3 well slides

The FIT-phalloidin-based F-actin red fluorescence (Abcam, Cambridge, UK) staining was done on HUVEC cells alone or HUVEC co-cultured with D283 and D425 cells on 3-well slides (ibidi, Fitchburg, WI, USA). The cells were grown for 24 h, fixed and stained according to the vendor’s instructions. Calcein AM green fluorescence (Invitrogen, Carlsbad, CA, USA) was used to stain D283 and D425 cells in the co-culture experiment. The images were captured with an Olympus BX61 Fluoview confocal microscope (Olympus, Center Valley, PA, USA) at 40× magnification. The immunostaining detection of B7-H3 protein on the MB tissue sections and cells was done following a standard protocol [21 (link)].

The FIT-phalloidin-based F-actin red fluorescence (Abcam, Cambridge, UK) staining was done on D283, D425, and D458 cells on 3-well slides (ibidi, Fitchburg, WI, USA) to label cell membrane-associated cytoskeletal elements. The cells were grown for 24 h, fixed and stained according to the vendor’s instructions. Calcein AM (2 µM) green fluorescence (Invitrogen) was used to stain D283 cell-derived B7-H3 OE exosomes. Isolated exosomes were diluted in PBS and coincubated with Calcein AM (0.5 µL of Calcein/15 µg of exosomes) at room temperature for 30 min. The Calcein AM stained exosomes (5 µg) were then incubated for 6 h on to the MB cells containing 1 mL of the complete media. The cells were fixed and stained with F-actin. The images were captured with an Olympus BX61 Fluoview confocal microscope (Olympus, Center Valley, PA, USA) at 40× magnification. The negative control to confirm nonspecific Calcein AM staining was done using SFM (processed in the same way as exosome extraction) overlaid on D458 cells followed by F-actin staining

To generate conditioned media for migration assays, cells were cultured in growth medium without FBS. After two days, the medium was aspirated, centrifuged at 1000× g, and stored in Protein LowBind^® tubes (Eppendorf, Hamburg, Germany) at −20 °C until use. For migration assays, 3.5 × 10⁵ cells/mL were cultured in removable 2-well culture inserts inside 3-well slides (both Ibidi, Gräfeling, Germany). After an adherence period of 24 h in the growth medium, cells were washed twice with serum-free medium and then cultured for 24 h in serum-free medium for FBS starvation. The 2-well chambers were removed and then washed with serum-free medium, and the experimental conditions were applied. For migration assays using MCF-7, 6 × 10⁵ cells/mL were seeded per chamber. After an adherence period of 24 h in growth medium, cells were washed twice with serum-free medium, and conditions were applied. CCR1-antagonist BX471 (Tocris Bioscience, Bristol, UK) was used at a concentration of 30 µM, while CCR5 antagonist TAK-779 (Merck, Darmstadt, Germany) was used at a concentration of 1 µM [30 (link)]. Gap closure was documented after 0 h and 48 h and the percentage of area covered was assessed using FastTrackAI (MetaVi Labs, Austin, TX, USA).