**5. Conclusions-Perspectives-Outlook**

Cancer immunotherapy has tremendous promise, but it has yet to be clinically applied in a wider variety of tumor situations. The main difficulties are toxicity and therapeutic responsiveness limited to a small subset of patients. The variation in patient response rates reflects the various paths tumors use to regulate the various immune-evasion mechanisms occurring in the tumor microenvironment. As a result, it appears that immunotherapy focused against one particular protumoral mechanism is not effective enough at producing a noticeable therapeutic impact. To ensure the creation of novel, efficient cancer treatments, it is extremely desirable to combine therapy approaches that simultaneously target several cancer immuno-evasion systems, albeit this may result in higher toxicity. In this way, immunogenic cell death (ICD)-based strategies have attracted a lot of scientific attention to address the current constraints in treating solid tumors. Indeed, ICD triggers the immune response against the tumor through the activation of dendritic cells, initiating a cascade process leading to an antigen-specific T-cell response. Even though ICD has the effect of boosting the immune system to eliminate the cancer cells, in many instances, the response is insufficient but has been shown to be significantly improved with immune checkpoint inhibitors. ICD can be induced by some chemotherapies (e.g., doxorubicin, 5-fluorouracil) and external beam therapies such as radiotherapy, photodynamic therapy, and hyperthermia.

On that basis, it seems that nanomedicines can offer the possibility of combining these different approaches in the same drug and thus considerably improve the effectiveness of cancer immunotherapy. Indeed, according to their design and the materials they are made of, NPs can act as drug-delivery vehicles and be sensitive to a physical stimulus for either diagnosis and/or therapy (theranostic potential). As vehicles for the precise delivery of tumor antigens and/or immunostimulatory molecules to specific cells located in lymphoid organs or in the tumor microenvironment, nanoparticle-based delivery systems have recently demonstrated a great potential to improve the effectiveness and safety profile of conventional immunotherapeutics. Among these nanomedicines, magnetic NPs such as SPIONs might have enormous potential for safe, more efficient, and individualized cancer treatment. SPIONs have strong biomedical potential because of their high stability, biocompatibility, and low toxicity. Like most nanomedicines, SPIONs enable localized delivery of payload drugs. They also allow us to perform a rational design of novel combinatorial therapies based on immunotherapeutic treatments. In this way, they can target the adaptive and/or innate immune system through their use with/as immunomodulatory therapies (e.g., M2-like TAMs polarization to M1-like phenotype), therapeutic vaccines, and adoptive cell therapies (e.g., cell tracking of chimeric antigen receptor (CAR) T cells). Moreover, and this is what differentiates them from other NPs, due to their distinct ability to react only to an applied external magnetic field, SPIONs are attracting a lot of attention. Indeed, this property is particularly intriguing for biomedical applications and has allowed the development of novel immunotherapeutic approaches that rely on heating capability (magnetic hyperthermia, thermoresponsive drug release), magnetically controlled navigation (i.e., to guide drugs and cell therapies at the target region under a magnetic field), and imageguided techniques, such as magnetic resonance imaging and magnetic particle imaging, a new SPION-based molecular imaging technique. Moreover, due to their versatility, SPIONs make it possible to perform multimodal imaging such as simultaneous PET-MRI, especially for cell tracking. Combining the two imaging modalities may provide at early time points the fast localization and absolute quantification of radiolabeled SPIONs using PET, while MRI gives high-resolution anatomical background information for long-term NP follow-up. This innovative simultaneous approach allows us to overcome the respective limitations of each modality (i.e., resolution for PET and sensitivity for MRI).

As soon as a nanoparticle must be designed, we have to consider that there must be an ad hoc specification, i.e., making this nanoparticle compatible with its further use as a drug or medical device. For an immunotherapeutic approach, due to the complexity of tumor biology, a disease-driven approach should be proposed for the rational design of SPIONs rather than the traditional formulation-driven approach ("one-size-fits-all"). The specification of tumor-targeted SPIONs with immunotherapeutic capabilities will depend on the application, and it is necessary to take into account their multimodal potential, especially for theranostics:


In spite of the strong theranostic potential of SPIONs, the limited quantity of SPIONbased nanomedicines in clinical trials and on the market demonstrates a number of challenges to be overcome in order to facilitate their translation from the bench to the bedside. The safety of metallic NPs remains a major concern. To evaluate SPION-based nanomedicine biocompatibility and enhance its therapeutic benefits, a detailed investigation of how it interacts with the host tissues is essential. Previous clinical use of SPION formulations that have received FDA/EMA approval has already shown their acceptable safety and biocompatibility, which is unmatched by other metal-based nanoparticle systems. This offers a benefit in using SPIONs as nanomedicines to boost therapeutic results as improvements in cancer immunotherapy are made. Nevertheless, there are still some major regulatory and industrial hurdles to be overcome prior to reaching the market, due to the complex nature of nanomedicine when compared to conventional pharmaceutical

products with a single agent. It is also important to consider the impacts of nanoparticles in general, and metallic NPs such as SPIONs in particular, on the environment, society, and ethics to make them acceptable in a biomedical context.

Overall, the unique properties and versatility of SPIONs pave the way for new approaches in the fields of drug delivery and theranostics for cancer immunotherapy, contributing to the personalization of treatments, especially to manage cancers with high unmet medical needs.

**Author Contributions:** Conceptualization, A.M.M.D., A.C. and B.C.; writing—original draft preparation, A.M.M.D., A.C. and B.C.; writing—review and editing, A.M.M.D., A.C., P.-S.B., E.K., A.O., P.-M.W. and B.C.; visualization, P.-E.D., C.P. and R.D.; supervision, B.C.; project administration, B.C.; funding acquisition, B.C. and A.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a French Government grant managed by the French National Research Agency under the program "Investissements d'Avenir" with reference ANR-10-EQPX-05- 01/IMAPPI Equipex. It was also supported by the Institut de Chimie Moléculaire de l'Université de Bourgogne (ICMUB UMR CNRS 6302) and by the Centre George-François Leclerc (Preclinical imaging and radiotherapy platform, nuclear medicine department).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was performed within Pharm'image, a regional center of excellence in pharmaco-imaging. Mélanie Guillemin and Marie Monterrat are gratefully and warmly thanked for their technical support. Jérémy Paris, CEO of SON SAS, is also thanked for his scientific advice.

**Conflicts of Interest:** The authors declare no conflict of interest. The company had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
