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. 2020 Aug 26;23(9):101499. doi: 10.1016/j.isci.2020.101499

Chemically Programmed Vaccines: Iron Catalysis in Nanoparticles Enhances Combination Immunotherapy and Immunotherapy-Promoted Tumor Ferroptosis

Ane Ruiz-de-Angulo 3,∗, Marc Bilbao-Asensio 1, James Cronin 4, Stephen J Evans 4, Martin JD Clift 4, Jordi Llop 2, Irene VJ Feiner 2, Rhiannon Beadman 4, Kepa Zamacola Bascarán 2, Juan C Mareque-Rivas 1,5,∗∗
PMCID: PMC7490994  PMID: 32919370

Summary

Immunotherapy has yielded impressive results, but only for a minority of patients with cancer. Therefore, new approaches that potentiate immunotherapy are a pressing medical need. Ferroptosis is a newly described type of programmed cell death driven by iron-dependent phospholipid peroxidation via Fenton chemistry. Here, we developed iron oxide-loaded nanovaccines (IONVs), which, chemically programmed to integrate iron catalysis, drug delivery, and tracking exploiting the characteristics of the tumor microenvironment (TME), improves immunotherapy and activation of ferroptosis. The IONVs trigger danger signals and use molecular disassembly and reversible covalent bonds for targeted antigen delivery and improved immunostimulatory capacity and catalytic iron for targeting tumor cell ferroptosis. IONV- and antibody-mediated TME modulation interfaced with imaging was important toward achieving complete eradication of aggressive and established tumors, eliciting long-lived protective antitumor immunity with no toxicities. This work establishes the feasibility of using nanoparticle iron catalytic activity as a versatile and effective feature for enhancing immunotherapy.

Subject Areas: Immunology, Biomaterials, Nanomaterials, Cancer, Therapy

Graphical Abstract

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Highlights

  • •

    Chemistry-enabled IONVs offer a generalizable approach for enhanced therapeutic effects

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    The IONVs are chemically programmed with catalytic iron and reversible bonds

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    IONVs take advantage of TME metabolism to increase ferroptosis in tumor cells

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    Immune checkpoint antibodies improve IONVs and vice versa

Immunology; Biomaterials; Nanomaterials; Cancer; Therapy

Introduction

It is becoming apparent that the immune system can be harnessed to treat cancer. One of the most promising new approaches is based on immune checkpoint inhibitors (ICIs), which re-invigorate the capacity of tumor antigen-specific T cells to attack the cancer (Wei et al., 2018). This immunotherapy has enabled some patients to produce an effective and durable antitumor response. However, the proportion of patients responding to single ICIs remains low (<20%) (Shields et al., 2017; Chen and Han, 2015). Despite efforts to develop combinatorial approaches that increase response rates, studies show minimal improvements in patient survival and/or greater toxicity, usually as a result of targeting redundant immune pathways (Moslehi et al., 2018; Messenheimer et al., 2017). Targeting immune checkpoints harnesses a pre-existing (ineffective) immune response of so-called immune “hot” tumors (Galon and Bruni, 2019) and is hampered by immune-related adverse effects (Friedman et al., 2016) and acquired drug resistance (Jenkins et al., 2018). Therefore, attractive strategies to improve the therapeutic index of ICI are based on the development of immunotherapy drug delivery technologies, and targeting non-redundant additional pathways, such as metabolic features of cancer, through combinatorial approaches (Riley et al., 2019; Doll et al., 2019).

Advances made in the understanding of the biology of cancer, biomedical engineering, and nanochemistry have enabled innovative treatment strategies, which when investigated in combination with immunotherapy show synergistic therapeutic effects in mouse models of cancer, but obstacles to their clinical translation remain. Combination with nanoparticle-enabled photodynamic therapy (PDT) (Ni et al., 2018) is limited by the light penetration depth, oxygen reliance, and systemic toxicity due to off-site drug accumulation (Lucky et al., 2015), whereas use of high-intensity focused ultrasound (HIFU) (Eranki et al., 2019) is also unsuitable for deep-seated tumors owing to attenuation of the ultrasound waves in deep tissue. Immunotherapy enhancement by photothermal ablation (PTA) and by magnetic hyperthermia (MHT) (Chao et al., 2019) require high local nanoparticle concentrations, which can cause severe damages to normal tissue in the tumor margins and induce tumor metastasis. Although iron oxide nanoparticle (IONP)-enabled ICI delivery applying external magnetic field has been reported in mice (Chiang et al., 2018), it is not currently feasible in targeting deep tumor tissue of human patients partly owing to lack of suitable magnetic systems (Shapiro et al., 2015). Consequently, there is an urgent need for developing approaches that address these challenges. Growing evidence implies that the tumor microenvironment (TME), which poses a significant obstacle to these approaches, may be targeted and harnessed to obtain substantial therapeutic benefits (Pitt et al., 2016; Anderson et al., 2017).

The immune component of the TME contains CD4+ and CD8+ T cells (regulatory T cells [Tregs] and exhausted effector T cells) and tolerogenic dendritic cells (DCs) and tumor-associated macrophages (Shiao et al., 2011; Gajewski et al., 2013). Intracellular communication in the TME is driven by a complex and dynamic network of signaling molecules such as cytokines and chemokines against a backdrop of specific metabolic alterations (hypoxia, low pH, nutrient deprivation, high content of H2O2, etc.). These features result in cancer cell metabolic reprogramming (Ward and Thompson, 2012) and an intrinsic vulnerability of cancer cells to a newly described form of regulated cell death termed ferroptosis (Yang and Stockwell, 2016). Ferroptosis, induced by oxidative stress and characterized by iron-dependent phospholipid peroxidation via chain reactions referred to as Fenton chemistry, offers a new way to target treatment-resistant cancers (Doll et al., 2019). We conceive here that iron oxide-filled nanovaccines (IONVs) can exploit the biochemical hallmarks of the TME for in situ “self-generation” of H2O2, which when converted by the IONV into highly toxic ROS, via Fenton chemistry and acting as peroxidase catalyst, drives and concentrates complementary or synergistic antitumor mechanisms in the TME. We chemically programmed the IONVs to provide (1) non-invasive imaging and effective delivery of combination therapy to the tumor and tumor draining lymph nodes (TDLNs), (2) nanoparticle-tuned disassembly to reveal “stealth” immunostimulatory features, (3) an iron oxide core that can artificially reprogram in situ pro-tumoral M2 macrophages into antitumor M1 macrophages (Li et al., 2019), and (4) improved tumor antigen cross-presentation by a pH-responsive hydrazone tumor antigen linkage strategy (Scheme 1). Importantly, since interferon gamma (IFN-γ) released from immunotherapy-activated CD8+ T cells has recently been shown to trigger ferroptosis-specific lipid peroxidation in tumor cells (Wang et al., 2019), we utilize the IONV iron content and peroxidase-like activity to sensitize tumor to ferroptosis, further stimulating the immunotherapy and overcoming immunotherapy resistance mechanisms.

Scheme 1.

Scheme 1

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Design, Mechanism and Features of IONVs

Design and mechanism of action of inherently therapeutic IONVs with chemically programmed multi-functional elements to activate and reprogram immune cells and cancer cells, harnessing the TME to sensitize tumors for immunotherapy and ferroptosis.

In aggressive and established melanomas, we performed a screening of different immune adjuvants in nanoparticle-free vaccines and then sought to systematically assess and quantify how specific nanoparticle engineering design elements impact the responses to therapies. Finally, the combination of IONV- and antibody-mediated nuclear imaging and modulation of cell populations in the TME resulted in complete eradication of established tumors in vivo, eliciting long-lived protective antitumor immunity in treated mice with no observed toxicities. This work provides proof-of-concept demonstration of how iron and IONP-dependent catalytic activity can harness TME biochemical features to improve vaccination and immunotherapy activation of ferroptosis and potentially become one of the centerpieces for immunotherapy enhancement with nanomedicine.

Results

Prophylactic and Therapeutic Implications of Different Toll-like Receptor Agonists

Toll-like receptor agonists (TLRa) have demonstrated potential for the treatment of cancer through direct and indirect activation of different leukocytes of the innate and adaptive immune systems (Schmidt, 2006). To use intrinsically therapeutic catalytic nanovaccines, we first performed a screening of different Tara’s against highly aggressive melanoma tumors, which are characteristically difficult to treat or “cure” once large visible tumors have developed (B16-F10 cells expressing the xeno-antigen ovalbumin [OVA]). The prophylactic implications and efficacy of the TLRa as a vaccine adjuvant was studied by subcutaneously administering low doses (1–5 μg) with full-length OVA. The studies revealed the efficacy of targeting synergistic TLR signaling, achieved by combining imiquimod (R837, synthetic small molecule serving as TLR7-specific ligand) and polyIC (synthetic double-stranded RNA serving as a TLR3 agonist) (Figures 1A–1C), as well as the anti-melanoma protection achieved with unmethylated CG-enriched oligodeoxynucleotides (CpG ODNs), a TLR9 ligand. In comparison, the TLR4 agonists Xcc-LOS (Xanthomonas campestris lipooligosaccharides) (Traini et al., 2019) and LPS (lipopolysaccharide), and the detoxified ligand MPLA (monophosphoryl lipid A), successfully used as adjuvant in clinically approved vaccines and tested in several clinical trials for anti-cancer vaccines, were less effective at generating protective antitumor immunity (Cluff, 2009). In order to study the in vivo antitumor effects of these adjuvants, we subcutaneously implanted the melanoma tumor and after the tumor had become established (ca. 7 days), we administered the best-performing adjuvants (polyIC-R837 or CpG ODNs) and OVA. The potential of cisplatin chemotherapy to convert the tumor into a permissive site for the activation of an adaptive immune response within the tumor (Kang et al., 2013), even without administration of adjuvant, was also studied. Co-treatment with chemotherapy and tumor antigen administration led to slower tumor growth and longer survival compared with monotherapy (Figures 1D and 1E), indicating that priming of the tumor-specific immune response with chemotherapy is possible with OVA antigen delivery into the TME. However, although the generation of antitumor effects achieved by CpG ODNs and polyIC-R837 was with complete absence of systemic toxicity, the chemotherapy acting as vaccine adjuvant is accompanied by systemic toxicity and rapid weight loss initiated following the first injection, an effect that exacerbated with each dose (Figure 1F).

Figure 1.

Figure 1

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In Vivo Evaluation of the Effect of Vaccination with Different TLR Agonists and Cisplatin Chemotherapy as Adjuvants

(A–C) In the prophylactic approach, C57BL/6 mice (n = 5) were immunized subcutaneously on days 0 and 14 with 5 μg of OVA free or in combination with TLR4 agonists Xcc-LOS, MPLA, LPS (1 μg/mouse), TLR3 agonist pIC (4 μg/mouse), TLR7 agonist R837 (2 μg/mouse), pIC-R837 or TLR6 agonist CpG ODNs (5 μg/mouse). Mice were challenged with 3 × 105 B16-F10(OVA) cells in the right back on day 21 after first immunization. (A) Average tumor growth curves and (B) Kaplan-Meier survival curves. (C) Comparison of tumor volume of different experimental groups at day 17 after tumor challenge. The data show mean ± SEM from a representative experiment.

(D–F) To exploit for cancer therapy C57BL/6 mice (n = 5) were treated on days 7, 10, and 13 after B16-F10(OVA) cells injection in the right back. Animals were subcutaneously administered with 10 μg of OVA free or in combination with pIC-R837 (4 and 2 μg, respectively), CpG ODNs (10 μg), and cisplatin (intraperitoneal, 100 μg). In this last group, cisplatin was administered with 60 μg of OVA antigen subcutaneously, in order to mimic the literature. (D) Average tumor growth curves, (E) Kaplan-Meier survival curves, and (F) animal body weight change over the experiment. Arrows indicate the days of therapy administration. Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-tailed unpaired Student's t test (A, D, and F), by log rank (Mantel-Cox) test against OVA (SC) group (B and E), and one-way ANOVA followed by Tukey's test (C). See also Figure S1.

Improvement of Vaccination Efficacy Derived from Nanocarrier Design

Delivery of tumor antigens and vaccine adjuvants to the tumor and TDLNs is important to prime antitumor immunity and fight against tumor immune escape mechanisms. Hence, for safe and robust vaccination a strategy is to engineer nanocarriers that enable vaccine effective delivery to the lymph node microenvironment (Irvine et al., 2013; Storni et al., 2005). The adjuvant can help to convert the immunodormant (“cold”) TME into an immunostimulatory (“hot”) one while taking advantage of tumor-associated antigen drainage. Since various physicochemical properties can be tailored to enable effective drug delivery to lymph nodes, nanocarrier design is critical (Schudel et al., 2019).

We created PEG-phospholipid (PEG-PL)-stabilized IONP-bound oleates carrying adjuvant/antigen (IONVs), which led to micelles <100 nm with good stability for up to 2 weeks (Figures S2–S5).(Carion et al., 2007; Cobaleda-siles et al., 2014) We included the cationic lipid DOTAP to self-assemble polyIC and CpG upon mixing via electrostatic interactions between the positive charges of the lipid and the negative charges of the nucleic acid-based TLRas. The incorporation of R837 to IONVs was conducted by its intercalation into the polyIC dsRNA chain, forming polyIC-R837 complexes as shown in our previous work (Bocanegra Gondan et al., 2018). The model antigen OVA was successfully adsorbed by iron oxide nanoparticle-filled micelles (mIONPs) containing DSPE-cPEG(2000)-PL with COOH end groups exploiting the concept of “nanoparticle-protein corona”(Nguyen and Lee, 2017). To investigate the delivery of the IONVs to lymphatic tissues and tumor, we 67Ga-radiolabeled the IONP surface exploiting the similarity between Ga(III) and Fe(III) as reported previously (Ruiz-De-Angulo et al., 2016). This smart organic synthesis-free process, which involves reaction of the readily obtained radiocation [67Ga][Ga(H2O)6]Cl3 (aq.) with the anionic IONVs at 70°C for 30 min followed by purification by ultracentrifugation, proceeded with very good yield (>80%; Figures 2A, 2B, and 2C). The 67Ga-doped IONVs showed excellent stability, with less than 10% of the bound 67Ga being released over 24 h even when challenged with 106-fold molar excess of DOTA chelator, and therefore suitability for in vivo tracking (Figure 2C).

Figure 2.

Figure 2

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Radiolabeling and SPECT/CT Tracking of IONVs Allow Non-invasive Monitoring of Therapy Outcome

(A and B) The radiocation [67Ga][Ga(H2O)6]Cl3(aq.) is loaded by heating with the anionic IONVs and following purification by ultrafiltration using a 100k membrane results in 67Ga-doped IONVs inradiolabeling yields > 80%. (C) The chelator DOTA (present in 106 molar excess relative to IONP) is able to remove <10% of the IONP-bound 67Ga over a 24-h period.

(D–F) Biodistribution study of 67Ga labeled mIONP-CpG ODNs (23.6 μg of magnetite, 43.4 MBq) injected in the hock of melanoma tumor-bearing mice. (D) SPECT/CT images 3, 24, and 48 h post injection (coronal and sagittal views). Ex vivo analysis by (E) SPECT/CT and (F) gamma counter 48 h after injection. T, tumor; bLN, brachial LN; aLN, axillary LN; Lu, lungs; L, liver; S, spleen; K, kidneys; iLN, iliac LN; sLN, sciatic LN; inLN, inguinal LN; pLN, popliteal LN; I, injection site. See also Figures S6 and S7.

SPECT/CT imaging results showed that these IONVs selectively accumulate in LNs and tumor up to 24 h (Figures 2D–2F and S6 and S7). Particles cleared from the injection site were excreted into the urine and accumulated at proximal and distal draining LNs within 3 h post injection (Figures 2D and S6), which is consistent with self-lymphatic delivery and confirms the lymphatic drainage from the tumor implantation site to tumor-draining lymph nodes (TDLNs). Therefore, the in vivo biodistribution experiments demonstrated IONVs are sufficiently small and stealthy to be rapidly taken up into lymphatic vessels and transit to the draining LN after subcutaneous (SC) injection.

To investigate the therapeutic efficacy of nanoparticle vaccination, mice were treated with non-covalently formed IONVs with the polyIC-R837 or CpG ODNs (Figure S8) adjuvant and OVA. The IONVs achieved significantly slower tumor growth relative to the treatment with free polyIC-R837 and OVA or to the clinical “gold standard” alum or Montanide™ ISA adjuvants. Only the treatment with the IONVs achieved 100% survival through the study endpoint at 30 days after tumor implantation, and the therapeutic effect was elicited with complete absence of systemic toxicity (Figure S8). Notably, the SC administration into the hock of mIONP-OVA without the TLRa adjuvant and even of mIONP alone showed an antitumor effect that is the same or superior to antigen administered intratumorally (IT), highlighting an important intrinsic IONP therapeutic contribution (Figure 3A).

Figure 3.

Figure 3

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In Vivo Evaluation of the Therapeutic Effect Derived from the “Stealth” Hydrophobic IONP Coating and IONP Size

C57BL/6 mice (n = 5) were treated on days 7, 10, and 13 after B16-F10(OVA) cells injection in the right back. (A) intrinsic adjuvant activity of mIONP was analyzed by treating animals with 10 μg of OVA antigen alone (IT) or in combination (as protein corona adsorbed by the nanoparticle) with mIONP (SC injection into the hock [ankle]; 61 μg magnetite); (B) nanoparticle size and exposure of coating was studied by injecting animals with different IONP-filled micelle designs (101 μg magnetite) peritumorally (SC, 2 cm from tumor); and (C) the effect of IONP coating nature was examined by comparing the co-administration (SC, right back) of B16-F10(OVA) cancer cells with IONPs with polar (ferumoxytol) or apolar (mIONP) coating (12 mg Fe/kg, 332 μg magnetite).

(A–C) Results are shown as average tumor growth curves (upper graphs) and Kaplan-Meier survival curves for each case group (lower graphs). Arrows indicate the days of therapy administration. Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 by two-tailed unpaired Student's t test (A–C upper graphs) and by log rank (Mantel-Cox) test against PBS/NaCl (0.9%) control group (A–C lower graphs).See also Figures S8–S10 for further in vivo studies and Figure S2 for characterization of the hydrophobic IONPs.

In line with these findings and to investigate whether IONVs promote a long-term immune response involving tumor antigen-specific CTL activation, we conducted prophylactic vaccination experiments. On day 70 after the last immunization, animals were rechallenged with melanoma cells in the contralateral side. Six weeks later, mice were sacrificed and blood and spleens were collected for immune analysis (Figure S9). IONVs were found to enable an enhanced overall specific immune response, resulting in higher percentages of OVA-specific CTLs that exhibited a multifunctional killing phenotype, as demonstrated by the increased frequency of IFN-γ and TNF-α-producing T cells and increased expression of the degranulation molecule CD107a. Moreover, higher levels of antigen-specific Th1-type CD4+ Th cells (producing IFN-γ and TNF-α Th1-type cytokines) and increased quality of the T cell response based on the analysis of effector (TEM) and central memory (TCM) CD8+ T cell percentages in spleen were also observed. The characterization of the immune responses confirmed the ability of IONVs to elicit potent and durable antitumor immunity.

To elucidate potential intrinsic adjuvant properties, we assessed the status of DCs after incubation with the designed mIONP alone and IONVs carrying TLRa adjuvants and OVA. The expression of the co-stimulatory signal molecules CD80 and CD86 as well as the major histocompatibility complex (MHC) class II, which are involved in the initiation of T cell-mediated immune responses against tumors, was observed with the mIONP alone and was comparable with the expression induced by stimulation with free TLRa agonists (Figure 4).

Figure 4.

Figure 4

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Comparison of DC Activation Elicited by mIONPs and Potent Immunostimulatory TLRa

Primary culture of BMDCs was incubated overnight with increasing concentrations of mIONPs (magnetite μg/mL) and standard amounts of TLR agonists (μg/mL) diluted in media.

(A) Activation and immunosuppressive marker expression measured by flow cytometry and expressed as relative mean fluorescent intensity (MFI).

(B and C) (B) Cytokine production on cell culture supernatant measured by ELISA and (C) cytotoxic effect of the different stimulus after overnight incubation (MTT assay). Results are shown as mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA (A and C) and by two-way ANOVA (B) followed by Tukey's test. See also Figure S3 for characterization of mIONPs

This result highlights the importance of the mIONPs toward promoting proper DC maturation for T cell clonal expansion and differentiation to CD8+ cytotoxic lymphocytes (CTLs) and improved co-presentation of antigen to CD4+ T helper (Th) cells. None of the mIONP formulations showed toxicity in DCs. Notably, the induction of the expression of PD-L1, which inhibits T cell effector function (via engagement of the PD-1 receptor expressed on activated T cells), was observed on DCs activated by the TLRa agonists and mIONP formulations (Figures 4 and 5). This result suggests that blocking PD-1/PD-L1 signaling could represent an opportunity to augment the antitumor effects of these systems.

Figure 5.

Figure 5

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Comparison of DC Activation with mIONP-OVA (Magnetite-Antigen, μg/mL) and OVA

(A–C) (A) Expression of surface markers measured by flow cytometry, (B) cytotoxic effect (MTT assay), and (C) cytokine production measured by ELISA. Results are shown as mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by one-way ANOVA (A and C) and by two-way ANOVA (B) followed by Tukey's test. See Figure S3 for mIONP-OVA characterization.

Hydrophobicity as ancient damage associated molecular pattern (DAMP) can be used to activate innate immune responses (Seong and Matzinger, 2004). To investigate if the micellar structure with “stealth” hydrophobic portions around IONPs (hydrophobic oleic acid coating covered by PEG-PL layer) contributes toward delaying tumor growth in vivo, mice with established tumors were treated with drug-free mIONPs differing in the amphiphilic organic coating and IONP core. If the concept that nanoparticle-tuned disassembly by amphiphilic polymer release from a mIONP system to reveal hydrophobic portions can be used as an IONV design principle to trigger an improved activation of an innate immune response capable of influencing tumor immunity and growth is correct, then modulating the stability of the intercalation with the IONP-bound oleates would be expected to affect the extent of tumor growth inhibition. Indeed, the results showed greater inhibition of tumor growth and prolonged survival with administration of mIONP coated with DPPE-mPEG(2000)-PL with methoxy end groups compared with PMAO-PEG as amphiphilic polymer coating (mIONP-trapped) (Figure 3B). This is consistent with the reported high stability of the PMAO-PEG coating (Yu et al., 2006). Using DPPE-mPEG(2000)-IONP micelles containing IONP core sizes of 20 nm (mIONP-big) instead of 6 nm the intrinsic therapy effects were abrogated, which could be derived from an inefficient migration toward TDLNs (Figures 3B and S10).

Administration of the US Food and Drug Administration (FDA)-approved iron supplement ferumoxytol has been found to promote tumor growth regression (Zanganeh et al., 2016). However, when the B16-F10(OVA) melanoma cells were implanted with or without ferumoxytol and compared with an equivalent dose of mIONPs, only the mIONP was able to suppress tumor growth highlighting the superior design features of the mIONPs (Figure 3C).

Antigen Attachment Mode Alters the Efficacy of IONVs

The concept of antigen-specific cancer immunotherapy is based on the generation of antigen-specific CTLs that recognize and destroy tumor cells that present antigen-derived peptides using cell surface MHC class I molecules (Embgenbroich and Burgdorf, 2018). However, many current strategies lead to the ineffective generation of antigen-specific CTLs after vaccination. Our approach to overcome this challenge is based on creating chemically engineered antigen-packed NPs, which can deliver antigen to DCs in the tumor and TDLNs and utilize pathogen-like endocytosis mechanisms. A suitably designed NP can promote the release of antigen in response to intracellular environmental stimuli (e.g., acidic pH) to promote cross-presentation (i.e., presentation of the internalized tumor antigen by means of the MHC-I pathway). Importantly, it has been postulated that lipid peroxidation in DCs can also cause endosomal antigen release for cross-presentation(Dingjan et al., 2016). Our converged solution is to exploit the peroxidative activity of the nanoparticle (vide infra) while optimizing NP-enhanced antigen delivery by means of a more efficient antigen chemical attachment mode. We developed a bis-aryl hydrazine linkage strategy for the coupling of mIONPs to the tumor antigen, which provides rapid, monitorable, and high yields of conjugation and stability at physiological pH, while enabling antigen release at endosomal pH for efficient cross-presentation (Traini et al., 2019).

To investigate the importance of the new antigen attachment mode, we treated mice with IONVs based on OVA adsorbed to the IONP micelles as a corona (mIONP-OVA(ads)) and its covalent conjugation by hydrazone linkages (mIONP-OVA(hyd)) (Figure 6). Although vaccination with adjuvant mIONP-polyIC-R837 co-administered with mIONP-OVA(ads) or mIONP-OVA(hyd) significantly suppressed tumor growth and the tumors were on average similar in size, only mice in the mIONP-OVA(hyd)-treated group survived tumor-free over a long period of monitoring (CR 40%) (Figure 6C). This result reveals that hydrazone bioconjugation chemistry optimizes therapeutic immune responses to the IONVs.

Figure 6.

Figure 6

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In Vivo Evaluation of the Therapeutic Efficacy Arising from an Optimized Antigen Nanoparticle Attachment and Adjuvant Core Material

C57BL/6 mice (n = 5) were treated on days 7, 10, and 13 after 3 × 105 B16-F10(OVA) cells injection in the right back with vehicle, 10 μg of OVA adsorbed by IONP (mIONP-OVA(ads)), conjugated to IONP by hydrazone linkages (mIONP-OVA(hyd)) alone or co-administered with adjuvants (mIONP-pIC-R837 or AuNP@PEI-pIC-R837 (4 μg of pIC and 2 μg of R837, 30 μg magnetite, and 5 μg of gold)).

(A–C) (A) Average tumor growth, (B) Kaplan-Meier survival plot, and (C) individual tumor growth curves. CR, complete rejection. Arrows indicate the days of therapy administration. Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired Student's t test between indicated groups (A) and by log rank (Mantel-Cox) test against AuNP@PEI-pIC-R837 group (B). See also Figures S3–S5 for IONV characterization, Figure S11 for AuNP@PEI characterization, and Figure S12 for further statistical analysis.

The Iron Oxide Core Elicits Antitumor Immunotherapy Efficacy

Having established several features that improve the IONV vaccination efficacy, we considered whether other biocompatible nanoparticles previously used for development of nanovaccines such as gold nanoparticles (AuNPs) (Almeida et al., 2014) might elicit similar antitumor efficacy. Studies have highlighted the potential of polyethylenimine (PEI) (Shen et al., 2017; Liang et al., 2018), one of the well-studied cationic polymers used as a transfection reagent for decades, for vaccine development. The mechanisms for the intrinsic immunoactivation function of PEI are diverse and include the triggering of “danger signals” in antigen-presenting cells and the ability to elicit ROS (Shen et al., 2017). We prepared AuNPs coated with PEI (Garaikoetxea Arguinzoniz et al., 2015) and by exploiting its positive charge, we developed PEI-modified AuNPs suitable for forming complexes with the anionic polyIC-R837 adjuvant. Moreover, the AuNPs@PEI-polyIC-R837 complexes had an optimal size for effective lymphatic delivery (Figure S11). Strikingly, the antitumor efficacy of this system was similar to that of mIONP-OVA(hyd) alone. Hence, the AuNP@PEI-polyIC-R837 complex is not able to replicate the adjuvant effects of mIONP-polyIC-R837 (Figure 6) owing to the intrinsic effects arising from the IONP core (see below).

Catalytic Properties and Cell Responses to mIONPs/IONVs: Creating the “Perfect Storm” and Catalytic Therapy

IONPs have been shown to perform peroxidase-like functions, which have led to proof-of-concept demonstrations of tumor-responsive catalytic nanomedicine (Huo et al., 2017; Lin et al., 2018). We reasoned that the unique antitumor effectiveness of the IONVs may be underpinned by its catalytic activity on iron and ROS-mediated processes in macrophages and cancer cells.

The peroxidase-like activity of mIONP was assayed by the colorimetric reaction of H2O2 with the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB), which upon oxidation turns blue (ε652 = 3.9 × 104 M−1 cm−1, Figure 7A). mIONP catalyzed the peroxidative reactions at the acidic conditions found in the TME and endosomal compartments to which the nanovaccines are targeted, whereas this activity was minimal at physiological pH. This activity was further confirmed by oxidation of o-phenylenediamine (OPD) (Figure 7B). UV-vis absorption time course curves indicated that the mIONPs had a higher catalytic activity than ferumoxytol and AuNPs under the same conditions (Figure 7A). The catalytic contribution of iron ions leached from mIONPs was found to be negligible (Figure 7A), confirming that the peroxidative activity is carried out by the nanoparticles.

Figure 7.

Figure 7

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Comparison of Catalytic Properties of mIONPs AuNPs and Ferumoxytol

(A and B) (A) Peroxidase-like activity of mIONPs at different pH values, AuNP@PEI, ferumoxytol, and released free iron cations in catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). (B) Catalytic oxidation of o-phenylenediamine (OPD). Concentrations of [Fe] = 150 μM for mIONPs and 550 μM for ferumoxytol were used.

(C and D) Michaelis-Menten kinetics plots for the oxidation of TMB in the presence of H2O2 at varying concentrations of (C) H2O2 and (D) TMB, based on data (mean ± SEM) from three independent experiments.

Varying the concentration of H2O2 and TMB, Michaelis-Menten steady-state kinetics were applied to determine the catalytic performance of the IONP micelles in terms of catalytic rate (kcat), maximal rate (Vmax), Michaelis constant (KM), and catalytic efficiency (kcat/KM) (Figures 7C and 7D). The kinetic parameters (KM = 0.338 mM, Vmax = 6.59 × 10−8 M−1 s−1) are indicative of strong catalytic performance (e.g., better than horseradish peroxidase [HRP] in terms of H2O2 affinity, for which the KM is 3.7 mM). It is known that, during tumor growth, the TME generates a substantial amount of H2O2 (∼50–100 μM) (Szatrowski and Nathan, 1991), and it is reasonable to expect that this H2O2 production will be significantly enhanced by the immunostimulatory TLRa-loaded IONVs. The data show that mIONPs catalyze the oxidation of TMB at iron concentration of 150 μM, which is below the concentration used in the in vivo and in vitro studies.

Tumor-associated macrophages (TAMs) often represent a dominant proportion of the immune cell infiltrate that can promote tumor progression, and suppress antitumor immune responses, but can be potentially harnessed to treat cancer (Bronte and Murray, 2015; Gabrilovich et al., 2012). Having demonstrated high catalytic peroxidative activity, we investigated the effects of mIONP on macrophages and cancer cells in direct cell-to-cell contact co-cultures. For comparison, the cells were also treated with cisplatin, polyIC, and R837. Although the widely used chemotherapy drug is not effective at killing the melanoma cells, it leads to complete macrophage obliteration (Figures 8A and 8B). PolyIC and R837 induced melanoma cell death but only when combined, synergizing to significantly enhance cytotoxicity in the melanoma cells with no toxic effects in macrophages. Free mIONPs were as effective at killing the melanoma cells as the combination of polyIC and R837. Notably, administration of mIONP-polyIC-R837 killed >90% of the target melanoma cells with >70% macrophage viability.

Figure 8.

Figure 8

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Cell Death Triggered by mIONPs and IONVs in Cell-to-Cell Contact Co-cultures of Melanoma Cells and Macrophages and Induction of Ferroptosis

(A and B) Cell viability was measured by flow cytometry following 24-h treatment (A) B16-F10(OVA) melanoma cells and (B) RAW 264.7 macrophages in cell-to-cell contact co-culture approach (77 μg/mL magnetite = 1 mM of Fe). Data are shown as mean ± SEM (n = 3).

(C) Effects of Erastin on the cell viability of RAW 264.7 (green bars) and B16-F10(OVA) (blue bars) in their respective mono-cultures, after 24 h of exposure determined by MTT assay. Data were normalized to the absence of treatment.

(D) Effects of mIONPs (0.5 mM of Fe) on the cell viability of B16-F10(OVA) after 24 h of exposure upon 24-h preconditioning with increasing concentrations of Erastin. Data were normalized to the absence of mIONP treatment for each Erastin concentration (solid green bars). Data are shown as mean ± SEM (n = 4).

(E) Reaction of Liperfluo with hydroperoxide lipids to generate fluorescence Liperfluo-Ox.

(F) B16-F10(OVA) incubated with IONPs (1 mM of Fe) for 24 h. Representative flow cytometric profiles are shown to demonstrate lipid peroxides with Liperfluo signals, where cells treated with cumene hydroperoxide are used as positive control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA followed by Tukey's test (A, B) and by two-way ANOVA followed by Sidak's test (C, D). See also Figure S13.

We then performed the studies with both types of cells in mono-cultures and in co-cultures separated by a porous membrane allowing free diffusion treatment and biochemical cell communication while restricting cell migration (Figure S13A). Again, mIONP induced more toxicity in melanoma cells than in macrophages and displayed an overall anticancer effect comparable with the highest dose of cisplatin (100 μM) (Figures 8A, 8B, S13B, and S13C). However, the results clearly highlighted the relevance of cell-to-cell contact interactions between the immune and melanoma cells to potentiate the mIONP immunotherapeutic effect.

We measured the production of IL-10, an effector molecule of M2 macrophages, which as an anti-inflammatory cytokine plays a critical role in limiting the host pro-inflammatory immune response and promoting tumor immune escape. Cultures treated with TLR ligands (including the TLR4 ligand LPS) or cisplatin exhibited high levels of IL-10, whereas treatment with IONP-filled micelles and mIONP-pIC-R837 led to essentially the same level of immunosuppressive IL-10 as the non-treatment control (Figure S13E). Thus, the mIONP-pIC-R837 provides effective melanoma cell killing overcoming mechanisms that impair antitumor immunity.

Finally, we sought to identify whether cancer cell death was due to ferroptosis. Using erastin as a ferroptosis activating agent, the results showed that the melanoma cells are much more susceptible to ferroptosis than macrophages (Figure 8C). This result is consistent with other studies showing that cells of the innate immune system display resistance to ferroptosis (Matsushita et al., 2015). Moreover, a recent study has shown that, antitumor M1 macrophages display resistance to ferroptosis, whereas M2 macrophages are sensitive to pro-ferroptosis stimulation and that this is linked to inducible nitric oxide synthase (iNOS)/NO• enrichment (Kapralov et al., 2020).

Importantly, mIONP by providing a source of iron, ROS, and catalytic activity can be used to trigger cancer cell ferroptosis (Figure 8D). Since one of the characteristics of ferroptosis is the accumulation of lipid hydroperoxides, measuring lipid peroxidation is important for evaluating whether ferroptosis occurs. To directly reveal whether lipid hydroperoxides are formed following incubation of the melanoma cells with mIONPs, we used Liperfluo, which reacting with hydroperoxides yields the fluorescent Liperfluo-Ox product (Figure 8E).(Yamanaka et al., 2012) The flow cytometry studies demonstrated the presence of hydroperoxide lipids in melanoma cells treated with mIONP even without erastin (Figure 8F).

Combination Immunotherapy with Checkpoint Inhibitors: “Pushing the Gas” and “Releasing the Brakes”

Immunotherapy with ICIs has created a paradigm shift in clinical oncology. The focus has been blocking inhibitory signals to release the brake on the activity of T cells present in the TME. Recently, several studies reported that this type of immunotherapy leads to production of IFN-γ, which plays a pivotal role in host antitumor immunity (Peng et al., 2012; Chen et al., 2009). However, IFN-γ is a potent inducer of PD-L1 expression and tumors exploit this as a strategy to escape antitumor immunity (Mandai et al., 2016). Importantly, a recent study demonstrates that immunotherapy-activated CD8+ T cells promote tumor cell lipid peroxidation and sensitize tumors to ferroptosis through IFN-γ (Wang et al., 2019). Hence, targeting and enhancing ferroptosis in tumors may be a promising new route to improve the efficacy of cancer immunotherapy (Wang et al., 2019) with additive or potentially synergistic interaction between immunotherapy and induction of ferroptosis. Immunotherapy studies are progressively being directed to immunostimulatory antibodies that instead of releasing the brakes on the immune system to boost antitumor immunity can promote activation pathways (i.e., “step on the gas”) (Mahoney et al., 2015). In this context, agonistic anti-OX40 antibodies are being tested in several phase I/II clinical trials either as monotherapy or in combination with other immune-modulating agents. They possess the dual function of enhancing the activation of the tumor infiltrating effector T cells (Teff) and inhibiting the immunosuppressive Treg cells (Aspeslagh et al., 2016). Recent studies have shown that, although systemic delivery of anti-OX40 may favor activation of peripheral lymphocytes to promote systemic toxicity, intratumoral administration is better for targeting tumor-infiltrating lymphocytes and to promote novel mechanisms of action such as depletion of intratumoral Treg and abscopal effects (i.e., a local low-dose immunotherapy eliciting a systemic antitumor immune response avoiding systemic toxic events) (Marabelle et al., 2013; Hebb et al., 2016).

On the basis of these studies and the results shown in this work, we explored combination of the IONVs with an antagonistic anti-PD-L1 (⟨PD-L1) and agonistic anti-OX40 antibodies (⟨OX40).

We first radiolabeled the immunostimulatory mAbs (monoclonal antibodies) with 89Zr by antibody modification with the bifunctional chelator p-isothiocyanatobenthyl-desferrioxamine and addition of [89Zr]Zr-oxalic acid solution (Vosjan et al., 2010). We then confirmed the expression of OX40 and PD-L1 in the TME by 89Zr-immuno-PET imaging and observed high uptake of both radiolabeled antibodies in the tumor and in the spleen (Figures 9A and 9B). The analysis of tumor-infiltrated immune cells by flow cytometry showed high levels of OX40+ T cells in the tumor and TDLNs. Notably, CD4+OX40+ T cells showed double-positive expression of T cell activation markers CD44 and CD25 (Figures 9C–9E), suggesting an optimal environment for the immunostimulatory mAbs-based therapies.

Figure 9.

Figure 9

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In Vivo Detection of the Expression of PD-L1 and OX40 Immune Receptors by Immuno-PET Imaging and Subsequent Analysis of Infiltrating Immune Cells Allowed Characterization of TME Status

(A) PET images were acquired 24, 48, and 72 h after administration of the radiolabeled anti-PD-L1 and anti-OX40 antibodies (80–100 μg, 1–7 MBq).

(B–E) (B) Antibody accumulation in tumor and spleen expressed as injected dose percentage per cm3 (% ID/cm3). Analysis of immune cells phenotype found in the spleen, TME and TDLN: (C) CD4+ and CD8+ T cell percentages, (D) OX40+ T cell0020percentages, and (E) activation profile of CD4+OX40+ T cells. Data are shown as mean ± SEM (n = 7).

To study the eradication of established tumors by the combination immunotherapy, mice were treated with IONVs, and, on days 1 and 4 after each vaccination, with ⟨PD-L1 administered (100 μg) intraperitoneally. Although IONVs alone induced delayed growth of the tumor, all animals eventually developed tumor. However, with the combination of vaccination and ⟨PD-L1 treatment in addition to animals showing partial response to the therapy, 40% of mice were free of tumor 90 days after the inoculation of the melanoma cells (Figures 10A–10C). To evaluate whether combination with ⟨OX40 monoclonal antibodies provides additional therapeutic benefit, mice were co-treated with an ultra-low dose of ⟨OX40 antibody (10 μg) delivered intratumorally and the IONVs administered subcutaneously at a tumor distant site. Again, the results consistently show 100% tumor eradication in mice and significantly reduced tumor progression with the combination treatment compared with the monotherapies or checkpoint-inhibition combination therapy with ⟨OX40 and ⟨PD-L1 antibodies (Figures 10A–10C). Moreover, when animals were re-challenged with the melanoma cells (90 days after first tumor challenge) they remained tumor-free for 30 days, compared with none for controls (Figures 10D and 10E).

Figure 10.

Figure 10

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In Vivo Evaluation of the Enhanced Effect of Combination Immunotherapy with IONVs and Immunostimulatory mAbs

C57BL/6 mice (n = 5) were inoculated with 3 × 105 B16-F10(OVA) cells and treated with nanovaccines (10 μg of OVA, 4 μg of pIC, and 2 μg of R837; 42 μg of magnetite) on days 4, 11, and 18. ICIs (100 μg of anti-PD-L1 (IP) and 10 μg of anti-OX40 (IT)) were administered on days 1 and 4 after each immunization.

(A–E) (A) Average tumor growth, (B) Kaplan-Meier survival plot, and (C) individual tumor growth curves. (D) Average tumor growth and (E) Kaplan-Meier survival plot following re-challenge of melanoma cells in the contralateral back of cured mice. CR, complete rejection. Arrows indicate the days of nanovaccines (black) and checkpoint inhibitors (blue) administration. Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student's t test between indicated groups (A) and by log rank (Mantel-Cox) test against mIONP-OVA(hyd) + mIONP-pIC-R837 group (B).

Discussion

Herein, we report the rationale for a systematic nanoparticle engineering design that exploits the iron content, nanoenzymatic activity, biochemical properties in TME, and chemical and structural features of IONVs as a generalizable therapeutic platform for cancer immunotherapy. A major finding was that iron and peroxidase-like activity of IONVs can be used to exploit and convert conditions in TME (often leading to therapy resistance) into a site that overcomes immunosuppressive mechanisms and elicits potent and durable antitumor responses that eradicate established tumors. The findings show how IONVs can unite and optimize concurrently effective vaccine delivery for inducing anticancer immunity, immune checkpoint blockade, and cancer cell ferroptotic-cell death to augment the therapeutic index of combination therapy and immunotherapy approaches.

Nanoparticles have been extensively studied to improve the delivery of chemotherapy drugs, but the efficacy of this approach has been plagued with disappointing results in clinical trials (Bourzac, 2016; Hare et al., 2017). In contrast, cancer immunotherapy using nanoparticle-based vaccines is a promising new area in which FDA-approved polymers and nanocarriers such as poly(lactic-coglycolic acid) (PLGA), liposomes, and phospholipid nanodiscs have already allowed various degrees of success (Maldonado et al., 2015; Rosalia et al., 2015; Varypataki et al., 2016; Kuai et al., 2017; Min et al., 2017; Xiaoding et al., 2017; Ling et al., 2019). It is becoming clear that nanomedicine can provide innovative solutions for immunotherapy (Irvine and Dane, 2020). Inorganic nanomaterials offer additional features for modulation of immune responses and non-invasive vaccine tracking owing to intrinsic adjuvant and imaging properties (Lee et al., 2012; Cobaleda-siles et al., 2014; Ruiz-De-Angulo et al., 2016; Wang et al., 2016; Bocanegra Gondan et al., 2018). In this study we show that engineered iron oxide nanoparticles (IONPs) can become a cornerstone for enhancing immunotherapy owing to the coupling of catalytic activity with immunomodulation. They are suitable for medical oncology use owing to excellent safety profiles, biodegradability and metabolization, facile synthesis and tuneability targeting enhancement of intrinsic therapy, and imaging properties (Revia and Zhang, 2016). An important target is to surpass the recently discovered antitumor activities of the FDA-approved IONP-based iron supplement ferumoxytol, which arise from activating and repolarizing macrophages via Fenton chemistry but requires administration of high doses (Zanganeh et al., 2016). Moreover, ferumoxytol administration has been shown to exert anti-leukemic effects exploiting the vulnerability of these cells to iron-based oxidative stress (Trujillo-Alonso et al., 2019). We show how adopting a systematic molecular engineering approach micelles filled with iron oxide nanoparticles can be engineered to significantly surpass the antitumor effects of ferumoxytol.

Consistent with recent studies showing that nanoparticles can induce ferroptosis in cancer cells and perform catalytic nanomedicine (Huo et al., 2017; Kim et al., 2016; Lin et al., 2018; Liu et al., 2018), the mIONP's iron-enabled and high peroxidase-like activity triggered selective cancer cell killing, but in particular in contact co-cultures with macrophages that alone could not induce cancer cell killing.

By targeting ferroptosis (Yang and Stockwell, 2016), which is induced by oxidative stress and characterized by iron-dependent accumulation of lipid hydroperoxides and can be enhanced by the action of immune checkpoint blockade-reinvigorated T cells (Wang et al., 2019), the IONVs combined with immunostimulatory mAbs can realize complete eradication of aggressive and established melanoma tumors in mice. When benchmarked against other treatments the melanomas proved to be essentially unresponsive to ferumoxytol treatment, adjuvant chemotherapy, vaccination with AuNPs, clinically approved aluminum hydroxide and Montanide-based adjuvants, and a combination of mAbs targeting co-stimulatory and coinhibitory signal in T cells (anti-OX40 and anti-PD-L1).

To target the sites of interest the inherently therapeutic IONVs incorporated a comprehensive and sophisticated delivery mechanism, which involves efficient delivery to TME and lymph nodes, efficient intracellular delivery to DCs by pathogen-mimicking endocytosis, and a chemically engineered conditional intracellular release and antitumor activity that exploits hydrazone bond cleavage and peroxidase-like catalysis in acidic endolysosomes. The radiolabeling of IONVs and ICIs confirmed the mechanistic basis for the combination therapy efficacy by enabling non-invasive monitoring of effective multi-pronged therapy delivery and real-time assessment of expression of blocking and activating immune checkpoint receptors.

Itemizing and evaluating the nanoparticle engineering features hypothesized to contribute to the enhancement of therapeutic outcomes allows a rational and on-demand design of IONVs. As expected, improvements were driven by several characteristics of the designed IONVs: (1) ability to locally deliver the multipronged therapeutic cargo to tumor and TDLNs enabled by IONVs size characteristics (<100 nm and spherical IONPs of 6 nm); (2) optimal tumor antigen delivery and cross-presentation through pH-responsive hydrazone conjugation and peroxidase-like activity; (3) nanoparticle-tuned disassembly to activate the “stealth” immunostimulation by the pathogen-mimicking hydrophobic nature of the nanoparticle core, which confers an intrinsic adjuvant activity to the nanosystem; and (4) iron and high catalytic activity of the iron oxide core that allows effective and stable radiolabeling with 67/68Ga for therapy tracking, reprogramming of macrophages, in situ generation of ROS, and further tumor cell sensitization to ferroptosis in the TME.

Although several externally triggered bioengineering approaches such as PTT, PDT, MHT, and magnetic navigation have demonstrated considerable promise for improving treatment outcomes with ICIs (Lucky et al., 2015; Ni et al., 2018; Chao et al., 2019; Eranki et al., 2019), most face challenges for clinical implementation owing to the intrinsic limitations of these techniques. In contrast, use and administration of iron and catalytically enhanced IONVs is easy to implement alongside chemotherapy, radiotherapy, and complementary immunotherapies. The effectiveness of tumor antigen hydrazone bond conjugation is demonstrated with full-length xeno-antigen OVA, yet this chemistry is readily applicable with peptide antigens and neoantigens that could be generated in controlled synthesis or on site (Min et al., 2017; Kuai et al., 2017). This offers a feasibility strategy to eventually achieve personalized treatments, which promises to be the next big immunotherapy and cancer treatment breakthrough.

In conclusion, the results presented here show how chemically programmed IONVs can be used as a platform for the engineering of vaccines that are intrinsically therapeutic exploiting TME reprogramming, and the tumor susceptibility to iron- and ROS-dependent processes and redox stress that could be clinically applicable during combination immunotherapy. Chemistry- and iron-enabled catalytic vaccines offer intriguing opportunities to also invigorate immunotherapy combinations with chemotherapy and radiotherapy and with other approaches such as PDT, PTT, MHT, and HIFU and magnetic navigation.

Limitations of the Study

Although the murine B16 melanoma model is widely used for preclinical assessment of immunotherapies, the therapeutic efficacy of the chemically programmed IONVs in clinically relevant models of human cancer needs to be undertaken. The intracellular mechanism(s) of antigen processing and presentation underpinning cross-presentation is not investigated. The potential role of lipid peroxidation induced by the IONVs also causing endosomal antigen release for cross-presentation is plausible but has not been investigated. Other immune cell populations around TME after the treatment can be investigated to provide a more comprehensive understanding of the IONV effects. How the mIONVs enhance ferroptosis and can be optimized for this purpose and the demonstration of the synergistic induction of ferroptosis by the checkpoint inhibitors will require additional studies.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Juan C. Mareque-Rivas (juan.mareque-rivas@swansea.ac.uk).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

This study did not generate/analyze any datasets/code.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

J.C.M.-R. and J.L. thank MediSURF project (PCIN2016-085) from the Spanish Ministry of Economy and Competitiveness (MINECO). This work was supported by an EPSRC PhD Studentship to M.B.-A. and R.B. (EP/N509553/1 and EP/R51312X/1). J.L. acknowledges financial support from the Spanish Ministry of Economy and Competitiveness (MINECO), grant number CTQ2017-87637-R. The authors would like to thank Dr. Vanessa Gómez-Vallejo and Víctor Salinas for 89Zr production, Zuriñe Baz for assistance in image quantification, April Rees for flow cytometry studies, and Dr. Asís Palazón (bioGUNE) and Dr. Nick Jones (Swansea Medical School) for critically reading the manuscript.

Author Contributions

A.R.d.A. prepared and characterized the nanovaccines, performed their radiolabeling, and designed and performed the tumor experiments, SPECT/CT imaging experiments, in vitro immune experiments (DCs and CD8+ T cells), and the phenotype analysis of OX40 T cells and contributed to the writing of the manuscript. M.B.-A. prepared and characterized the nanovaccines, performed the catalytic activity assays and the co-culture experiments, and contributed to the writing of the manuscript. J.C.M.-R. oversaw the research project and contributed to planning experiments and interpreting the data and wrote the manuscript. J.L. and I.V.J.F. performed radiolabeling and nuclear imaging studies. K.Z.B. provided valuable technical assistance during the in vivo studies. S.J.E and M.J.D.C. supported the in vitro cellular assays. J.C and R.B. designed and performed the ferroptosis studies. All authors reviewed the manuscript.

Declaration of Interests

The authors declare no competing interests.

Published: September 25, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101499.

Contributor Information

Ane Ruiz-de-Angulo, Email: aruizdeangulo@cicbiogune.es.

Juan C. Mareque-Rivas, Email: juan.mareque-rivas@swansea.ac.uk.

Supplemental Information

Document S1. Transparent Methods and Figures S1–S14
mmc1.pdf (3MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods and Figures S1–S14
mmc1.pdf (3MB, pdf)

Data Availability Statement

This study did not generate/analyze any datasets/code.

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