Impact of doxorubicin-loaded ferritin nanocages (FerOX) vs. free doxorubicin on T lymphocytes: a translational clinical study on breast cancer patients undergoing neoadjuvant chemotherapy.

Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity.Our findings underscore the importance of optimizing DOX delivery to enhance its antitumor efficacy while minimizing adverse effects, highlighting the pivotal role played by FerOX in mitigating DOX-induced toxicity towards T-cells, thereby positioning it as a promising DOX formulation. This study contributes valuable insights to modern cancer therapy and immunomodulation.

Pyruvate transamination and NAD biosynthesis enable proliferation of succinate dehydrogenase-deficient cells by supporting aerobic glycolysis.

Succinate dehydrogenase (SDH) is the mitochondrial enzyme converting succinate to fumarate in the tricarboxylic acid (TCA) cycle. SDH acts as a tumor suppressor with germline loss-of-function mutations in its encoding genes predisposing to aggressive familial neuroendocrine and renal cancer syndromes. Lack of SDH activity disrupts the TCA cycle, imposes Warburg-like bioenergetic features, and commits cells to rely on pyruvate carboxylation for anabolic needs. However, the spectrum of metabolic adaptations enabling SDH-deficient tumors to cope with a dysfunctional TCA cycle remains largely unresolved. By using previously characterized Sdhb-deleted kidney mouse cells, here we found that SDH deficiency commits cells to rely on mitochondrial glutamate-pyruvate transaminase (GPT2) activity for proliferation. We showed that GPT2-dependent alanine biosynthesis is crucial to sustain reductive carboxylation of glutamine, thereby circumventing the TCA cycle truncation determined by SDH loss. By driving the reductive TCA cycle anaplerosis, GPT2 activity fuels a metabolic circuit maintaining a favorable intracellular NAD+ pool to enable glycolysis, thus meeting the energetic demands of SDH-deficient cells. As a metabolic syllogism, SDH deficiency confers sensitivity to NAD+ depletion achieved by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. Beyond identifying an epistatic functional relationship between two metabolic genes in the control of SDH-deficient cell fitness, this study disclosed a metabolic strategy to increase the sensitivity of tumors to interventions limiting NAD availability.

HER-2-Targeted Nanoparticles for Breast Cancer Diagnosis and Treatment.

Human epidermal growth factor receptor-2 (HER-2) overexpressing breast cancer is a breast cancer subtype characterized by high aggressiveness, high frequency of brain metastases and poor prognosis. HER-2, a glycoprotein belonging to the ErbB receptor family, is overexpressed on the outer membrane of cancer cells and has been an important therapeutic target for the development of targeted drugs, such as the monoclonal antibodies trastuzumab and pertuzumab. These therapies have been available in clinics for more than twenty years. However, despite the initial enthusiasm, a major issue emerged limiting HER-2 targeted therapy efficacy, i.e., the evolution of drug resistance, which could be tackled by nanotechnology. The aim of this review is to provide a first critical update on the different types of HER-2-targeted nanoparticles that have been proposed in the literature in the last decade for therapeutic purposes. We focus on the different targeting strategies that have been explored, their relative outcomes and current limitations that still need to be improved. Then, we review the nanotools developed as diagnostic kits, focusing on the most recent techniques, which allow accurate quantification of HER-2 levels in tissues, with the aim of promoting more personalized medicinal approaches in patients.

Protein-Based Nanoparticles for the Imaging and Treatment of Solid Tumors: The Case of Ferritin Nanocages, a Narrative Review.

Protein nanocages have been studied extensively, due to their unique architecture, exceptional biocompatibility and highly customization capabilities. In particular, ferritin nanocages (FNs) have been employed for the delivery of a vast array of molecules, ranging from chemotherapeutics to imaging agents, among others. One of the main favorable characteristics of FNs is their intrinsic targeting efficiency toward the Transferrin Receptor 1, which is overexpressed in many tumors. Furthermore, genetic manipulation can be employed to introduce novel variants that are able to improve the loading capacity, targeting capabilities and bio-availability of this versatile drug delivery system. In this review, we discuss the main characteristics of FN and the most recent applications of this promising nanotechnology in the field of oncology with a particular emphasis on the imaging and treatment of solid tumors.

Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity.

BACKGROUND: Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments. METHODS: TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis. RESULTS: Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro. In vivo, the intratumoral synergistic combination of poly(I:C)+R848 significantly prevented tumor growth and metastasis in lung cancer and fibrosarcoma immunocompetent murine models. Regressing tumors showed increased infiltration of macrophages with a higher M1:M2 ratio, recruitment of CD4+ and CD8+ T cells, accompanied by a reduction of immunosuppressive CD206+ TAMs and FOXP3+/CD4+ T cells. The depletion of both CD4+ and CD8+ T cells resulted in complete loss of treatment efficacy. Treated mice acquired systemic antitumoral response and resistance to tumor rechallenge mediated by boosted macrophage cytotoxic activity and T-cell proliferation. Proteomic experiments validate the superior activation of innate immunity by poly(I:C)+R848 combination versus single treatments or poly(I:C)+R837, and protein-protein-interaction network analysis reveal the key activation of the STAT1 pathway. DISCUSSION: These findings demonstrate the antitumor immune responses mediated by macrophage activation on local administration of poly(I:C)+R848 combination and support the intratumoral application of this therapy to patients with solid tumors in the clinic.

Nanobiotechnology and Immunotherapy: Two Powerful and Cooperative Allies against Cancer.

A number of novel cancer therapies have recently emerged that have rapidly moved from the bench to the clinic. Onco-immunotherapies, such as immune checkpoint blockade inhibitors and adoptive cell therapies, have revolutionized the field, since they provide a way to induce strong anti-tumor immune responses, which are able to fight cancer effectively. However, despite showing great efficacy in hematological and some solid tumors, unresponsiveness, development of therapy resistance and the development of serious adverse effects, limit their capacity to impact the vast majority of tumors. Nanoparticle-based delivery systems are versatile vehicles for a wide variety of molecular cargoes and provide an innovative strategy to improve conventional onco-immunotherapies. They can be finely tuned to release their contents in the tumor microenvironment, or to deliver combinations of adjuvants and antigens in the case of nanovaccines. In this review, we summarize the recent advancements in the field of nanobiotechnology, to remodel the tumor microenvironment and to enhance immunotherapies.

D-mannose suppresses macrophage IL-1ß production.

D-mannose is a monosaccharide approximately a hundred times less abundant than glucose in human blood. Previous studies demonstrated that supraphysiological levels of D-mannose inhibit tumour growth and stimulate regulatory T cell differentiation. It is not known whether D-mannose metabolism affects the function of non-proliferative cells, such as inflammatory macrophages. Here, we show that D-mannose suppresses LPS-induced macrophage activation by impairing IL-1ß production. In vivo, mannose administration improves survival in a mouse model of LPS-induced endotoxemia as well as decreases progression in a mouse model of DSS-induced colitis. Phosphomannose isomerase controls response of LPS-activated macrophages to D-mannose, which impairs glucose metabolism by raising intracellular mannose-6-phosphate levels. Such alterations result in the suppression of succinate-mediated HIF-1α activation, imposing a consequent reduction of LPS-induced Il1b expression. Disclosing an unrecognized metabolic hijack of macrophage activation, our study points towards safe D-mannose utilization as an effective intervention against inflammatory conditions.

Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors.

Background: Tumor-associated macrophages (TAMs), with M2-like immunosuppressive profiles, are key players in the development and dissemination of tumors. Hence, the induction of M1 pro-inflammatory and anti-tumoral states is critical to fight against cancer cells. The activation of the endosomal toll-like receptor 3 by its agonist poly(I:C) has shown to efficiently drive this polarization process. Unfortunately, poly(I:C) presents significant systemic toxicity, and its clinical use is restricted to a local administration. Therefore, the objective of this work has been to facilitate the delivery of poly(I:C) to macrophages through the use of nanotechnology, that will ultimately drive their phenotype toward pro-inflammatory states. Methods: Poly(I:C) was complexed to arginine-rich polypeptides, and then further enveloped with an anionic polymeric layer either by film hydration or incubation. Physicochemical characterization of the nanocomplexes was conducted by dynamic light scattering and transmission electron microscopy, and poly(I:C) association efficiency by gel electrophoresis. Primary human-derived macrophages were used as relevant in vitro cell model. Alamar Blue assay, ELISA, PCR and flow cytometry were used to determine macrophage viability, polarization, chemokine secretion and uptake of nanocomplexes. The cytotoxic activity of pre-treated macrophages against PANC-1 cancer cells was assessed by flow cytometry. Results: The final poly(I:C) nanocomplexes presented sizes lower than 200 nm, with surface charges ranging from +40 to -20 mV, depending on the envelopment. They all presented high poly(I:C) loading values, from 12 to 50%, and great stability in cell culture media. In vitro, poly(I:C) nanocomplexes were highly taken up by macrophages, in comparison to the free molecule. Macrophage treatment with these nanocomplexes did not reduce their viability and efficiently stimulated the secretion of the T-cell recruiter chemokines CXCL10 and CCL5, of great importance for an effective anti-tumor immune response. Finally, poly(I:C) nanocomplexes significantly increased the ability of treated macrophages to directly kill cancer cells. Conclusion: Overall, these enveloped poly(I:C) nanocomplexes might represent a therapeutic option to fight cancer through the induction of cytotoxic M1-polarized macrophages.

Lipid and Polymer-Based Nanoparticle siRNA Delivery Systems for Cancer Therapy.

RNA interference (RNAi) uses small interfering RNAs (siRNAs) to mediate gene-silencing in cells and represents an emerging strategy for cancer therapy. Successful RNAi-mediated gene silencing requires overcoming multiple physiological barriers to achieve efficient delivery of siRNAs into cells in vivo, including into tumor and/or host cells in the tumor micro-environment (TME). Consequently, lipid and polymer-based nanoparticle siRNA delivery systems have been developed to surmount these physiological barriers. In this article, we review the strategies that have been developed to facilitate siRNA survival in the circulatory system, siRNA movement from the blood into tissues and the TME, targeted siRNA delivery to the tumor or specific cell types, cellular uptake, and escape from endosomal degradation. We also discuss the use of various types of lipid and polymer-based carriers for cancer therapy, including a section on anti-tumor nanovaccines enhanced by siRNAs. Finally, we review current and recent clinical trials using NPs loaded with siRNAs for cancer therapy. The siRNA cancer therapeutics field is rapidly evolving, and it is conceivable that precision cancer therapy could, in the relatively near future, benefit from the combined use of cancer therapies, for example immune checkpoint blockade together with gene-targeting siRNAs, personalized for enhancing and fine-tuning a patient's therapeutic response.

The Tricarboxylic Acid Cycle at the Crossroad Between Cancer and Immunity.

Significance: The tricarboxylic acid (TCA) cycle is a housekeeping metabolic pathway essential for generation of energy and biosynthetic intermediates. Alterations of the TCA cycle play a pivotal role in oncogenesis and inflammation. As such, some metabolic vulnerabilities, imposed by TCA cycle dysfunction in cancer, have been identified. Similarly, the TCA cycle appeared as an actionable pathway in immunopathologies. Recent Advances: Metabolic changes accompanying cell transformation have been usually considered as adaptive mechanisms to malignant transformation. The identification of oncogenic mutations in some TCA cycle enzymes changed this view, indicating altered mitochondrial metabolism as an instrumental mechanism for cancer initiation. Similarly, the observation that TCA cycle-derived metabolites have multiple signaling roles in immune cells supports the idea of this pathway as a metabolic rheostat of immune responses. Critical Issues: This review summarizes the crucial role of the TCA cycle in pathophysiology describing the post-translational and epigenetic impact of oncometabolites accumulation in cancer and immune cells. Future Directions: Additional studies will be necessary to further explore the role of oncometabolites in paracrine signaling and to identify genuine metabolic and nutritional liabilities imposed by TCA cycle dysfunction in cancer, hardly to be escaped by resistance mechanisms.

MIS416 as a siRNA Delivery System with the Ability to Target Antigen-Presenting Cells.

MIS416 is a microparticulate formulation derived from propionibacterium acnes cell wall skeletons with intrinsic adjuvant activity. Conjugates of MIS416-SS-peptide containing a disulfide linkage facilitate the cytoplasmic delivery and release of peptides in antigen-presenting cells (APCs). We hypothesized that MIS416-siRNA (small interfering RNA) conjugates, containing a disulfide linkage between MIS416 and the siRNA, would allow cytoplasmic release of siRNA in APCs. MIS416-SS-siStat3 conjugates added to cell culture medium of monolayers of DCs in culture flasks successfully targeted Stat3 mRNA in DCs in vitro without transfection, downregulating Stat3 mRNA and protein levels. These results suggest that MIS416-SS-siRNA conjugates can be used as a novel siRNA delivery system for the knockdown of mRNA levels in APCs.

Bridging Small Molecules to Modified Bacterial Microparticles Using a Disulphide Linkage: MIS416 as a Cargo Delivery System.

MIS416 is an intact minimal cell wall skeleton derived from Proprionibacterium acnes that is phagocytosed by antigen presenting cells, including dendritic cells (DCs). This property allows MIS416 to be exploited as a vehicle for the delivery of peptide antigens or other molecules (for example, nucleic acids) to DCs. We previously showed that covalent (non-cleavable) conjugation of OVA, a model antigen derived from ovalbumin, to MIS416 enhanced immune responses in DCs in vivo, compared to unconjugated MIS416 and OVA. Intracellular trafficking promotes the lysosomal degradation of MIS416, leading to the destruction of MIS416 plus the associated cargos conjugated to MIS416. However, lysosomal degradation of cargo may not be desired for some MIS416 conjugates. Here we have investigated whether a cleavable linkage could facilitate release of the cargo in the cytoplasm of DCs to avoid lysosomal degradation. DCs were treated in vitro with disulfide-containing conjugates, and as hypothesised faster release of SIINFEKL peptide in the cytoplasm of DCs was observed with the inclusion of a disulfide bond between MIS416 and cargo. The inclusion of a cleavable disulfide bond in the conjugates did not significantly alter the amount of SIINFEKL antigens presented on MHC I molecules on DCs as compared with conjugates without a disulfide bond. However, the conjugates containing disulfide-linkages performed either slightly better (p<0.05) than, or the same as conjugates without a disulfide bond with respect to in vitro OT-1 T-cell proliferation induced by the presentation of SIINFEKL antigens on DCs, or DC activation studies, respectively. However, disulfide-containing conjugates were less effective than conjugates without a disulfide bond in in vivo cytotoxicity assays. In conclusion, inclusion of a disulfide bond in MIS416-peptide conjugates was associated with efficient release of peptides in the cytoplasm of DCs, an important consideration for MIS416-mediated delivery of degradation-sensitive cargoes. However, treatment of DCs with disulfide-containing conjugates did not significantly alter the presentation of peptide antigens on MHC class I molecules to T-cells, or greatly enhance antigen-associated T-cell proliferation in vitro.

Polymeric vesicles loaded with gadoteridol as reversible and concentration-independent magnetic resonance imaging thermometers.

The use of temperature sensitive liposomes (TSLs) loaded with paramagnetic Gd(III) complexes have been explored to develop MRI agents able to provide a imaging guide to heating-based therapies. Though the performance of such probes has been already demonstrated in vivo at preclinical level, further improvements (e.g., concentration independent image response, reversibility of the sensor) are necessary to increase the accuracy of the temperature readout. This work reports for the first time, the potential of Gd-loaded polymersomes (bilayered vesicles made of amphiphilic di-block copolymers) as improved thermosensitive MRI probes. Differently from conventional TSLs, such probes do not display a defined gel-to-liquid temperature transition and, therefore, they did not release their content in a wide temperature range, thereby allowing reversible temperature readouts. Moreover, a ratiometric approach based on the measurement of the ratio between transverse and longitudinal water protons relaxation rates (R2/R1) allows a temperature readout independent of the probe concentration. The imaging performance of temperature sensitive polymersomes prepared in this work was tested both in vitro and in vivo after subcutaneous injection in healthy mice.

Improved paramagnetic liposomes for MRI visualization of pH triggered release.

This work aims at assessing the in vitro potential of paramagnetic pH sensitive liposomes as imaging tools for visualizing drug-delivery and release processes by Magnetic Resonance Imaging (MRI). pH sensitive liposomes (pSLs) were formulated using the fusogenic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), the membrane stabilizer D-α-tocopherol-hemisuccinate (THS), and were loaded with several paramagnetic complexes including the clinically approved Gadoteridol (marketed as ProHance™). The proposed formulation allows the fast and full release of Gadoteridol at pH 5.5. The leakage of the imaging reporter from the vesicles was associated with a relaxivity enhancement that allowed its visualization by MRI. It was observed that the release mechanism implies the protonation of the THS basic sites that leads to vesicle aggregation, thus enabling the expression of the fusogenic property of POPE. Attempts for improving the MRI properties of pSLs were pursued through the encapsulation of imaging agents with higher relaxivity than Gadoteridol, but it was observed that the release kinetic can be significantly affected by the probe size. Aiming at preparing stealth pSLs, PEG chains were conjugated to the external surface of the vesicles via cleavable disulphide bridges. Such nanomedicines do not release their content at acidic pH as long as the coating polymer is not removed from the surface. The results obtained suggest that the liposomal formulation investigated in this work has the potential for visualizing drug-delivery and release processes by in vivo MRI preclinical studies.

In vivo MRI multicontrast kinetic analysis of the uptake and intracellular trafficking of paramagnetically labeled liposomes.

This work aims at developing a MRI method that allows to get more insight into the understanding of the in vivo fate of liposomes and their payload. The method relies on the temporal assessment of the contrast changes induced by the presence of a classical relaxation agent versus the effect induced by a CEST (chemical exchange saturation transfer) agent. Liposomes were loaded with the paramagnetic complexes, Gd-HPDO3A and [Tm-DOTMA](-) [Na](+), in order to endow the nanovesicles with the characteristic properties of T(1)/T(2) and CEST/T(2) MRI agents, respectively. The paramagnetically loaded liposomes were injected directly into the tumor (B16 melanoma xenograft in mice) where they generate T(1), T(2), and CEST MR contrasts that were quantitatively monitored over time (0-48h). The kinetic of each contrast enhancement reports about peculiar properties relative to the fate of the liposomes in the tumor environment. A kinetic model has been set-up to fit the experimental multicontrast data in order to extract the relevant information about the cellular uptake of the liposomes and the release of their payload. Upon comparing conventional stealth liposomes with pH-sensitive ones, it has been shown that the latter ones differ essentially in the step associated with the release of the drug that is likely occurring in the endosomal acidic vesicles.