Melatonin is a molecule with different antitumor actions in breast cancer and has been described as an inhibitor of vascular endothelial growth factor (VEGF). Despite the recognition of the key role exerted by VEGF in tumor angiogenesis, limitations arise when developing models to test new antiangiogenic molecules. Thus, the aim of this study was to develop rapid, economic, high capacity and easy handling angiogenesis assays to test the antiangiogenic effects of melatonin and demonstrate its most effective dose to neutralize and interfere with the angiogenic sprouting effect induced by VEGF and MCF-7. To perform this, 3D endothelial cell (HUVEC) spheroids and a chicken embryo chorioallantoic membrane (CAM) assay were used. The results showed that VEGF and MCF-7 were able to stimulate the sprouting of the new vessels in 3D endothelial spheroids and the CAM assay, and that melatonin had an inhibitory effect on angiogenesis. Specifically, as the 1 mM pharmacological dose was the only effective dose able to inhibit the formation of ramifications around the alginate in the CAM assay model, this inhibition was shown to occur in a dose-dependent manner. Taken together, these techniques represent novel tools for the development of antiangiogenic molecules such as melatonin, with possible implications for the therapy of breast cancer.

Breast cancer (BC) continues to pose a significant burden on global cancer-related morbidity and mortality, primarily driven by metastasis. However, the combined influence of microRNAs (miRNAs) and intratumoral microbiota on BC metastasis remains largely unexplored. In this study, we aimed to elucidate the interplay between intratumoral microbiota composition, miRNA expression profiles, and their collective influence on metastasis development in BC patients by employing 16S rRNA sequencing and qPCR methodologies. Our findings revealed an increase in the expression of miR-149-5p, miR-20b-5p, and miR-342-5p in metastatic breast cancer (Met-BC) patients. The Met-BC patients exhibited heightened microbial richness and diversity, primarily attributed to diverse pathogenic bacteria. Taxonomic analysis identified several pathogenic and pro-inflammatory species enriched in Met-BC, contrasting with non-metastatic breast cancer (NonMet-BC) patients, which displayed an enrichment in potential probiotic and anti-inflammatory species. Notably, we identified and verified a baseline prognostic signature for metastasis in BC patients, with its clinical relevance further validated by its impact on overall survival. In conclusion, the observed disparities in miRNA expression and species-level bacterial abundance suggest their involvement in BC progression. The development of a prognostic signature holds promise for metastasis risk assessment, paving the way for personalized interventions and improved clinical outcomes in BC patients.

Emerging evidence suggests a bidirectional relationship between gut microbiota, melatonin synthesis, and breast cancer (BC) development in hormone receptor-positive patients (HR+HER2+ and HR+HER2-). This study investigated alterations in gut microbiota composition, the serum serotonin–N-acetylserotonin (NAS)–melatonin axis, fecal short-chain fatty acids (SCFAs) and beta-glucuronidase (βGD) activity, and serum zonulin in HR+ BC patients compared to healthy controls. Blood and fecal samples were analyzed using mass spectrometry for serotonin, NAS, melatonin, and SCFAs; ELISA for AANAT, ASMT, 14-3-3 protein, and zonulin; fluorometric assay for βGD activity; and 16S rRNA sequencing for gut microbiota composition. HR+ BC patients exhibited gut dysbiosis with reduced Bifidobacterium longum and increased Bacteroides eggerthii, alongside elevated fecal βGD activity, SCFA levels (e.g., isovaleric acid), and serum zonulin, indicating increased intestinal permeability. Serum serotonin and N-acetylserotonin (NAS) levels were elevated, while melatonin levels were reduced, with a higher NAS/melatonin ratio in BC patients. AANAT levels were increased, and ASMT levels were decreased, suggesting disrupted melatonin synthesis. Bifidobacterium longum positively correlated with melatonin and negatively with βGD activity, while Bacteroides eggerthii showed a positive correlation with βGD activity. These findings suggested that gut microbiota alterations, disrupted melatonin synthesis, microbial metabolism, and intestinal permeability may contribute to BC pathophysiology. The NAS/melatonin ratio could represent a potential biomarker, necessitating further mechanistic studies to confirm causality and explore therapeutic interventions.

Background This study aimed to investigate the intricate relationship between the gut microbiota and serum melatonin levels in hormonal receptor-positive breast cancer (BC) patients, focusing on alterations in microbial composition, the melatonergic pathway, and their implications for BC development. Methods Serum and fecal samples were obtained from hormonal receptor-positive BC patients and healthy controls. Mass spectrometry was used to measure the serum levels of serotonin, N-acetylserotonin (NAS), and melatonin and the fecal levels of short-chain fatty acids (SCFAs). Beta-glucuronidase (βGD) activity was quantified using a fluorometric assay kit, while arylalkylamine N-acetyltransferase (AANAT), acetylserotonin-O-methyltransferase (ASMT), and zonulin were assessed via ELISA. The gut microbiota composition was evaluated using 16S rRNA sequencing. Results We identified significant alterations in the gut microbiota composition and melatonin production of BC patients compared to healthy controls. This dysbiosis is characterized by heightened serum serotonin, N-acetylserotonin (NAS), and fecal β-glucuronidase (βGD) activity, concomitant with diminished serum melatonin levels in BC patients. Moreover, increased fecal levels of isovaleric acid (IVA) and isobutyric acid (IBA), coupled with increased serum zonulin levels, highlight intestinal permeability alterations that could facilitate the translocation of gut bacteria and inflammatory compounds, predisposing individuals to cancer development. Notably, we observed reduced gut microbiota diversity and significant shifts in predominant bacterial taxa, withBacteroides eggerthiienrichment and a reduction in beneficialBifidobacterium longumpositively associated with serum melatonin levels, suggesting potential roles in BC development. Dysregulation of the serotonin-NAS-melatonin axis, along with perturbed expression of enzymes involved in the melatonergic pathway, underscores their implications in BC. Finally, we propose the NAS/melatonin ratio as a potential diagnostic biomarker for discriminating hormonal receptor-positive BC patients from healthy individuals, offering promising avenues for clinical management strategies. Conclusions Overall, our findings shed valuable light on the contributions of the gut microbiota and the melatonergic pathway to the development of hormonal receptor-positive BC, warranting further research into potential therapeutic targets.

Breast cancer-related lymphedema (BCRL) is closely associated with obesity, yet the gut microbiota remains poorly characterized in this population. We longitudinally evaluated gut microbiota profiles in 48 women with overweight or obesity and BCRL who were randomized to either a multimodal lifestyle intervention combining an energy-restricted Mediterranean-style diet and exercise (n = 28) or control care (n = 20). Fecal samples collected at baseline, 3 months, and 6 months (144 samples) underwent 16S rRNA gene sequencing. The intervention resulted in greater reductions in body weight, body mass index, and fat mass over time (group × time p < 0.001, p = 0.001, and p = 0.002, respectively). No significant group × time interaction was observed for alpha diversity or overall microbial community composition based on Bray–Curtis dissimilarity (PERMANOVA R² = 0.006, F = 0.459, p = 0.324). Exploratory DESeq2 differential abundance analyses identified FDR-significant taxon-specific differences, including higher abundances of Barnesiellaceae and Desulfovibrionaceae in the intervention group and of Prevotella_7 and Lachnospiraceae_UCG_010 in controls (FDR-adjusted p < 0.05). These DESeq2 findings were not confirmed in mixed-effects sensitivity analyses after FDR correction. Prevotella_7 was more abundant in controls at baseline and at 6 months. Participant-blocked group × time models, which were given inferential priority, yielded FDR-significant baseline-to-3-month interaction tests for Izemoplasmatales and Frisingicoccus, although the corresponding shrunken effect estimates were close to zero. Secondary, hypothesis-generating analyses based on supplementation status, supplement adherence, and clinically relevant weight-loss response identified additional taxon-specific patterns associated with supplementation and adherence, including a tentative species-level association involving Bifidobacterium bifidum among supplemented participants, whereas no significant microbiota differences were observed by weight-loss response. Overall, these findings provide no evidence of broad restructuring of the gut microbial community but identify exploratory taxon-specific signals that warrant confirmation in larger longitudinal studies using strain-resolved and functional approaches.

Background: Obesity is a major modifiable risk factor for endometrial cancer (EC), yet whether body mass index (BMI) shapes the tumour-resident microbiota and its relationship with host tumour transcription remains unclear. We investigated whether EC tumours from lean and overweight/obese postmenopausal women harbour distinct microbial and molecular profiles.Methods: We conducted a cross-sectional observational study of 53 postmenopausal women with histologically confirmed EC. Formalin-fixed paraffin-embedded tumours were classified as Lean (BMI ≤25 kg/m²; n=33) or Overweight/Obese (BMI >25 kg/m²; n=20). Intratumoral microbiota was profiled by 16S rRNA sequencing, differential abundance was assessed using edgeR and centred log-ratio (CLR) models, RNA sequencing was performed in 14 tumours for paired microbiome–transcriptome integration, and microbial functional capacity was inferred with PICRUSt2.Findings: Ow_Ob tumours displayed distinct microbial community composition (Bray–Curtis PERMANOVA, p=0.0001), characterised by enrichment of Propionibacterium acnes and depletion of Ruminococcus bromii, consistently identified by both analytical approaches. These microbial differences were accompanied by 987 BMI-associated genes, with enrichment of immune pathways among upregulated genes and mitochondrial and cell-cycle pathways among downregulated genes. Paired analyses linked microbial abundance with BMI-adjusted host gene expression and identified BMI-associated inferred microbial functions.Interpretation: BMI is associated with distinct intratumoral microbial features in EC, including a reproducible species-level signature linked to immune and metabolic transcriptional programmes, supporting tumour-associated microbiota as a potential molecular interface between obesity and EC biology.