Af114

To detect the contribution of EVs toward the formation of metastatic niche in vivo, lung tissues were probed for the differential expression of fibronectin and CD45 in the C3H/HeJ or C57BL/6J (The Jackson Laboratory) mice receiving either PBS or EVs isolated from control or IGF2BP1-depleted SW1 or PVMM (syngeneic to C57BL/6J mice) cells, respectively. EVs were isolated from SW1 cells with either method of purification mentioned above. The mice were treated with 20 μg of EVs per mouse via tail-vein injection every alternate day for a period of 21 days. The mice were sacrificed one week after the last EV injection and their lungs were harvested. The 4% paraformaldehyde-fixed lung tissues were paraffin-embedded and sectioned, after which fluorescence immunohistochemistry was performed. In brief, the sections were deparaffinized, and antigen retrieval was done with 10 mM citrate buffer (pH 6). Following blocking, the lung tissue sections were incubated overnight with primary antibodies against fibronectin (Abcam #ab2413) or CD45 (Novus Biologicals #AF114). Fluorescently labeled secondary antibodies (Jackson Laboratories) were used in conjunction with nuclear dye DAPI to visualize the distribution of fibronectin and CD45 in the lung tissues. The in vivo experiment was repeated twice.