Journal: Breast Cancer Research : BCR

Article Title: Plasma extracellular vesicles reflect response and prognosis in patients with breast cancer undergoing neoadjuvant treatment

doi: 10.1186/s13058-025-02209-0

Figure Lengend Snippet: Study design and characterization of plasma-derived extracellular vesicles. A Schematic illustration of the study design and workflow. Fifty-nine patients with breast cancer were included in the study. Blood samples were collected at two time points: before NST initiation (PRE) and after completion of NST, prior to surgery (POST). Pathological response was evaluated post-surgery, and patients were classified as either achieving pathological complete response (pCR) or having residual disease (non-pCR). Patients were stratified into breast cancer subtypes (Luminal B-like (LUMB), HER2-positive, and triple-negative breast cancer (TNBC)), and clinical data were recorded. EVs were isolated from 1 mL of plasma using size exclusion chromatography (SEC) followed by ultrafiltration. Downstream EV characterization and analysis included transmission electron microscopy (TEM), western blotting, nanoparticle tracking analysis (NTA), Qubit protein quantification, and MACSPlex surface marker profiling. B TEM image showing vesicle-like structures with typical morphology and diameters below 200 nm. Smaller, bright particles were also observed, likely corresponding to co-isolated lipoproteins or other non-vesicular components. C Western blot analysis of EV preparations from three patients (PRE and POST). The EV markers CD9 and Flotillin were detected in the EV samples, while the cellular marker Calnexin was absent. The plasma protein ApoA1 was present in all EV preparations, indicating co-isolation of plasma components. Cell lysate was used as a control for CD9, Flotillin and Calnexin, while plasma was used for ApoA. Stain-free SDS PAGE gels used to verify equal loading for Western blot analysis are shown in Additional file

Article Snippet: Surface protein profiling of EVs was performed using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer’s overnight protocol for the MACSPlex Filter Plate with minor modifications.

Techniques: Clinical Proteomics, Derivative Assay, Isolation, Size-exclusion Chromatography, Transmission Assay, Electron Microscopy, Western Blot, Marker, Control, Staining, SDS Page

Journal: Breast Cancer Research : BCR

Article Title: Plasma extracellular vesicles reflect response and prognosis in patients with breast cancer undergoing neoadjuvant treatment

doi: 10.1186/s13058-025-02209-0

Figure Lengend Snippet: Pre-treatment EV surface marker profiles across breast cancer subtypes and treatment response groups. Surface marker expression in plasma-derived EVs collected before neoadjuvant systemic treatment (PRE), analyzed using the MACSPlex assay. Values represent CD9/CD63/CD81-normalized median fluorescence intensity (MFI, APC channel) for markers retained after threshold filtering (MFI > 0.5 in ≥ 50% of samples). A Marker expression grouped by breast cancer subtype: Luminal B (LUMB), HER2-positive (HER2 +), and triple-negative breast cancer (TNBC). B Marker expression grouped by treatment response: pathological complete response (pCR) vs. non-pCR. Note that all EV samples were collected before treatment, while response was evaluated after completion of NST. Bars represent mean values with standard deviation (SD). Statistical comparisons were performed using one-way ANOVA or unpaired t-tests, with multiple comparisons corrected using the Holm method. A p -value < 0.05 was considered statistically significant and is indicated by *; non-significant results are labeled as “ns”. Individual marker data are shown in Additional files (subtypes) and (treatment response)

Article Snippet: Surface protein profiling of EVs was performed using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer’s overnight protocol for the MACSPlex Filter Plate with minor modifications.

Techniques: Marker, Expressing, Clinical Proteomics, Derivative Assay, Fluorescence, Standard Deviation, Labeling

Journal: Breast Cancer Research : BCR

Article Title: Plasma extracellular vesicles reflect response and prognosis in patients with breast cancer undergoing neoadjuvant treatment

doi: 10.1186/s13058-025-02209-0

Figure Lengend Snippet: EV surface marker expression changes in response to neoadjuvant systemic treatment. Surface marker expression in plasma-derived EVs before and after neoadjuvant systemic treatment (NST), assessed using MACSPlex analysis. For each patient and marker, data are presented as the POST/PRE ratio of CD9/CD63/CD81-normalized APC signal, representing the individual fold change. A All patients combined. B Stratified by treatment response: pathological complete response (pCR) vs. non-pCR. Graphs show show box plots with median, interquartile range, and full range. All data are plotted on a log scale; values > 1 indicate increased expression post-treatment, and values < 1 indicate decreased expression. Statistical comparisons were performed using paired t-tests with Holm correction. Colored asterisks indicate significant within-group changes relative to baseline (PRE), red asterisks indicate significant upregulation post-treatment, and blue asterisks indicate significant downregulation post-treatment. *Significance level: * p < 0.05

Article Snippet: Surface protein profiling of EVs was performed using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer’s overnight protocol for the MACSPlex Filter Plate with minor modifications.

Techniques: Marker, Expressing, Clinical Proteomics, Derivative Assay