactive species are produced, which can overwhelm the antioxidant defenses of the cell, generating oxidative stress and cell death [26]. This approach has been shown to induce ICD in specific conditions. The type and concentration of the photosensitizer [27,106], as well as the light dose [26], can significantly affect its ICD-inducing capacity. Moreover, oxygen supply in the target tissue can also affect PDT outcomes.

Doix et al. (2019) reported that the combination of a therapeutical vaccine based on DCs stimulated with PDT-killed cells and radiotherapy can delay the development of experimental squamous cell carcinoma in mice. Firstly, the authors showed that the non-porphyrinic photosensitizer OR141, at low doses, induced ICD in SCC7 squamous cell carcinoma cells in vitro. These PDT-killed SCC7 cells were then used to prime and stimulate the maturation of DCs in vitro, which were then employed as a therapeutic vaccine against xenografts of SCC7 cells developed in the flank of C3H mice. This DC vaccine was subcutaneously injected three times at one-week intervals near the tumordraining lymph node. One week later, radiotherapy mediated by a Cesium-137 γ-ray irradiator was applied onto the tumors. Occasionally, a fourth injection of the DC vaccine was performed at the time of radiotherapy application (peri-radiotherapy). It was observed that the tumor growth was delayed only when the DC vaccine was applied during the peri-radiotherapy period.

Rodrigues et al. (2022) showed that PDT using the photosensitizer aluminum-phthal ocyanine mainly induced necrosis at the highest photosensitizer concentration and light dose, while milder protocols of PDT were able to efficiently induce ICD in both colorectal CT26 and mammary 4T1 murine adenocarcinoma cells [26]. In vitro, the milder PDT protocol consisted of 12.2 nM aluminum-phthalocyanine for colorectal adenocarcinoma CT26 cells and 9.0 nM aluminum-phthalocyanine for breast adenocarcinoma 4T1 cells, both irradiated with 25 J/cm<sup>2</sup> red light dose. The exposure or release of calreticulin, HSP70, HSP90 and HMGB-1 close to that promoted by this PDT protocol was comparable to that observed with mitoxantrone treatment. Increasing the photosensitizer concentration and light dose resulted in a reduction on the release of these DAMPs. This shows that the PDT regimen has to be fine-tuned to induce ICD.

As commented previously, there are several ICD inducers described in the literature. Among them are classical approaches, such as chemotherapy, PDT and radiotherapy. These classical therapies are usually aimed at tumor destruction, mainly by inducing direct cytotoxicity to target tumor cells, regardless of the death mechanism underlying this effect. This cytotoxicity induced in a noncontrolled fashion can fail to trigger immune activation. Immune-targeted cytotoxic cancer treatments have to thus be designed to induce ICD, representing an adaptation in former protocols in order to increase the immunogenicity of cancer treatments.

Several ICD inducers are used in well-recognized therapies. Thus, we understand that the translation of this knowledge to clinical protocols can be easier if compared to the implementation of new anticancer drugs which are currently under development. As previously discussed, the literature shows evidence of abscopal effects in radiotherapy and PDT protocols, for example, that could be due to ICD. However, due to the complexity of cancer cells and tumor tissues, it is probable that personalized approaches are necessary to the successful use of immune-activating strategies in the clinic. Another barrier to the development of clinical protocols for ICD induction is the lack of standardization in ICD studies, as evidenced in this section. Thus, the use of widely recognized, bona-fide parameters that demonstrate that an immunogenic response is being induced is essential for translating research into clinical protocols [107]. This can be supported by guidelines proposed in the literature [29].
