Triple-negative breast cancer is an aggressive breast cancer subtype characterized by the absence of human epidermal growth factor receptor 2, estrogen and progesterone receptors, limiting targeted therapy options. Cisplatin, a chemotherapeutic agent, induces DNA damage and exhibits some efficacy against triple-negative breast cancer, but its effectiveness is often reduced by chemoresistance and systemic toxicity. To optimize this strategy in a biocompatible and precise manner, we developed a nanoplatform based on red blood cell-derived extracellular vesicles for the combined delivery of Cetuximab and cisplatin, enabling immune evasion, and the possibility of autologous personalization and GMP-compliant production. Owing to their DNA-free lumen and lack of EGFR, RBC-EVs preserve cisplatin activity and prevent interference with cetuximab. This formulation enhances cisplatin's cytotoxicity by up to 50

Extracellular vesicles (EVs) are a subgroup of the circulating particles, released by cells in both normal and diseased states, carrying active biomolecules. They have gained significant attention as potential cancer biomarkers, particularly in breast cancer (BC). Previous research showed variations in EVs content and quantity between BC patients and healthy controls (HC). However, studying the biochemical profile of EVs remains challenging due to their low abundance and complex composition. Additionally, EVs may interact with other plasma components, like lipoproteins (LPs), forming a so called "biomolecular corona" that further complicates their analysis. Here, Raman spectroscopy (RS) is proposed as a fast tool to obtain the biochemical profile of circulating EVs in the context of BC. RS was employed to differentiate various extracellular particles (EPs) in blood, including LPs and EVs. The study also evaluated RS's capability to quantify major classes of biomolecules and compared these results with those obtained by traditional biochemical assays. Finally, compositional differences in large EVs (lEVs) and small EVs (sEVs) were assessed between HC and BC patients. RS revealed the existence of distinct biochemical signatures associated with BC, highlighting increased levels of nucleic acids and lipids in the BC group.

Extracellular vesicles (EVs) offer a promising avenue for non-invasive, real-time monitoring of metastatic breast cancer (mBC), but clinical application as a liquid biopsy is hindered by their heterogeneity and low abundance. Here we present a Single Molecule Array (SiMoA) platform enhanced by membrane sensing peptides (MSP) for the highly sensitive detection of HER2 on EV membranes (EVs-HER2) and general EVs population (CD9+) directly from plasma samples of mBC patients. The MSP-based SiMoA assay demonstrated superior sensitivity and specificity compared to conventional antibody-based assays, allowing the detection of lower amounts of EVs and discriminating EVs derived from breast cancer patient-derived organoids (BC-PDO) from healthy control-derived organoids (HC-PDO). Concerning the analysis of EVs in plasma samples (n=49 mBC patients, n=30 healthy controls), we observed significantly lower CD9+ EVs levels in mBC patients relative to healthy controls, a trend consistently confirmed across assays. Notably, EVs-HER2 levels were significantly enriched in HER2-positive patients and correlated with clinical HER2 status assessed by immunohistochemistry. Besides, lower CD9+ EVs levels were associated with poorer clinical outcomes, highlighting the potential prognostic utility of EV quantification. Our findings underscore the potential of MSP-enhanced SiMoA platforms for accurate, minimally invasive monitoring of EVs-HER2 in mBC and for monitoring CD9+ EVs levels to assess disease progression. ### Competing Interest Statement The authors declare the following potential conflicts of interest: Alessandro Gori (A.G) is listed as co-inventor on European patent application EP4025911A1 (Conjugates composed of membrane-targeting peptides for extracellular vesicles isolation, analysis and their integration thereof). The patent covers the peptide-based probes and methods described in this study. All other authors declare no competing interests. Ministero della Salute, https://ror.org/00789fa95, ricerca corrente Ministero dell’università e della ricerca, https://ror.org/0341vw408, PRIN: progetti di ricercar di rilevante interesse nazionale- Bando 2022, Prot. 2022CS9H53”

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.

BackgroundA reliable preclinical model of patient-derived organoids (PDOs) was developed in a case study of a 69-year-old woman diagnosed with breast cancer (BC) to investigate the tumour evolution before and after neoadjuvant chemotherapy and surgery. The results were achieved due to the development of PDOs from tissues collected before (O-PRE) and after (O-POST) treatment.MethodsPDO cultures were characterized by histology, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), confocal microscopy, flow cytometry, real-time PCR, bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and drug screening.ResultsBoth PDO cultures recapitulated the histological and molecular profiles of the original tissues, and they showed typical mammary gland organization, confirming their reliability as a personalized in vitro model. Compared with O-PRE, O-POST had a greater proliferation rate with a significant increase in the Ki67 proliferation index. Moreover O-POST exhibited a more stem-like and aggressive phenotype, with increases in the CD24low/CD44low and EPCAMlow/CD49fhigh cell populations characterized by increased tumour initiation potential and multipotency and metastatic potential in invasive lobular carcinoma. Analysis of ErbB receptor expression indicated a decrease in HER-2 expression coupled with an increase in EGFR expression in O-POST. In this context, deregulation of the PI3K/Akt signalling pathway was assessed by transcriptomic analysis, confirming the altered transcriptional profile. Finally, transcriptomic single-cell analysis identified 11 cell type clusters, highlighting the selection of the luminal component and the decrease in the number of Epithelial-mesenchymal transition cell types in O-POST.ConclusionNeoadjuvant treatment contributed to the enrichment of cell populations with luminal phenotypes that were more resistant to chemotherapy in O-POST. PDOs represent an excellent 3D cell model for assessing disease evolution.

Background: Lipofilling (LF) is a widely employed technique for breast reconstruction following surgery in breast cancer (BC) patients. However, recent research has shed light on a potential tumorigenic role of adipocytes and adipose-derived stem cells (ASCs), prompting concerns on the safety of LF for cancer patients.

HER2 overexpression/amplification (HER2+) occurs in 15-20% of breast cancer (BC) and identifies a clinically aggressive BC subtype. Although the introduction of effective anti-HER2 drugs remarkably improved the prognosis of HER2+ BC patients, primary and acquired tumor resistance to anti-HER2 treatments underscores the need for novel therapies for HER2+ BC patients. Tumor cells exhibit unique metabolic alterations that are responsible for primary or acquired tumor resistance to standard therapies, and which are increasingly emerging as potential targets for novel treatment strategies. In particular, the dysregulation of fatty acid β-oxidation (FAO), a catabolic process that produces energy (ATP and NADPH production) to sustain cancer growth, survival and aggressiveness, is associated with therapy resistance in diverse malignancies including BC. Specifically, carnitine palmitoyltransferase 1A (CPT1A), the key rate-limiting enzyme of mitochondrial FAO, facilitates cancer metabolic adaptation, thus representing a promising target for cancer therapy. In this context, gene set enrichment analysis of profiled matched HER2+ BC samples (n=33) collected before and after trastuzumab-based neo-adjuvant biochemotherapy (trastuzumab plus taxanes) revealed that the Fatty Acids (FA) metabolism gene set was one of the most significantly Hallmark enriched pathway (NES=1.57; FDR=0.0039) in post- compared with pre-treated HER2+ BC samples, implying that trastuzumab plus chemotherapy treatment induces an enhancement of FAO activity. Accordingly, CPT1A transcript levels were found to be inversely associated with lapatinib (L) susceptibility in HER2+ BC cell lines and patient-derived organoids resistant to anti-HER2 therapy versus L-sensitive cell models. Additionally, Seahorse Analysis revealed higher baseline oxidation of the FA palmitate in intrinsically L-resistant HER2+ BC cells as compared with L-sensitive cells, thus supporting the hypothesis that FAO could represent the major lipid metabolic pathway sustaining the energetic needs of HER2+ BC cells resistant to anti-HER2 therapy for survival and growth. Further, the pharmacological dual blockade of HER2 and CPT1A significantly increases cytotoxicity of L-resistant cell models versus single agents, corroborating the candidacy of CPT1A as a potential metabolic vulnerability to be targeted to overcome refractoriness to HER2-targeted therapy in HER2+ BC. Our data support the hypothesis that the inhibition of FAO sensitizes HER2+ BC cells to anti-HER2 drugs, thus representing a potential strategy to overcome drug resistance in HER2+ BC and paving the way to investigate CPT1A inhibitors with anti-HER2 drugs to improve the clinical outcomes of HER2+ BC patients with acquired resistance to standard anti-HER2 therapies. Citation Format: Alma Franceschini, Lorenzo T Castagnoli, Tiziana IINDT Triulzi, Paola Antonia Corsetto, Matteo Dugo, Antonino Belfiore, Andrea Vingiani, Lorena Signati, Serena Mazzucchelli, Fabio Corsi, Francesca Ligorio, Elda Tagliabue, Claudio Vernieri, Serenella M. Pupa. Carnitine palmitoyltransferase IA: an emerging potential metabolic target to counteract HER2-targeted therapy resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A039.

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.

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.

Background Breast cancer Patient Derived Organoids (PDO) have been demonstrated to be a reliable model to study cancer that promised to replace and reduce the use of animals in pre-clinical research. They displayed concordance with the tissue of origin, resuming its heterogenicity and representing a good platform to develop approaches of personalized medicines. Although obtain PDOs from mammary tumour, was a very challenging process, several ongoing studies evaluated them as a platform to study efficacy, sensitivity and specificity of new drugs and exploited them in personalized medicine. Despite tissue organization represented a crucial point to evaluate in a 3-dimensional model, since it could influence drug penetration, morphology of breast cancer PDOs has not been analysed yet. Here, we proposed a complete ultrastructural analysis of breast PDOs obtained from tumour and healthy tissues to evaluate how typical structures observed in mammary gland were resumed in this model. Methods 81 samples of mammary tissue (healthy or tumour) resulting from surgical resections have been processed to obtain PDO. The resulting PDOs embedded in matrigel drop have been processed for transmission electron microscopy and analysed. A comparison between ones from healthy and ones from cancerous tissue has been performed and PDOs derived from tumour tissue have been stratified according to their histological and molecular subtype. Result The morphological analysis performed on 81 PDO revealed an organized structure rich in Golgi, secretion granules and mitochondria, which was typical of cells with a strong secretory activity and active metabolism. The presence of desmosomes, inter and intracellular lumens and of microvilli and interdigitations signified a precise tissue-organization. Each PDO has been classified based on whether or not it possessed (i) peripheral ridges in mitochondria, (ii) intracellular lumens, (iii) intercellular lumens, (iv) micro-vesicles, (v) open desmosomes, (vi) cell debris, (vii) polylobed nuclei, (viii) lysosomes and (ix) secretion granules, in order to identify features coupled with the cancerous state or with a specific histological or molecular subtype. Conclusion Here we have demonstrated the suitability of breast cancer PDO as 3-dimensional model of mammary tissue. Besides, some structural features characterizing cancerous PDO have been observed, identifying the presence of distinctive traits.

BackgroundPatient-derived organoids (PDO) technology represents an emerging tool for the study of tumor biology and drug responsiveness, thus being useful to design personalized medicine approaches. Despite several studies and clinical trials are ongoing using PDO from colorectal and pancreatic cancer, only few research papers have been published exploiting PDO from breast cancer. Here, we have developed a new protocol to establish PDO from surgical and biopsy samples. Furthermore, we have set up also the methodologies adopted for culture and morphological evaluations.ResultsSurgical and core biopsy specimens collected from 33 patients with diagnosis of breast cancer have been processed using the protocols here described obtaining PDO from cancerous and healthy mammary tissue (when available) in a quick and easy way with good yields. The more critical aspects influencing the yield were the characteristic of the tissue of origin (healthy vs tumor tissue) and the amount of material obtained after enzymatic digestion process. Success rate from healthy samples was about 20,83%, while this percentage was higher in samples from cancer tissue (i.e. 87,5%). Also the morphological characterization of breast cancer PDO by brightfield and transmission electron microscopy has been reported.ConclusionsDespite obtaining some organoids from a surgical or biopsy specimen is not a difficult procedure, the establishment of a stable organoid line able to grow and replicate, suitable for long-term biobank storage, is not so obvious. A novel, simple and quick procedure to obtain PDO from surgical and biopsy samples is here proposed to achieve high success rate .