High-Resolution Raman Imaging of >300 Patient-Derived Cells from Nine Different Leukemia Subtypes: A Global Clustering Approach.

Leukemia comprises a diverse group of bone marrow tumors marked by cell proliferation. Current diagnosis involves identifying leukemia subtypes through visual assessment of blood and bone marrow smears, a subjective and time-consuming method. Our study introduces the characterization of different leukemia subtypes using a global clustering approach of Raman hyperspectral maps of cells. We analyzed bone marrow samples from 19 patients, each presenting one of nine distinct leukemia subtypes, by conducting high spatial resolution Raman imaging on 319 cells, generating over 1.3 million spectra in total. An automated preprocessing pipeline followed by a single-step global clustering approach performed over the entire data set identified relevant cellular components (cytoplasm, nucleus, carotenoids, myeloperoxidase (MPO), and hemoglobin (HB)) enabling the unsupervised creation of high-quality pseudostained images at the single-cell level. Furthermore, this approach provided a semiquantitative analysis of cellular component distribution, and multivariate analysis of clustering results revealed the potential of Raman imaging in leukemia research, highlighting both advantages and challenges associated with global clustering.

Novel loading protocol combines highly efficient encapsulation of exogenous therapeutic toxin with preservation of extracellular vesicles properties, uptake and cargo activity.

Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.

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.

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.

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.

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.

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.

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.

Lipofilling in Breast Oncological Surgery: A Safe Opportunity or Risk for Cancer Recurrence?

Lipofilling (LF) is a largely employed technique in reconstructive and esthetic breast surgery. Over the years, it has demonstrated to be extremely useful for treatment of soft tissue defects after demolitive or conservative breast cancer surgery and different procedures have been developed to improve the survival of transplanted fat graft. The regenerative potential of LF is attributed to the multipotent stem cells found in large quantity in adipose tissue. However, a growing body of pre-clinical evidence shows that adipocytes and adipose-derived stromal cells may have pro-tumorigenic potential. Despite no clear indication from clinical studies has demonstrated an increased risk of cancer recurrence upon LF, these observations challenge the oncologic safety of the procedure. This review aims to provide an updated overview of both the clinical and the pre-clinical indications to the suitability and safety of LF in breast oncological surgery. Cellular and molecular players in the crosstalk between adipose tissue and cancer are described, and heterogeneous contradictory results are discussed, highlighting that important issues still remain to be solved to get a clear understanding of LF safety in breast cancer patients.

Stable and scalable SERS tags conjugated with neutravidin for the detection of fibroblast activation protein (FAP) in primary fibroblasts.

SERS tags are a class of nanoparticles with great potential in advanced imaging experiments. The preparation of SERS tags however is complex, as they suffer from the high variability of the SERS signals observed even at the slightest sign of aggregation. Here, we developed a method for the preparation of SERS tags based on the use of gold nanostars conjugated with neutravidin. The SERS tags here obtained are extremely stable in all biological buffers commonly employed and can be prepared at a relatively large scale in very mild conditions. The obtained SERS tags have been used to monitor the expression of fibroblast activation protein alpha (FAP) on the membrane of primary fibroblasts obtained from patients affected by Crohn's disease. The SERS tags allowed the unambiguous identification of FAP on the surface of cells thus suggesting the feasibility of semi-quantitative analysis of the target protein. Moreover, the use of the neutravidin-biotin system allows to apply the SERS tags for any other marker detection, for example, different cancer cell types, simply by changing the biotinylated antibody chosen in the analysis.

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin.

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

Selective Targeting of Cancer-Associated Fibroblasts by Engineered H-Ferritin Nanocages Loaded with Navitoclax.

Cancer-associated fibroblasts (CAFs) are key actors in regulating cancer progression. They promote tumor growth, metastasis formation, and induce drug resistance. For these reasons, they are emerging as potential therapeutic targets. Here, with the aim of developing CAF-targeted drug delivery agents, we functionalized H-ferritin (HFn) nanocages with fibroblast activation protein (FAP) antibody fragments. Functionalized nanocages (HFn-FAP) have significantly higher binding with FAP+ CAFs than with FAP- cancer cells. We loaded HFn-FAP with navitoclax (Nav), an experimental Bcl-2 inhibitor pro-apoptotic drug, whose clinical development is limited by its strong hydrophobicity and toxicity. We showed that Nav is efficiently loaded into HFn (HNav), maintaining its mechanism of action. Incubating Nav-loaded functionalized nanocages (HNav-FAP) with FAP+ cells, we found significantly higher cytotoxicity as compared to non-functionalized HNav. This was correlated with a significantly higher drug release only in FAP+ cells, confirming the specific targeting ability of functionalized HFn. Finally, we showed that HFn-FAP is able to reach the tumor and to target CAFs in a mouse syngeneic model of triple negative breast cancer after intravenous administration. Our data show that HNav-FAP could be a promising tool to enhance specific drug delivery into CAFs, thus opening new therapeutic possibilities focused on tumor microenvironment.

Prediction of nodal staging in breast cancer patients with 1-2 sentinel nodes in the Z0011 era.

The aim of this study was to provide an innovative nomogram to predict the risk of >2 positive nodes in patients fulfilling the Z0011 criteria with 1-2 sentinel lymph nodes (SLNs) only retrieved.From 2007 to 2017, at the Breast Unit of ICS Maugeri Hospital 271 patients with 1-2 macrometastatic SLNs, fulfilling the Z0011 criteria, underwent axillary dissection and were retrospectively reviewed.A mean of 1.5 SLNs per patient were identified and retrieved. One hundred eighty-seven (69.0%) had 1-2 positive nodes, and 84 (31.0%) had >2 metastatic nodes. Independent predictors of axillary status were: positive SLNs/retrieved SLNs ratio (odds ratio [OR] 10.95, P = .001), extranodal extension (OR 5.51, P = .0002), and multifocal disease (OR 2.9, P = .003). A nomogram based on these variables was constructed (area under curve after bootstrap = 0.74).The proposed nomogram might select those patients fulfilling the Z0011 criteria, with 1-2 SLNs harvested, in whom a high axillary tumor burden is expected, aiding to guide adjuvant treatments.

Development of Tumor-Targeted Indocyanine Green-Loaded Ferritin Nanoparticles for Intraoperative Detection of Cancers.

Indocyanine green (ICG) is a fluorescent dye with a strong emission in the near-infrared spectral range that allows deep signal penetration and minimal interference of tissue autofluorescence. It has been employed in clinics for different applications, among which the more interesting is certainly near-infrared fluorescence image-guided surgery. This technique has found wide application in surgical oncology for lymph node mapping or for laparoscopic surgery. Despite ICG being useful for tracking loco-regional lymph nodes, it does not provide any information about cancer involvement of such lymph nodes or lymphatic vessels, lacking any tumor-targeting specificity. However, the clinical need in surgical oncology is not only a specific tracking of metastatic nodes but also the intraoperative detection of micrometastatic deposits. Here, we have exploited a nanotechnological solution to improve ICG usefulness by its encapsulation in H-ferritin (HFn) nanocages. They are natural protein-based nanoparticles that exhibit some very interesting features as delivery systems in oncological applications because they display specific tumor homing. We show that HFn loaded with ICG exhibits specific uptake into different cancer cell lines and is able to deliver ICG to the tumor more efficiently than the free dye in an in vivo model of TNBC. Our results pave the way for the application of ICG-loaded HFn in fluorescence image-guided surgery of cancer.

Raman Analysis Reveals Biochemical Differences in Plasma of Crohn's Disease Patients.

BACKGROUNDS AND AIMS: There is no accurate and reliable circulating biomarker to diagnose Crohn's disease [CD]. Raman spectroscopy is a relatively new approach that provides information on the biochemical composition of samples in minutes and virtually without any sample preparation. We aimed to test the use of Raman spectroscopy analysis of plasma samples as a potential diagnostic tool for CD. METHODS: We analysed by Raman spectroscopy dry plasma samples obtained from 77 CD patients [CD] and 45 healthy controls [HC]. In the dataset obtained, we analysed spectra differences between CD and HC, as well as among CD patients with different disease behaviours. We also developed a method, based on principal component analysis followed by a linear discrimination analysis [PCA-LDA], for the automatic classification of individuals based on plasma spectra analysis. RESULTS: Compared with HC, the CD spectra were characterised by less intense peaks corresponding to carotenoids [p <10-4] and by more intense peaks corresponding to proteins with ß-sheet secondary structure [p <10-4]. Differences were also found on Raman peaks relative to lipids [p = 0.0007] and aromatic amino acids [p <10-4]. The predictive model we developed was able to classify CD and HC subjects with 83.6% accuracy [sensitivity 80.0% and specificity 85.7%] and F1-score of 86.8%. CONCLUSIONS: Our results indicate that Raman spectroscopy of blood plasma can identify metabolic variations associated with CD and it could be a rapid pre-screening tool to use before further specific evaluation.