Fig 1: Lactadherin blockade inhibits sEV-MDA231-mediated ascites formation and mesenteric/peritoneal tumor micronodules formation in an in vivo peritoneal carcinomatosis murine model. NOD/SCID mice (two separated experiments) were inoculated intraperitoneally (ip) with 2.0 × 106 MDA-MB-231, either alone (saline group) or together with sEV-MDA231, anti-lactadherin antibody or sEV-MDA231 + anti-lactadherin antibody. Seven days after tumor cells inoculation, mice were treated again on days 7, 9, 11, 15, 17 and 19 (7 total doses). Mice were euthanized on day 21 post-cell injection and organs were collected in the necropsy (A). When present, malignant ascites were also collected and total cell number was determined by trypan blue exclusion assay (B-C). For tumor micronodules measurement, tumor tissues were fixed and paraffin-embedded. Histologic sections of mesenteric tissue were first stained with HE. Images of HE-stained sections were gray scale-transformed to define a hyperdense area. Tumor micronodules number in mesenteric tissues of the peritoneal cavity were evaluated by purple hyperchromia. Tumor nodules and tumor area were also measured by gray range density/intensity, which coincides with the denser nuclear staining of tumor cells (D-E), and the quantified. Analyses were made using ImageJ software and corroborated using mathworks software (https://es.mathworks.com). E Tumor nodules number (upper graph) and area (lower graph) are shown. The final in vivo evaluation was performed in a double-blinded fashion and corroborated by anatomopathological analysis
Fig 2: Lactadherin is differentially expressed in human BC cell lines. A Lactadherin (MFGE8) mRNA expression was measured in different human BC cell lines and in mammary epithelial breast cells through RT-qPCR. Lactadherin (MFGE8) mRNA expression was normalized against GAPDH; B Lactadherin protein was detected through WB analysis. Data was normalized against GAPDH detection for quantification; C Confocal microscopy evaluation of lactadherin in different BC cell lines. 120X, z-stack augmented images are shown. Scale bar represents 20 µm; D-F Lactadherin detection in BC cells by flow cytometry. Cell surface D and intracellular E, F histograms and quantifications are shown. The gating strategy is depicted in Additional file 1: Figure S2. mRNA A and protein B levels are shown as mean ± SEM. Images and data are representative of 3–4 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001
Fig 3: Lactadherin levels are associated with more aggressive BC subtypes. Analysis of TCGA data show that MFGE8 expression is higher in ER- and PR- BC patients and in more aggressive basal and normal-like PAM50 subtypes. A-D BC patients’ TCGA transcriptomic data were obtained from UCSC Xena database and MFGE8 expression were associated with ER, PR and HER2 histologic status. E–F Transcriptomic and proteomic data from another cohort of BC patients were downloaded directly from TCGA and association analyses of lactadherin levels with tumor stage E and PAM50 subtypes were performed F. G-I TCGA transcriptomic data from BC cell lines were obtained from UCSC Xena database and MFGE8 expression were associated with ER, PR and HER2 histologic status. Mann–Whitney test statistical analysis was performed A-C; Kruskall Wallis test and Dunn’s multiple comparisons correction were performed D; Group comparisons between stages I-II vs III-IV were carried out using Wilcoxon Rank Sum Test (Mann–Whitney U-test) E; Comparisons between PAM50 tumor subtypes were performed with Kruskal–Wallis test, followed by pairwise comparisons using Wilcoxon Rank Sum Test. P-value adjustment was performed using Holm’s method F; Kruskal–Wallis test statistical analysis was performed G-I
Fig 4: MFGE8 expression is associated with worse survival in BC advanced stages and more aggressive PAM50 subtypes. A Harmonized survival data from Liu et al. [59] was used to perform univariate survival analysis between advanced (stages III-IV) and early (stages I-II) BC patients, using progression-free interval as primary outcome, as recommended by TCGA. B Sub-analysis between advanced and early BC by PAM50 subtype was also performed. Samples were labeled as MFGE8-high and low using a median cutoff value. All survival analyses were conducted using RNA-seq UQ-FPKM values, through implementation of the survival and survminer R packages
Supplier Page from Abcam for Human MFGE8 ELISA Kit (Lactadherin)