Fig 2: Challenge of SKBR3-based 3D models of the immune microenvironment of HER2-OE BC with anti-HER2 antibodies induced cancer cell apoptosis and downmodulation of CD16 in NK cells. (A) Viability assessment with the fluorescent probes Mitoview (live cells, red) and Nucview (apoptotic cells, green) revealed an increase in apoptosis in SKBR3 aggregates upon 4 days of single (trastuzumab, Tmab) or dual (trastuzumab plus pertuzumab, Tmab + Pmab) anti- epidermal growth factor receptor 2 (HER2) antibody challenge. Co-cultures were supplemented with IL-15. Representative pictures depict one of 11 independent experiments, performed with different immune donors. Scale bars: 150 µm. Full panel for 2 timepoints and all culture conditions in Supplementary Figure 3 . (B) Percentage of NK cells (CD3−CD56+) and CD16+ NK cells (CD3−CD56+CD16+) within the viable immune cell population (CD45+ DAPI−), detected by flow cytometry. NK cells representativeness was not impacted by the 4-day single and dual anti-HER2 antibody challenges but the CD16+ NK subset was partially depleted. Co-cultures were supplemented with IL-15. (C) Percentage of the two major NK cell subsets (CD3−CD56dimCD16+ and CD3−CD56+CD16–) in the viable immune cell population (CD45+DAPI−), detected by flow cytometry. Both NK cell subsets were modulated upon the 4-day single and dual anti-HER2 antibody challenges. Co-cultures were supplemented with IL-15. (B, C) Cell populations were identified and quantified according to gating strategy supplied in Supplementary Figure 2A (Panel 2). Bars represent mean ± S.D. of N = 11 independent experiments, performed with different immune donors. Pairwise statistical comparisons, relative to co-cultures with IL-15 and without antibody challenge, evaluated by a paired t-test: #, p<0.05, ##, p<0.01, ###, p<0.001.

Fig 3: Sc-RNA sequencing analysis reveals cell type composition and key alterations in MDD. (A, B) UMAP visualization of single-cell clusters (A) before and (B) after cell type annotation; (C) Dot plot of canonical marker gene expression used for cell type identification. Dot size represents the percentage of cells within a cluster expressing the indicated gene, and color intensity reflects the average expression level; (D) Bar graph comparing the proportional abundance of a key cell subtype between Control and MDD groups. Statistical significance (*p < 0.05). (E) Based on the expression of CD16 (FCGR3A) and CD56 (NCAM1), NK cells were classified into two subsets, CD16+NK cells and CD56+NK cells. (F) The proportions of CD3-CD56+ and CD3-CD16+ cells were analyzed by flow cytometry and visualized through scatter plots. (G) The proportions of CD3-CD56+ cells and CD3-CD16+ cells in the whole blood of normal individuals and MDD patients were presented in a bar chart. Normal group vs. MDD group, **P<0.01, **P<0.001.

Fig 4: pEVs differentially shape monocyte adhesion markers and subset distribution according to fetal sex. Monocytes from non-pregnant donors were cultured o.n. in basal conditions (-) or with M-pEVs or F-pEVs and analyzed by flow cytometry for CD11c (A), CD11b (B), and CD16 expression and subset distribution (C). The percentage (%) of double-positive cells and mean fluorescence intensity (M.F.I.) were evaluated. Representative histograms and dot plots of live cells are shown. Results are presented as mean ± SEM; each point represents an independents donor, and pEV conditions correspond to the mean of ≥ 2 independent pEV preparations. Gray: M-pEVs; Orange: F-pEVs. Statistical analyses were performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. Adjusted P values derived from Tukey’s multiple comparisons test were used to control the family-wise error rate (FWER). *P < 0.05 was considered statistically significant.