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.

Ferritin nanoconjugates guide trastuzumab brain delivery to promote an antitumor response in murine HER2 + breast cancer brain metastasis.

Brain metastasis (BM) represents a clinical challenge for patients with advanced HER2 + breast cancer (BC). The monoclonal anti-HER2 antibody trastuzumab (TZ) improves survival of BC patients, but it has low central nervous system penetrance, being ineffective in treating BM. Previous studies showed that ferritin nanoparticles (HFn) may cross the blood brain barrier (BBB) through binding to the transferrin receptor 1 (TfR1). However, whether this has efficacy in promoting the trans-BBB delivery of TZ and combating BC BM was not studied yet. Here, we investigated the potential of HFn to drive TZ brain delivery and promote a targeted antitumor response in a murine model of BC BM established by stereotaxic injection of engineered BC cells overexpressing human HER2. HFn were covalently conjugated with TZ to obtain a nanoconjugate endowed with HER2 and TfR1 targeting specificity (H-TZ). H-TZ efficiently achieved TZ brain delivery upon intraperitoneal injection and triggered stable targeting of cancer cells. Treatment with H-TZ plus docetaxel significantly reduced tumor growth and shaped a protective brain microenvironment by engaging macrophage activation toward cancer cells. H-TZ-based treatment also avoided TZ-associated cardiotoxicity by preventing drug accumulation in the heart and did not induce any other major side effects when combined with docetaxel. These results provided in vivo demonstration of the pharmacological potential of H-TZ, able to tackle BC BM in combination with docetaxel. Indeed, upon systemic administration, the nanoconjugate guides TZ brain accumulation, reduces BM growth and limits side effects in off-target organs, thus showing promise for the management of HER2 + BC metastatic to the brain.

Low neutrophil-to-lymphocyte ratio and pan-immune-inflammation-value predict nodal pathologic complete response in 1274 breast cancer patients treated with neoadjuvant chemotherapy: a multicenter analysis.

Background: Systemic inflammatory markers draw great interest as potential blood-based prognostic factors in several oncological settings. Objectives: The aim of this study is to evaluate whether neutrophil-to-lymphocyte ratio (NLR) and pan-immune-inflammation value (PIV) predict nodal pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) in node-positive (cN+) breast cancer (BC) patients. Design: Clinically, cN+ BC patients undergoing NAC followed by breast and axillary surgery were enrolled in a multicentric study from 11 Breast Units. Methods: Pretreatment blood counts were collected for the analysis and used to calculate NLR and PIV. Logistic regression analyses were performed to evaluate independent predictors of nodal pCR. Results: A total of 1274 cN+ BC patients were included. Nodal pCR was achieved in 586 (46%) patients. At multivariate analysis, low NLR [odds ratio (OR) = 0.71; 95% CI, 0.51-0.98; p = 0.04] and low PIV (OR = 0.63; 95% CI, 0.44-0.90; p = 0.01) were independently predictive of increased likelihood of nodal pCR. A sub-analysis on cN1 patients (n = 1075) confirmed the statistical significance of these variables. PIV was significantly associated with axillary pCR in estrogen receptor (ER)-/human epidermal growth factor receptor 2 (HER2)+ (OR = 0.31; 95% CI, 0.12-0.83; p = 0.02) and ER-/HER2- (OR = 0.41; 95% CI, 0.17-0.97; p = 0.04) BC patients. Conclusion: This study found that low NLR and PIV levels predict axillary pCR in patients with BC undergoing NAC. Registration: Eudract number NCT05798806.

Quantification and prospective evaluation of serum NfL and GFAP as blood-derived biomarkers of outcome in acute ischemic stroke patients.

Identification of reliable and accessible biomarkers to characterize ischemic stroke patients' prognosis remains a clinical challenge. Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) are markers of brain injury, detectable in blood by high-sensitive technologies. Our aim was to measure serum NfL and GFAP after stroke, and to evaluate their correlation with functional outcome and the scores in rehabilitation scales at 3-month follow-up. Stroke patients were prospectively enrolled in a longitudinal observational study within 24 hours from symptom onset (D1) and monitored after 7 (D7), 30 ± 3 (M1) and 90 ± 5 (M3) days. At each time-point serum NfL and GFAP levels were measured by Single Molecule Array and correlated with National Institute of Health Stroke Scale (NIHSS), modified Rankin scale (mRS), Trunk Control Test (TCT), Functional Ambulation Classification (FAC) and Functional Independence Measure (FIM) scores. Serum NfL and GFAP showed different temporal profiles: NfL increased after stroke with a peak value at D7; GFAP showed an earlier peak at D1. NfL and GFAP concentrations correlated with clinical/rehabilitation outcomes both longitudinally and prospectively. Multivariate analysis revealed that NfL-D7 and GFAP-D1 were independent predictors of 3-month NIHSS, TCT, FAC and FIM scores, with NfL being the biomarker with the best predictive performance.

Novel Bioengineering Strategies to Improve Bioavailability and In Vivo Circulation of H-Ferritin Nanocages by Surface Functionalization.

Due to its unique architecture and innate capability to specifically target cancer cells, ferritin has emerged as an attractive class of biomaterials for drug delivery. In many studies, various chemotherapeutics have been loaded into ferritin nanocages constituted by H-chains of ferritin (HFn), and their related anti-tumor efficacy has been explored by employing different strategies. Despite the multiple advantages and the versatility of HFn-based nanocages, there are still many challenges to face for their reliable implementation as drug nanocarriers in the process of clinical translation. This review aims at providing an overview of the significant efforts expended during recent years to maximize the features of HFn in terms of increased stability and in vivo circulation. The most considerable modification strategies explored to improve bioavailability and pharmacokinetics profiles of HFn-based nanosystems will be discussed herein.

Determination of the quality of lipoproteins by Raman spectroscopy in obese and healthy subjects.

Lipoproteins (LPs) are multimolecular complexes of lipids and proteins responsible for transporting fatty acids, cholesterol, and micronutrients (carotenoids) through the body. The quantification of triglycerides and cholesterol carried by lipoproteins is a leading clinical parameter to assess the increased risk of cardiovascular events. However, in recent times, the study of the overall "quality" of lipoproteins, defined by their biochemical composition and oxidation state, has emerged as necessary to improve the definition of the cardiovascular risk. In this work, we present Raman spectroscopy (RS) as an effective method to immediately detect the functional groups relative to the principal biochemical components and the level of unsaturated lipids present in LPs. Furthermore, we show how RS can reveal the differences in the biochemical composition and oxidation state of LPs extracted from a cohort of obese patients (Ob) and a control group of healthy subjects (HC). In particular, RS revealed how low-density lipoproteins (LDLs) from obese patients are enriched in triglycerides and more oxidized than those from the control group, while high-density lipoproteins (HDLs) from Ob patients were depleted in cholesterol and phospholipids. RS analysis also allowed the study of the relationship between the levels of carotenoids present in the different classes of LPs highlighting how this parameter depends on the disease severity. Overall, these results demonstrated that RS is a viable approach for quickly and effectively gaining information on LPs' biochemical composition and oxidation state, providing an immediate measure of their quality. Besides, RS further proved the role of LPs in obesity and metabolic dysfunctions.

Neurofilament-light chain quantification by Simoa and Ella in plasma from patients with dementia: a comparative study.

Neurofilament light chains (NfL) are neuron-specific cytoskeletal proteins whose plasmatic concentrations have been explored as a clinically useful marker in several types of dementia. Plasma concentrations of NfL are extremely low, and just two assays are commercially available for their study: one based on the SiMoA technology and one based on Ella. We thus studied plasma levels of NfL with both platforms to check the correlation between them and to assess their potential in the diagnosis of neurodegeneration. Plasma NfL levels were measured on 50 subjects: 18 healthy controls, 20 Alzheimer's disease, and 12 frontotemporal dementia patients. Ella returned plasmatic NfL levels significantly higher than SiMoA, however the results were strongly correlated (r = 0.94), and a proportional coefficient of 0.58 between the two assays was calculated. Both assays detected higher plasma NfL levels in patients with dementia than in the control group (p < 0.0001) and allowed their discrimination with excellent diagnostic performance (AUC > 0.95). No difference was found between Alzheimer's and Frontotemporal dementia either using SiMoA or Ella. In conclusion, both the analytical platforms resulted effective in analysing plasma levels of NfL. However, the correct interpretation of results requires the precise knowledge of the assay used.

In Vitro Immunoreactivity Evaluation of H-Ferritin-Based Nanodrugs.

Biological nanoparticles, such as proteins and extracellular vesicles, are rapidly growing as nanobased drug-delivery agents due to their biocompatibility, high loading efficiency, and bioavailability. However, most of the candidates emerging preclinically hardly confirm their potential when entering clinical trials. Among other reasons, this is due to the low control of synthesis processes and the limited characterization of their potential immunoreactivity profiles. Here, we propose a combined method that allow us to fully characterize H-ferritin nanoparticles' immunoreactivity during their production, purification, endotoxin removal, and drug loading. H-Ferritin is an extremely interesting nanocage that is being under evaluation for cancer therapy due to its innate cancer tropism, favorable size, and high stability. However, being a recombinant protein, its immunoreactivity should be carefully evaluated preclinically to enable further clinical translation. Surprisingly, this aspect is often underestimated by the scientific community. By measuring proinflammatory cytokine release as a function of endotoxin content, we found that even removing all pyrogenic contaminants from the nanocage, a mild immunoreactivity was still left. When we further purified H-ferritin by loading doxorubicin through a highly standardized loading method, proinflammatory cytokine release was eliminated. This confirmed the safety of H-ferritin nanocages to be used for drug delivery in cancer therapy. Our approach demonstrated that when evaluating the safety of nanodrugs, a combined analysis of acute toxicity and immunoreactivity is necessary to guarantee the safety of newly developed products and to unveil their real translational potential.

Extensive Intraductal Component in Breast Cancer: What Role in Disease-Free Survival?

INTRODUCTION: Extensive intraductal component (EIC) associated to early breast cancer could increase the risk locoregional recurrence, but its impact on distant metastases is still unclear. The aim of the present study was to assess the role of EIC on 5-year survival outcomes in patients affected by early breast cancer treated with breast-conserving surgery. METHODS: A total of 414 consecutive patients with a minimum follow-up of 60 mo were collected from January 2007 to December 2015. Disease-free survival (DFS), distant metastasis-free survival (DMFS), and locoregional recurrence-free survival at 5 y were assessed considering the presence or absence of EIC and other clinical and pathological features. RESULTS: Absence of EIC was independently associated with worse 5-year DFS (hazard ratio [HR] 1.68, P = 0.008) and 5-year DMFS (HR 1.93, P = 0.007), whereas 5-year locoregional recurrence-free survival was not affected (HR 1.50, P = 0.16). Five-year DFS was increased by EIC in T1 patients (P = 0.03) but not in T2 stage. Moreover, EIC was associated to better DFS in G2 (P = 0.03) and G3 patients (P = 0.01) but not in G1 cases. CONCLUSIONS: Our results suggest that EIC is independently correlated with increased 5-year DFS and in particular with 5-year DMFS.

Prognostic Potential of Immune Inflammatory Biomarkers in Breast Cancer Patients Treated with Neoadjuvant Chemotherapy.

Immune inflammatory biomarkers are easily obtained and inexpensive blood-based parameters that recently showed prognostic and predictive value in many solid tumors. In this study, we aimed to investigate the role of these biomarkers in predicting distant relapse in breast cancer patients treated with neoadjuvant chemotherapy (NACT). All breast cancer patients who referred to our Breast Unit and underwent NACT were retrospectively reviewed. The pre-treatment neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), and pan-immune-inflammation value (PIV) were calculated from complete blood counts. The primary outcome was 5-year distant-metastasis-free survival (DMFS). In receiver operating characteristic analyses, the optimal cutoff values for the NLR, PLR, MLR, and PIV were determined at 2.25, 152.46, 0.25, and 438.68, respectively. High levels of the MLR, but not the NLR, PLR, or PIV, were associated with improved 5-year DMSF in the study population using both univariate (HR 0.52, p = 0.03) and multivariate analyses (HR, 0.44; p = 0.02). Our study showed that the MLR was a significant independent parameter affecting DMFS in breast cancer patients undergoing NACT. Prospective studies are required to confirm this finding and to define reliable cutoff values, thus leading the way for the clinical application of this biomarker.

Bisdemethoxycurcumin (BDC)-Loaded H-Ferritin-Nanocages Mediate the Regulation of Inflammation in Alzheimer's Disease Patients.

BACKGROUND: Bisdemethoxycurcumin (BDC) might be an inflammation inhibitor in Alzheimer's Disease (AD). However, BDC is almost insoluble in water, poorly absorbed by the organism, and degrades rapidly. We thus developed a new nanoformulation of BDC based on H-Ferritin nanocages (BDC-HFn). METHODS: We tested the BDC-HFn solubility, stability, and ability to cross a blood-brain barrier (BBB) model. We tested the effect of BDC-HFn on AD and control (CTR) PBMCs to evaluate the transcriptomic profile by RNA-seq. RESULTS: We developed a nanoformulation with a diameter of 12 nm to improve the solubility and stability. The comparison of the transcriptomics analyses between AD patients before and after BDC-HFn treatment showed a major number of DEG (2517). The pathway analysis showed that chemokines and macrophages activation differed between AD patients and controls after BDC-HFn treatment. BDC-HFn binds endothelial cells from the cerebral cortex and crosses through a BBB in vitro model. CONCLUSIONS: Our data showed how BDC-Hfn could improve the stability of BDC. Significant differences in genes associated with inflammation between the same patients before and after BDC-Hfn treatment have been found. Inflammatory genes that are upregulated between AD and CTR after BDC-HFn treatment are converted and downregulated, suggesting a possible therapeutic approach.

Fast quantification of extracellular vesicles levels in early breast cancer patients by Single Molecule Detection Array (SiMoA).

PURPOSE: Preliminary reports suggest that extracellular vesicles (EVs) might be a promising biomarker for breast cancer (BC). However, the quantification of plasmatic levels of EVs is a complex task. To overcome these limitations, we developed a new, fast, and easy to use assay for the quantification of EVs directly in plasma based on the use of Single-Molecule Array (SiMoA). METHODS: By using SiMoA to identify CD9+/CD63+ EVs, we analyzed plasma samples of 181 subjects (95 BC and 86 healthy controls, HC). A calibration curve, made of a serial dilution of lyophilized standards from human plasma, was used in each run to ensure the obtainment of quantitative results from the assay. In a subgroup of patients, EVs concentrations were estimated in plasma before and after 30 days from cancer surgery. Additional information on the size of EVs were also acquired using a Nanosight system to obtain a clearer understanding of the mechanism underlying the releases of EVs associated with the presence of cancer. RESULTS: The measured levels of EVs resulted significantly higher in BC patients (median values 1179.1 ng/µl vs 613.0 ng/µl, p < 0.0001). ROC curve was used to define the optimal cut-off level of the test at 1034.5 ng/µl with an AUC of 0.75 [95% CI 0.68-0.82]. EVs plasmatic concentrations significantly decreased after cancer surgery compared to baseline values (p = 0.014). No correlation was found between EVs concentration and clinical features of BC. CONCLUSION: SiMoA assay allows plasmatic EVs levels detection directly without any prior processing. EVs levels are significantly higher in BC patients and significantly decreases after cancer surgery.

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.

Preoperative Systemic Inflammatory Biomarkers Are Independent Predictors of Disease Recurrence in ER+ HER2- Early Breast Cancer.

The host's immune system plays a crucial role in determining the clinical outcome of many cancers, including breast cancer. Peripheral blood neutrophils and lymphocytes counts may be surrogate markers of systemic inflammation and potentially reflect survival outcomes. The aim of the present study is to assess the role of preoperative systemic inflammatory biomarkers to predict local or distant relapse in breast cancer. In particular we investigated ER+ HER2- early breast cancer, considering its challenging risk stratification. A total of 1,763 breast cancer patients treated at tertiary referral Breast Unit were reviewed. Neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR) and lymphocyte-to-monocyte (LMR) ratios were assessed from the preoperative blood counts. Multivariate analyses for 5-years locoregional recurrence-free (LRRFS), distant metastases-free (DMFS) and disease-free survivals (DFS) were performed, taking into account both blood inflammatory biomarkers and clinical-pathological variables. Low NLR and high LMR were independent predictors of longer LRRFS, DMFS and DFS, and low PLR was predictive of better LRRFS and DMFS in the study population. In 999 ER+ HER2- early breast cancers, high PLR was predictive of worse LRRFS (HR 0.42, p=0.009), while high LMR was predictive of improved LRRFS (HR 2.20, p=0.02) and DFS (HR 2.10, p=0.01). NLR was not an independent factor of 5-years survival in this patients' subset. Inflammatory blood biomarkers and current clinical assessment of the disease were not in agreement in terms of estimate of relapse risk (K-Cohen from -0.03 to 0.02). In conclusion, preoperative lymphocyte ratios, in particular PLR and LMR, showed prognostic relevance in ER+ HER2- early breast cancer. Therefore, they may be used in risk stratification and therapy escalation/de-escalation in patients with this type of tumor.

Circulating Fibroblast Activation Protein as Potential Biomarker in Patients With Inflammatory Bowel Disease.

A major concern in the management of Inflammatory Bowel Disease (IBD) is the absence of accurate and specific biomarkers to drive diagnosis and monitor disease status timely and non-invasively. Fibroblast activation protein (FAP) represents a hallmark of IBD bowel strictures, being overexpressed in stenotic intestinal myofibroblasts. The present study aimed at evaluating the potential of circulating FAP (cFAP) as an accessible blood biomarker of IBD. Quantitative determination of cFAP was performed by enzyme-linked immunosorbent assay on plasma samples prospectively collected from patients with IBD and control subjects. A discrimination model was established on a training set of 50% patients and validated on independent samples. Results showed that cFAP concentration was reduced in patients with IBD when compared to controls (p < 0.0001). Age, sex, smoking, disease location and behavior, disease duration and therapy were not associated with cFAP. The sensitivity and specificity of cFAP in discriminating IBD from controls were 70 and 84%, respectively, based on the optimal cutoff (57.6 ng mL-1, AUC = 0.78). Predictions on the test set had 57% sensitivity, 65% specificity, and 61% accuracy. There was no strong correlation between cFAP and routine inflammatory markers in the patients' population. A subgroup analysis was performed on patients with Crohn's disease undergoing surgery and revealed that cFAP correlates with endoscopic mucosal healing. In conclusion, cFAP deserves attention as a promising blood biomarker to triage patients with suspected IBD. Moreover, it might function as a biomarker of post-operative remission in patients with Crohn's disease.

Full-Length Recombinant hSP-D Binds and Inhibits SARS-CoV-2.

SARS-CoV-2 infection of host cells is driven by binding of the SARS-CoV-2 spike-(S)-protein to lung type II pneumocytes, followed by virus replication. Surfactant protein SP-D, member of the front-line immune defense of the lungs, binds glycosylated structures on invading pathogens such as viruses to induce their clearance from the lungs. The objective of this study is to measure the pulmonary SP-D levels in COVID-19 patients and demonstrate the activity of SP-D against SARS-CoV-2, opening the possibility of using SP-D as potential therapy for COVID-19 patients. Pulmonary SP-D concentrations were measured in bronchoalveolar lavage samples from patients with corona virus disease 2019 (COVID-19) by anti-SP-D ELISA. Binding assays were performed by ELISAs. Protein bridge and aggregation assays were performed by gel electrophoresis followed by silver staining and band densitometry. Viral replication was evaluated in vitro using epithelial Caco-2 cells. Results indicate that COVID-19 patients (n = 12) show decreased pulmonary levels of SP-D (median = 68.9 ng/mL) when compared to levels reported for healthy controls in literature. Binding assays demonstrate that SP-D binds the SARS-CoV-2 glycosylated spike-(S)-protein of different emerging clinical variants. Binding induces the formation of protein bridges, the critical step of viral aggregation to facilitate its clearance. SP-D inhibits SARS-CoV-2 replication in Caco-2 cells (EC90 = 3.7 µg/mL). Therefore, SP-D recognizes and binds to the spike-(S)-protein of SARS-CoV-2 in vitro, initiates the aggregation, and inhibits viral replication in cells. Combined with the low levels of SP-D observed in COVID-19 patients, these results suggest that SP-D is important in the immune response to SARS-CoV-2 and that rhSP-D supplementation has the potential to be a novel class of anti-viral that will target SARS-CoV-2 infection.

Raman analysis of microcalcifications in male breast cancer.

Microcalcifications (MCs) are important disease markers for breast cancer. Many studies were conducted on their characterization in female breast cancer (FBC), but no information is available on their composition in male breast cancer (MBC). Raman spectroscopy (RS) is a molecular spectroscopy that can rapidly explore the biochemical composition of MCs without requiring any staining protocol. In this study, we optimized an algorithm to identify the mineral components present in MCs from Raman images. The algorithm was then used to study and compare MCs identified on breast cancer pieces from male and female patients. In total, we analyzed 41 MCs from 5 invasive MBC patients and 149 MCs from 13 invasive FBC patients. Results show that hydroxyapatite is the most abundant type of calcium both in MBC and FBC. However, some differences in the amount and distribution of calcium minerals are present between the two groups. Besides, we observed that MCs in MBC have a higher amount of organic material (collagen) than FBC. To the best of our knowledge, this study provides the first overview of the composition of MCs present in MBC patients; and suggests that these patients have specific features that differentiate them from the previously studied FBC. Our result support thus the need for studies designed explicitly to the understanding of MBC.