EP4735010A1 - Dendritic cells loaded with lysates from late ferroptotic cells to treat cancer - Google Patents

Abstract

The present invention relates to the field of immunotherapeutical compositions which can be used to treat cancer. More in particular, the present invention discloses dendritic cells loaded with lysates derived from late ferroptotic cancer cells which are useful to treat cancer.

Description

DendriƟc cells loaded with lysates from late ferroptoƟc cells to treat cancer Technicalfield of the invenƟon The present invenƟon relates to thefield of immunotherapeuƟcal composiƟons which can be 5 used to treat cancer. More in parƟcular, the present invenƟon discloses dendriƟc cells loaded with lysates derived from late ferroptoƟc cancer cells which are useful to treat cancer. Background art According to World Health Organization (WHO), cancer is the second leading cause of death globally, accounting for an estimated 9.6 million deaths, or one in six deaths, in 2019. 10 Cutaneous melanoma is the neoplasm originated from the melanocytes of the epidermis, and, although it corresponds to only 4% of skin related cancers, it is the causal agent for 80% of deaths from dermatological cancer (Miller et al., 2019). Unlike other tumor types, melanoma incidence and its mortality rate increased each year, an event associated with excesses in sun exposure and the progressive loss of the ozone layer (Chang et al., 2014). There are nearly 15 324.635 new cases of melanoma worldwide and 57.043 deaths (Sung et al., 2021). When surgical excision is performed on tumors with early diagnosis, the average survival rate at 10 years is 80%. However, in the case of metastatic melanoma, survival decreases to <10%. New insights in immuno-oncology and the subsequently developed immunotherapies have caused a major breakthrough in the management of metastatic melanoma in the last decade, 20 creating the hope of curing (metastatic) cancer. Despite encouraging results, the proportion of patients experiencing a long-term/durable response is still limited. Therefore, the major challenge focuses on designing new therapies to treat melanoma in advanced stages with systemic dispersion. To improve response rates multiple combination strategies are currently being explored in clinical trials. However, the number of combinations that has demonstrated 25 clear advantage over monotherapy and has entered clinical practice is scarce. In melanoma the combination of anti-PD1 and anti-CTLA4 immunotherapy is superior to anti-PD1 monotherapy at the cost of considerable toxicity: approximately 60% of the patients experience grade 3 to 4 immune related adverse events (Larkin et al., 2019). These data indicate that there is a need for new immunotherapeutic strategies that could further improve efficacy and toxicity profiles. Gliomas, the most frequent intrinsic type of primary tumors of the central nervous system (CNS) in adults, are associated with significant morbidity and mortality (Miller et al., 2021). 5 According to the newest World Health Organization classification of tumors of the CNS (Torp et al., 2022), gliomas, glioneuronal tumors, and neuronal tumors are divided into six different families. Among these are adult-type diffuse gliomas (i.e., most adult patients with primary brain tumors, e.g., glioblastoma (GBM), IDH- wildtype), pediatric-type diffuse low-grade gliomas (with favorable prognoses), and pediatric-type diffuse high-grade gliomas (with poor 10 prognoses) (Louis et al., 2021). The pediatric and adult types of gliomas are distinctively different biologically and genetically. Of note, pediatric-type diffuse gliomas have been subdivided into low-grade gliomas (LGG) and high-grade gliomas (HGG) (Komori, 2022). GBM is classified as a grade 4 malignancy; it is the most aggressive type of cancer of the central nervous system and has a poorer prognosis (Louis et al., 2021; Tesileanu et al., 2020; Weller 15 et al., 2021). Despite the development of novel, complex, multidisciplinary, targeted therapies, such as focal radiotherapy and adjuvant chemotherapeutics in combination with surgical resection, glioblastoma therapy has not progressed much over the last decades (Lieberman, 2017). The median survival of patients diagnosed with glioblastoma is 12–15 months, with a five-year survival rate of 5% (Ostrom et al., 2015; Stupp et al., 2009). 20 Therefore, there is an urgent need to develop novel patient-adjusted anticancer immunotherapies that actively stimulate antitumor T cells, generate long-term memory, and result in significant clinical benefits. Several recent, novel, therapeutic approaches have emerged that rely on vaccination to activate the patient’s own immune system and to induce a potent and long-lasting immune 25 response against cancer antigens. Dendritic cells are key to initiating and directing immune responses (Harari et al., 2020; O’Keeffe et al., 2015), and one of these approaches involves the use of dendritic cells loaded with antigenic material derived from or based on the autologous tumor. One such approach is based on the identification of neo-antigens, but it has low efficacy due to the high antigenic heterogeneity of glioma (e.g., GBM) (Touat et al., 2020). Moreover, this approach is complex, labor-intensive, and costly. In contrast, the preparation of cancer cell lysate from the glioma tissue of a patient is less complex and the lysate includes neo-antigens as well as non-mutated tumor antigens, which can result in a broader immune response. However, though the immunogenicity of the lysate loaded in the 5 dendritic cells is important (Garg et al., 2016; Goldbrunner et al., 2021; Vandenberk et al., 2016), whole glioma and melanoma cells are usually killed by freeze-thawing (F/T) (Belmans et al., 2017; Hunn et al., 2015; Salcedo et al., 2006), which induces an accidental and unregulated form of necrotic cell death of low immunogenicity (Aaes et al., 2016; Gamrekelashvili et al., 2012; Turubanova et al., 2019). One way to increase the 10 immunogenicity of the lysate is to kill the glioma and melanoma cells by a method that induces immunogenic cell death (ICD) (Galluzzi et al., 2020). ICD has recently been shown to be a prerequisite for the activation of the patient’s immune system. Thus, induction of ICD provides two benefits, effectively killing cancer cells and activating an immune response specific to the cancer cells. ICD is characterized by the release 15 or surface exposure of damage-associated molecular patterns (DAMPs), which function as adjuvants to activate strong anticancer immunity (Fucikova et al., 2020; Krysko et al., 2012). In addition, ICD is characterized by the expression of tumor-specific antigens (antigenicity). The combination of this antigenicity and adjuvanticity allows efficient induction of anti-tumor immunity. 20 Triggering apoptotic cell death with anti-cancer drugs is one of the principal approaches for killing cancer cells. However, the effectiveness of apoptosis induction in tumors is limited, due to the acquired or intrinsic resistance of cancer cells to apoptosis (Holohan et al., 2013; Okada & Mak, 2004; Su et al., 2016). Therefore, exploiting other forms of non-apoptotic cell death opens new therapeutic avenues for eliminating cancer cells and limiting the survival of drug- 25 resistant clones. Ferroptosis is a form of regulated cell death that is characterized by the accumulation of iron and the production of reactive oxygen species in cells. It is morphologically, biochemically, and genetically distinct from other well-known forms of regulated cell death such as apoptosis and necroptosis (Friedmann Angeli et al., 2019). Ferroptosis is marked by the oxidative modification of phospholipid membranes via an iron- dependent mechanism. An initial characterization of this pathway demonstrated that cysteine depletion, which leads to the exhaustion of the intracellular pool of glutathione (reduced) (GSH) specifically triggers this form of cell death. The requirement for GSH to protect from ferroptosis was later related to the optimal activity of the enzyme glutathione 5 peroxidase 4 (GPX4), a selenoprotein required for the efficient reduction of peroxidized phospholipids and to suppress the activation of arachidonic acid (AA)-metabolizing enzymes, which may contribute to the process of phospholipid peroxidation. Since then, a complex interplay between lipid, iron, and cysteine metabolism has emerged as an important regulator of this cell death pathway (Friedmann Angeli et al., 2019). It has been previously shown that 10 only early ferroptotic fibrosarcoma cells are immunogenic in vitro and in vivo in the tumor prophylactic vaccination mice model, while late ferroptotic cells were not immunogenic in vitro and in vivo (Efimova et al., 2020). More recently it has been reported that ferroptotic cancer cells are also not immunogenic (Wiernicki et al., 2022). In this study, the authors co- cultured ferroptotic cancer cells with dendritic cells (DCs) and found that ferroptotic cells are15 poorly engulfed and even decreased the maturation of DCs, and dampened antigen cross- presentation. Importantly that DCs derived from spleen (CD11C⁺XCR1⁺TAMRA⁺) loaded with ferroptotic, in contrast to necroptotic, cancer cells fail to protect against melanoma B16 subcutaneous tumors growth. Taken together, there is still an urgent need to develop novel and/or alternative therapeutical 20 methods to treat cancer. Brief descripƟon offigures Figure 1. Cell death measured byflow cytometry or by MTS of the GL261 cells sƟmulated either with RSL3 or Sulfasalazine (SAS). QuanƟficaƟon was done by Sytox Blue (Sytox) staining 25 or by MTS. For ferroptosis inducƟon, GL261 cells were treated with RSL3 at a dose from 1 µM to 3 µM for 24h or 48h. The values are the means ± SEM and represent three and more independent experiments. Normality of distribuƟon was assessed using the Shapiro-Wilk test. If the distribuƟon was normal, ANOVA was used; if the distribuƟon was not normal, the Kruskal-Wallis test was used. A: Cell death measured byflow cytometry or by MTS of the GL261 cells sƟmulated with RSL3. *p < 0.05, **p < 0.01, ***p < 0.001, Kruskal-Wallis test, n ≥ 3. B: Cell death measured byflow cytometry or by MTS of the GL261 cells sƟmulated with Sulfasalazine (SAS). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test, n ≥ 3. C: The analysis of ferroptoƟc cell death of GL261 cells treated with RSL3 using the MTS 5 assay. GL261 cells were treated with specific cell death inhibitors for 1 h and then with a mixture of RSL3 (2,5 µM) and respecƟve cell death inhibitor. The following concentraƟons of inhibitors were used: 50 µM zVAD-fmk (apoptosis), 20 µM Nec-1s (necroptosis), 50 µM Vit E (ferroptosis), 10 µM DFO (ferroptosis) and 1 µM Fer-1 (ferroptosis). ****p < 0.0001, ANOVA, n = 4. D: The analysis of late ferroptoƟc cell death of GL261 cells treated with SAS using the 10 MTS assay. GL261 cells were treated with cell death inhibitors for 1 h and then with a mixture of SAS (300 µM) and respecƟve inhibitor. The following concentraƟons of inhibitors were used: 50 µM zVAD-fmk (apoptosis), 20 µM Nec-1s (necroptosis), 50 µM Vit E (ferroptosis), 10 µM DFO (ferroptosis) and 1 µM Fer-1 (ferroptosis). ****p < 0.0001, ANOVA, n = 4. Figure 2. Prophylactic vaccination of mice with DC-based vaccines loaded with glioma GL261 15 lysates obtained from late ferroptotic cells. A: Experimental setup. Dendritic cells (DCs) were loaded with late ferroptotic lysates (DC-GL261-RSL3) as described in the materials and methods. For late ferroptosis induction, GL261 cells were treated with RSL3 at a dose of 2.5 µM for 24h. As controls, we used DC-based vaccines loaded with lysates GL261 cells subjected to F/T cycles (accidental necrosis, non-immunogenic cell death, negative control, DC-LG261- 20 FT) or DC alone or mice were injected with PBS. The mice were injected with respective DC vaccines 7 and 14 days before intracranially injection with viable GL261 cells using stereotactic coordinates. B: Survival of mice vaccinated and challenged with GL261 cells as described in (A). *p < 0.05, **p < 0.01, Mantel-Cox logarithmic test, n = 6-8. C: Representative T1- tomograms of layer-by-layer frontal brain sections on day 23. D: The temporal progression of 25 neurological deficits in mice treated as described in (A) is shown for each group. *p < 0.05, Unpaired t-test, n = 6-8. Figure 3. Therapeutic vaccination of mice with DC-based vaccines loaded with glioma GL261 lysates obtained from late ferroptotic cells increased the mice survival. A: Experimental setup. DCs were loaded with late ferroptotic lysates (DC-GL261-RSL3 and DC-GL261-SAS) as 30 described in the materials and methods. For induction of late ferroptosis, GL261 cells were treated with RSL3 at a dose of 2.5 or with SAS at a dose of 300 µM for 24h. As controls, we used DC alone or mice were injected with PBS. The mice were vaccinated with DC vaccines 2, 9, and 16 days after intracranially injection with viable GL261 cells using stereotactic coordinates. B: Survival of mice vaccinated with DC-GL261-RSL3 (lysates derived from late 5 ferroptotic cells) and challenged with viable GL261 cells as described in (A). *p < 0.05, Mantel- Cox logarithmic test, n = 12. C: The temporal progression of neurological deficits in mice treated with DC-GL261-RSL3 as described in (A) is shown for each group. *p < 0.05, Unpaired t-test, n = 12. D: Survival of mice vaccinated with DC-GL261-SAS (lysates derived from late ferroptotic cells) and challenged with viable GL261 cells as described in (A). *p < 0.05, Mantel- 10 Cox logarithmic test, n = 9-12. E: The temporal progression of neurological deficits in mice treated with DC-GL261-SAS as described in (A) is shown for each group. *p < 0.05, Unpaired t-test, n = 9-12. Figure 4. Cell death measured by MTS of the CT2a cells stimulated with RSL3. Quantification was done by MTS. For late ferroptosis induction, GL261 CT2a were treated with RSL3 at a dose 15 from 0,25 µM to 3 µM for 24h or 48h. The values are the means ± SEM and represent three and more independent experiments. A: Cell death measured by MTS of the CT2a cells stimulated with RSL3. ****p < 0.0001, ANOVA, n = 3. B: The analysis of late ferroptotic cell death of CT2a cells treated with RSL3 using the MTS assay. CT2a cells were treated with respective specific cell death inhibitors for 1 h and then with a mixture of RSL3 (0,5 µM) and 20 respective cell death inhibitor. The following concentrations of inhibitors were used: 50 µM zVAD-fmk (apoptosis), 20 µM Nec-1s (necroptosis), 50 µM Vit E (ferroptosis), 10 µM DFO (ferroptosis) and 1 µM Fer-1 (ferroptosis). ***p < 0.001, ****p < 0.0001, ANOVA, n = 3. Figure 5. Therapeutic vaccination of mice with DC-based vaccines loaded with lysates of CT2a cells derived from late ferroptotic cells increased the mice survival. A: Experimental 25 setup. DCs were loaded with late ferroptotic lysates (DC-CT2a-SAS) as described in the materials and methods. For late ferroptosis induction, CT2a cells were treated with RSL3 at a dose of 0.5 µM for 24h. As controls, we used DCs alone or mice were injected with PBS. The mice were vaccinated with DC vaccines 2, 9, and 16 days after intracranially injection with viable GL261 cells using stereotactic coordinates. B: Survival of mice vaccinated with DC-CT2a- RSL3 and challenged with viable CT2a cells as described in (A). *p < 0.05, **p < 0.01, Mantel- Cox logarithmic test, n = 15-16. C: The temporal progression of neurological deficits in mice treated with DC-CT2a-RSL3 as described in (A) is shown for each group (n = 15-16). *p < 0.05, Unpaired t-test, n = 15-16. 5 DescripƟon of the invenƟon The present invenƟon is based on the surprisingfinding that when (autologous) dendriƟc cells are loaded with lysates from late ferroptoƟc cancer cells and are administered to cancer- bearing mice that this leads to a superior anƟ-cancer immunoprotecƟve effect resulƟng in less 10 tumor growth and significantly increased survival of these mice. Therefore, the present invenƟon relates infirst instance to dendriƟc cells loaded with lysates derived from late ferroptoƟc cancer cells for use to prevent or treat cancer. With the term ‘dendriƟc cells’ are meant the well-known anƟgen-presenƟng cells (also known as accessory cells) of the mammalian immune system. A dendriƟc cell's main funcƟon is to 15 process anƟgen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and adapƟve immune systems. DendriƟc cells can be obtained via any method known in the art. DendriƟc cells can -for example- be obtained from bone marrow that is isolated from Ɵbias and femurs, and, can then be grown on in a culture medium. The laƩer dendriƟc cells can be ‘loaded’ by bringing protein obtained from 20 lysates from late ferroptoƟc cancer cells into a suspension of dendriƟc cells so that said dendriƟc cells (which can be acƟvated with -for example- LPS) can engulf and uptake said proteins so that said that fragments of said proteins can be presented to T cells. ‘Late ferroptoƟc cancer cells’ can be obtained by sƟmulaƟng tumor cells with a ferroptosis- inducing agent such as RSL3, sulfasalazine (SAS), ML-162, sorafenib, altretamine, withaferin A 25 and ErasƟn, silica nanoparƟcles, photoacƟvaƟon or by using other methods aiming to inacƟvate, deplete or affect (pharmacologically of geneƟcally) glutathione peroxidases (GPXs, e.g., GPX4), squalene synthase, HMG-CoA reductase, ND dehydrogenase, glutathione S- transferase, glutamate-cysteine ligase, acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphaƟdylcholine acyltransferase 3 or inhibiƟng the import of cysƟne (e.g., by affecƟng the system XC-), or affecƟng metabolism of polyunsaturated faƩy acids (D’Herde & Krysko, 2017; Dierge et al., 2021; Hassannia et al., 2019). With the term ‘ferroptoƟc’ is meant a cancer cell which undergoes an iron-catalyzed regulated 5 necroƟc cell death (induced pharmacologically or geneƟcally) that occurs through excessive non-enzymaƟc or auto-oxidaƟon of lipids in which reacƟve oxygen species iniƟate the oxidaƟon of polyunsaturated faƩy acids containing phospholipids (Dixon et al., 2012) leading to the loss of cellular redox homeostasis and to accumulaƟon of toxic lipid peroxidaƟon products (e.g., oxidized phosphaƟdylethanolamines). The deleterious effects of lipid 10 peroxidaƟon in ferroptoƟc cancer cells can be neutralized or blocked by iron chelators such as deferoxamine (DFO), and a wide variety of lipophilic radical traps such as Vitamin E (Vit E), ferrostaƟn-1 (Fer-1), and liproxstaƟn-1 (Hassannia et al., 2019). With the term ‘late’ ferroptoƟc cancer cells is meant a cell death aŌer at least 24 hours of sƟmulaƟon or more with a ferroptosis inducer. In this case the proporƟon of cells that stain 15 posiƟvely for Sytox is approximately 80% or more. While “early” ferroptoƟc cancer cells aŌer sƟmulaƟon with a ferroptosis inducer for 1 hour are defined as cells stain with approximately 20% or less for Sytox which indicates 20% or less of dead cells as is described by Efimova et al. (2020). In other words, ‘early ferroptoƟc cancer cells’ comprise a maximum of 20% death cells aŌer sƟmulaƟon whereas ‘late ferroptoƟc cancer cells’ comprise a minimum of 80% death 20 cancer cells aŌer sƟmulaƟon. ‘Lysates’ can be obtained by subjecƟng cells to six cycles of freezing (–80 °C) and thawing (+37 °C), mechanical disrupƟon (e.g., sonicaƟon), heat shock or hyperthermia (from +40°C to +60°C), electroporaƟon, gamma irradiaƟon, high-intensity focused ultrasound, photoacƟvaƟon, photoporaƟon alone or in combinaƟon with nanoparƟcles and 25 nanomaterials, acidic buffers (e.g., the citrate-phosphate buffer pH 3.3) or enzymaƟc digesƟon. The term ‘cancer’ can be any type of cancer such as melanoma, glioma, glioblastoma, fibrosarcoma etc. The term ‘cancer’ specifically relates to glioblastoma and melanoma Hence, and more in parƟcular, the present invenƟon relates to dendriƟc cells for use as described above wherein said cancer is glioblastoma or melanoma. Moreover, the present invenƟon relates to dendriƟc cells for use as described above wherein said lysate is obtained by freezing and thawing said late ferroptoƟc cancer cells. 5 Furthermore, the present invenƟon relates to dendriƟc cells for use as described above wherein said late ferroptoƟc cancer cells are obtained by inducing late ferroptosis ex vivo via contacƟng said cancer cells with a ferroptosis inducer. In parƟcular, the present invenƟon relates to dendriƟc cells for use as described above wherein said loaded with lysates is obtained by contacƟng said dendriƟc cells with said lysates. 10 The present invenƟon further relates to method to treat a cancer paƟent in need thereof comprising: - collecƟng dendriƟc cells, -collecƟng cancer cells, -inducing late ferroptosis of said cancer cells via contacƟng said cancer cell with a ferroptosis 15 inducer, -lysing said late ferroptoƟc cancer cells, -loading said lysates into said dendriƟc cells, and -administering an effecƟve amount of said dendriƟc cells loaded with lysates derived from late ferroptoƟc cancer cells to said paƟent in need thereof. 20 More specially, the present invenƟon relates to a method to treat as described above wherein said dendriƟc cells and/or said cancer cells are collected from said paƟent and are thus ‘autologous’ cells. ‘Heterologous’ or homologous anƟgens, tumor associated anƟgens or neo-anƟgens derived from ferroptoƟc cancer cells can be used as well for loading onto dendriƟc cell vaccines. 25 Furthermore, the present invenƟon discloses a method as described above wherein said cancer is glioblastoma or melanoma. The present invenƟon further relates to a method as described above wherein said lysate is obtained by freezing and thawing said late ferroptoƟc cancer cells. The present invenƟon further relates to a method as described above wherein said late ferroptoƟc cancer cells are obtained by inducing late ferroptosis ex vivo via contacƟng said 5 cancer cells with a ferroptosis inducer. The present invenƟon further relates to a method as described above wherein said loaded with lysates is obtained by contacƟng said dendriƟc cells with said lysates. The present invenƟon thus discloses a pharmaceuƟcal composiƟon comprising dendriƟc cells loaded with lysates derived from late ferroptoƟc cancer cells. 10 More in parƟcular, the present invenƟon discloses a pharmaceuƟcal composiƟon as described above wherein said cancer cells are glioblastoma or melanoma cells. The term ‘pharmaceuƟcal composiƟon’ or ‘medicament’ relates to a substance used in therapy or treatment. Moreover, the present invenƟon relates to a pharmaceuƟcal composiƟon comprising loaded 15 dendriƟc cells as described above. The term ‘a pharmaceuƟcal composiƟon’ relates to a composiƟon comprising dendriƟc cells as described above and which is further formulated to be compaƟble with its intended route of administraƟon. Hence, suitable diluents, solvents, anƟoxidants, chelaƟng agents, buffers, carriers, isotonic agents, binding agents, growth factors, mRNA, cytokines and chemokines, adjuvants, toll-like receptor agonists, immune 20 checkpoints inhibitors,flavoring agents, propellants, detergents, nanomedicine (liposomes, nanoparƟcles, nanomaterials) and the like which are described in detail in, for example, WO 03/004989, can be added to the dendriƟc cells as described above. The present invenƟon also relates to a method to treat or prevent cancer of a subject in need thereof comprising administering a therapeuƟcally effecƟve amount of dendriƟc cells as 25 described above or of a pharmaceuƟcal composiƟon as described above. By the term ‘method to treat’ or ‘treatment’ is meant the medical management of a paƟent with the intent to cure, ameliorate, stabilize, or prevent cancer. It is further understood that appropriate doses of said compounds (which can also be denominated as drugs, medicaments or pharmaceuƟcal composiƟons) depends upon a number of factors within the knowledge of 10 the ordinary skilled physician. The dose of these compounds will vary, for example, depending upon the idenƟty, size, and condiƟon of the paƟent being treated, upon the route of administraƟon of said compounds (i.e., parenteral (intravenous, intradermal, subcutaneous), oral, transdermal, transmucosal or rectal) and upon the effect which the skilled physician 5 desires the compound to have. Examples Results Ferroptosis-based DC vaccines induce significant protective immunity against glioma in the 10 prophylactic and therapeutic orthotopic mice models Firstly, the capacity of RSL3 and Sulfasalazine (SAS) at varying concentrations to induce cell death in GL261 glioma cells was analysed (Figure 1A,B). This was followed by confirmation that the cell death observed is late ferroptosis (Figure 1C,D). The extent of cell death was quantified by flow cytometry or the MTS assay. 15 We then evaluated the immunogenic potential of DC vaccines based on lysates derived from late ferroptotic cells (DC-GL261-RSL3) and the ability of such DC vaccines to trigger anti-glioma protective immunity in the prophylactic settings. For this, we examined whether this DC immunotherapy could protect mice against intracranial tumor challenge with viable GL261 cells. First, mice had two prophylactic vaccination of DC vaccine at 14 and 7 days before 20 intracranial tumor inoculation with viable GL261 cells (Figure 2A). As the negative control, we used PBS or non-loaded DCs or DCs loaded ex vivo with F/T glioma GL261 cells (non- immunogenic accidental necrosis; DC-GL261-FT). For each vaccine, GL261 cell lysates were prepared from pre-stimulated glioma cells. After the injection of DC vaccines, all the mice were intracranially inoculated with 2 × 10⁴ live GL261 glioma cells and then monitored the 25 development of symptoms of neurological deficit and mice survival. The mice vaccinated with DC-GL261-RSL3 (lysates derived from late ferroptotic cells) demonstrated a significant increase in median survival compared to mice injected with non-loaded DCs (25,5 days versus 63 days, p < 0.01) or with DCs loaded with F/T GL261 (24 days versus 63 days, p < 0.01) (Figure 2B). In the DC-GL261-RSL3 group, mice had a smaller tumor volume (Figure 2C) and showed a slower development of neurological deficit (Figure 2D). In conclusion, late ferroptosis-based DC vaccines induce significant protective immunity against glioma in the prophylactic model. Prophylactic vaccination is a convenient way to analyze molecular mechanisms and 5 determine the ability to induce an immune response. However, in clinical practice, it is not possible to give prophylactic vaccination, since it is required to cure the patient of an already existing tumor. Therefore, we tested the efficacy of DC-GL261-RSL3 vaccines derived from lysates of late ferroptotic cells in a therapeutic orthotopic intracranial glioma mouse model. As a negative control, we used non-loaded DCs (DC-PBS). We found that three consecutive 10 vaccinations of DC-GL261-RSL3 (derived from lysates of late ferroptotic cells) at 2, 9, and 16 days after tumor inoculation (Figure 3A) significantly increased survival compared to mice injected with non-loaded DCs (23.5 days versus 25 days, p < 0.05) (Figure 3B). Three times DC- GL261-RSL3 vaccine delayed the development of symptoms of neurological deficit and protected against brain damage, which begins to appear at day 20 but this difference 15 disappeared by day 28 after tumor inoculation relative to control group (Figure 3C). Vaccinations of DC vaccine, based on glioma cells induced with another late ferroptosis inducer Sulfasalazine (SAS) DC-GL261-SAS significantly increased survival compared to mice injected with non-loaded DCs (23.5 days versus 26 days, p < 0.05) (Figure 3D). The development of symptoms of neurological deficit was significantly delayed from 20^th to 28^th 20 day (Figure 3E). To comprehensively understand how different types of glioma cells respond to ferroptosis induction and produce antitumour immune response we used another glioma CT2a cell line, which is low immunogenic glioma cells (Belmans et al., 2017; Khalsa et al., 2020). First, the capacity of the ferroptosis inducer to induce cell death in this cell line was evaluated (Figure 25 4A) and the late ferroptosis was confirmed (Figure 4B). Subsequently, a therapeutic vaccination was performed utilising CT2a glioma. Initially, 5*10⁵ living CT2a glioma cells were injected into the animal's brain. On days 2, 9 and 16 following intracranial inoculation, a DC vaccine based on lysates derived from late ferroptotic CT2a cells was administered (Figure 5A). In order to serve as negative controls, PBS and non-loaded dendritic cells (DC-PBS) were employed. The survival of animals in the DC-CT2a-RSL3 group was found to be significantly greater than that observed in the PBS tumour inoculation control (26 days versus 30 days, p < 0.01) and with non-loaded DCs (26.5 days versus 30 days, p < 0.01) (Figure 5B). The development of symptoms of neurological deficit was delayed from 24^th to 45^th day 5 comparing with PBS group and from 26^th to 45^th day comparing with DC-PBS group (Figure 5C). Materials and Methods Cell lines 10 High immunogenic murine glioma GL261 cells were cultured at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose and supplemented with 2 mM glutamine, 100 µM sodium pyruvate, 100 units/ml penicillin, 100 µg/L streptomycin and 10% fetal bovine serum. Low immunogenic murine glioma CT2a cells were cultured at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 15 4.5 g/L glucose and supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 µg/L streptomycin and 10% fetal bovine serum. GL261 glioma shares many characteristics with human glioblastomas, including mutations such as p53 and K-ras, PTEN deficiency, positive staining for vimentin, and high expression levels of MHC I, indicating immunogenicity. However, it does not express co-stimulatory20 markers of T-cell activation such as B7-1 and B7-2 (Oh et al., 2014). GL261 is classified as non- metastatic and shares similarities with ependymoblastoma (Belmans et al., 2017). CT2A glioma, which originates from a malignant astrocytoma, is categorized as p53wt and marked by PTEN deficiency, a high mitotic index, and an invasive nature. Furthermore, it expresses the stem cell marker CD133 and replicates the histological features of high-grade gliomas 25 (Binello et al., 2012). Although CT2A is considered to have low immunogenicity, it is responsive to immunotherapy and demonstrates immune cell infiltration into the tumour microenvironment (Belmans et al., 2017; Khalsa et al., 2020). Mice experiments 5 Female C57BL/6J mice (6–8 weeks old) were housed in specific pathogen-free conditions. The mouse experiments were performed according to the guidelines of the local Ethics Committees of and the Faculty of Medicine and Health Sciences of Ghent University (Belgium; ECD 22-12aan, ECD-23-73). Generation of bone-marrow-derived dendritic cells (DCs) 10 Bone marrow was isolated from tibias and femurs in RPMI medium (GIBCO) supplemented with 5% heat-inactivated fetal calf serum, 20 ng/ml murine GM-CSF (UGent-IRC-VIB Protein Core Facility), 1% L-glutamine, and 50 μM 2-mercapthoethanol. The bone marrow was suctioned with a 25 G needle (0.5 × 25 mm), resuspended, and coarse debris was filtered through a Cell Strainer 70 μm (Falcon). The suspension was cleared of erythrocytes with a 15 lysing solution. The cells were grown for up to 10 days. Fresh culture medium was added on day 3, and on day 6 the medium was fully refreshed. Cell death stimulation and cell death assay GL261 tumor cells were stimulated with 2.5 µM RSL3 or 300 µM Sulfasalazine (SAS) and incubated for 24 h in order to induce late ferroptosis with about 70-80% of cell death. CT2a 20 tumor cells were stimulated with 0.5 µM RSL3 to induce late ferroptosis with about 70-80% of cell death. The cells were stained with Sytox Blue Nucleic Acid Stain and analysed on a BD FACS LSR II flow cytometer or stained with CellTiter 96 AQ_ueous One Solution Reagent (MTS) and analysed on Tecan Spark microplate fluorescence reader. DC vaccines 25 DCs were collected on day 8. The GL261 or CT2a cells after respective treatment were then subjected to six cycles of freezing (–80 °C) and thawing (+37 °C). Total protein in the cell lysate was measured with a commercial BCA Protein Assay. Two mg of protein was added to a suspension of 10 × 10⁶ DCs for 90 min. To activate the DCs, they were treated with lipopolysaccharide (5 μg/ml) for 24 h. In some experiments, PBS or DCs co-cultured with GL261 or CT2a glioma cell lysates subjected to several freeze/thaw cycles to induce accidental necrosis were used as controls. Prophylactic protocol Female C57BL/6j mice (6–8 weeks old) were injected intraperitoneally twice seven days apart with a suspension containing 1 × 10⁶ prepared DCs. Seven days after the last injection, 2 × 10⁴ viable GL261 glioma cells were injected intracranially. All animals were anesthetized with a mixture of medical oxygen and isoflurane (induction: 5%; maintenance: 2%) and immobilized in a stereotaxic frame. The injection was performed using a stereotactic device 2 mm lateral and 2 mm posterior to the bregma and 3 mm below the dura mater. The skin was sutured, and meloxicam was administered subcutaneously (1 mg/kg) to manage post-operative pain. Therapeutic protocol Female C57BL/6j mice (6–8 weeks old) were anesthetized with a mixture of medical oxygen and isoflurane (induction: 5%; maintenance: 2%) and intracranially injected with 2 × 10⁴ viable GL261 glioma cells or 5 × 10⁵ viable CT2a glioma cells. The mice were injected intraperitoneally with a suspension containing 1 × 10⁶ of the prepared DCs (as described above) three or four times on days 2, 9, 16 after intracranial injection of viable glioma cells. Neurological status assessment After intra-cranial inoculation with glioma cells and/or DC vaccines, the mice were monitored three times per week and clinical symptoms were scored with a neurological deficit grading scale (Garg et al., 2016). The dynamics of the functional state of the central nervous system was evaluated on a scale to assess the severity of neurological deficit, with modifications for mice. The scale includes several tests of motor activity, coordination, reflexes, muscle tone, ptosis, and exophthalmos. Each test was scored 2 points for no reaction, 0 for good/normal reaction, and –1 for some disturbances. The values were summed up and interpreted as death (20 points), severe central nervous system damage (14─19 points), moderate damage (8─13 points), or light damage (2─7 points). The neurological score was evaluated by a blinded investigator. Magnetic resonance imaging 5 To assess the dynamics of intracranial tumor growth in the prophylactic model, magnetic resonance imaging (MRI) was applied using a high-field magnetic resonance tomograph, Agilent Technologies DD2-4009.4 T (400 MHz) with a volume coil M2M (Н1). The animals were anesthetized with a mixture of medical oxygen and isoflurane (induction: 5%; maintenance: 2%) in a fixed position inside the magnet tunnel for 40 min. The VnmrJ program 10 was used to obtain and process data. T1-tomograms of layer-by-layer frontal brain sections weighted by proton density were obtained using the multi gradient-echo multi slice (MGEMS) pulse sequence with the following parameters: TR = 1000 ms, TE = 1.49 ms, 6 echoes, FOV 20 × 20 mm, matrix 128 × 128 and aŌer −256 × 256, slice thickness 1 mm, 15 slices, 17 min and 4 s scanning time. 15 Statistical Analysis Statistics were calculated in GraphPad Prism (V.9.2). In graphs showing cell death, the values are the means ± SEM and represent three and more independent experiments. Normality of distribution was assessed using the Shapiro-Wilk test. If the distribution was normal, ANOVA was used; if the distribution was not normal, the Kruskal-Wallis test was used. Kaplan–Meier 20 survival curves show the timeline of tumor development. Survival in the low-risk and high-risk groups was analyzed by log-rank Mantel–Cox test. The level of neurological deficit was analysed by Unpaired t-test.

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Claims

Claims 1. DendriƟc cells loaded with lysates derived from late ferroptoƟc cancer cells for use to prevent or treat cancer. 2. DendriƟc cells for use according to claim 1 wherein said cancer is glioblastoma or 5 melanoma. 3. DendriƟc cells for use according to claims 1-2 wherein said lysate is obtained by freezing and thawing said ferroptoƟc cancer cells. 4. DendriƟc cells for use according to claims 1-3 wherein said late ferroptoƟc cancer cells are obtained by inducing ferroptosis ex vivo via contacƟng said cancer cells with a ferroptosis inducer, wherein said ferroptosis inducer is chosen from the list of: RSL3, sulfasalazine, ML- 162, sorafenib, altretamine, withaferin A, ErasƟn, silica nanoparƟcles, photoacƟvaƟon, or, by using methods aiming to inacƟvate, deplete or affect glutathione peroxidases, squalene synthase, HMG-CoA reductase, ND dehydrogenase, glutathione S-transferase, glutamate- cysteine ligase, acyl-CoA synthetase long-chain family member 4 and lysophosphaƟdylcholine acyltransferase 3, or, by inhibiƟng the import of cysƟne, or, by affecƟng metabolism of polyunsaturated faƩy acids. 5. DendriƟc cells for use according to claims 1-4 wherein said loaded with lysates is obtained by contacƟng said dendriƟc cells with said lysates. 6. A method to treat a cancer paƟent in need thereof comprising: - collecƟng dendriƟc cells, -collecƟng cancer cells, -inducing late ferroptosis of said cancer cells via contacƟng said cancer cell with a ferroptosis inducer, -lysing said late ferroptoƟc cancer cells, -loading said lysates into said dendriƟc cells, and -administering an effecƟve amount of said dendriƟc cells loaded with lysates derived from late ferroptoƟc cancer cells to said paƟent in need thereof, wherein said ferroptosis inducer is chosen from the list of: RSL3, sulfasalazine, ML-162, sorafenib, altretamine, withaferin A, ErasƟn, silica nanoparƟcles, photoacƟvaƟon, or, by using methods aiming to inacƟvate, deplete or affect glutathione peroxidases, squalene synthase, HMG-CoA reductase, ND dehydrogenase, glutathione S-transferase, glutamate-cysteine 5 ligase, acyl-CoA synthetase long-chain family member 4 and lysophosphaƟdylcholine acyltransferase 3, or, by inhibiƟng the import of cysƟne, or, by affecƟng metabolism of polyunsaturated faƩy acids. 7. A method to treat according to claim 6 wherein said dendriƟc cells and/or said cancer cells are collected from said paƟent. 8. A method according to claim 6-7 wherein said cancer is glioblastoma or melanoma. 9. A method according to claims 6-8 wherein said lysate is obtained by freezing and thawing said late ferroptoƟc cancer cells. 10. A method according to claims 6-9 wherein said late ferroptoƟc cancer cells are obtained by inducing ferroptosis ex vivo via contacƟng said cancer cells with a ferroptosis inducer, wherein said ferroptosis inducer is chosen from the list of: RSL3, sulfasalazine, ML-162, sorafenib, altretamine, withaferin A, ErasƟn, silica nanoparƟcles, photoacƟvaƟon, or, by using methods aiming to inacƟvate, deplete or affect glutathione peroxidases, squalene synthase, HMG-CoA reductase, ND dehydrogenase, glutathione S-transferase, glutamate-cysteine ligase, acyl-CoA synthetase long-chain family member 4 and lysophosphaƟdylcholine acyltransferase 3, or, by inhibiƟng the import of cysƟne, or, by affecƟng metabolism of polyunsaturated faƩy acids. 11. A method according to claims 6-10 wherein said loaded with lysates is obtained by contacƟng said dendriƟc cells with said lysates. 12. A pharmaceuƟcal composiƟon comprising dendriƟc cells loaded with lysates derived from late ferroptoƟc cancer cells. 13. A pharmaceuƟcal composiƟon according to claim 12 wherein said cancer cells are glioblastoma or melanoma cells.