Fig. 8
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