Fig. 3: EVs derived from ITGBL1-overexpressing CRC cells induced the liver and lung pre-metastatic niche formation. | Nature Communications

Fig. 3: EVs derived from ITGBL1-overexpressing CRC cells induced the liver and lung pre-metastatic niche formation.

From: Primary tumors release ITGBL1-rich extracellular vesicles to promote distal metastatic tumor growth through fibroblast-niche formation

Fig. 3

a Biodistribution of cancer-derived EVs in the potential metastatic organs of naive mice. Fluorescence microscopy analysis of the PKH26-labeled EVs (red) in the liver, lung, brain, and bone marrow of mice injected with SW620-EVs. Normal colonic epithelial cells NCM-460-EVs were used as the controls. White arrows indicate EVs foci. For each group, three mice were used for analysis. Scale bar, 50 μm. b Interaction between cancer-derived EVs and resident cells. NCM-460-EVs were used as the controls. Immunofluorescence analysis of resident tissue-specific stomal cells in the lung and liver of mice after injection of PKH26-labeled EVs (red). The 6 μm O.C.T. (Optimum Cutting Temperature) tissue cryosections were stained with antibodies against α-SMA, S100A4, F4/80, and CD31. Secondary antibodies conjugated to Alexa Fluor 488 (green) were used for imaging. Nuclear staining was done with DAPI (40, 6-diamidino-2-phenylindole). Top two: representative images of immunofluorescence microscopy of NCM-460-EVs or SW620-EVs co-staining with α-SMA, S100A4, F4/80, and CD31 in liver tissue sections from mice. Bottom two: representative images of immunofluorescence microscopy of NCM-460-EVs or SW620-EVs co-staining with α-SMA, S100A4, F4/80, and CD31 in lung tissue sections from mice. For each group, three mice were used for analysis. Scale bar, 25 μm. c, d Indicated genes (IL-6, IL-8, IL-1β, α-SMA, TGF-β, and CXCL12) expressions in the lung fibroblasts and liver stellate cells of mice in b were detected by qRT-PCR analysis. e, f Representative immunofluorescence images of α-SMA and FN expression in arbitrary units (a.u.), F4/80+ cells, and Ly6G+ cells in the liver and lung of the mice educated with no EVs (control), NCM-460-EVs, SW480-pcDNA3.1-EVs, SW480-pcDNA3.1-ITGBL1-EVs, SW620-shRNA-NC-EVs, and SW620-shRNA-ITGBL1-EVs. For α-SMA and F4/80 detection, secondary antibodies conjugated to Alexa Fluor 594 (red) were used. For FN and Ly6G detection, secondary antibodies conjugated to Alexa Fluor 488 (green) were used. Nuclear staining was done with DAPI. Scale bar, 100 μm. Each experiment was performed at least in triplicate. Student’s t-test was used to analyze the data; *p < 0.05, **p < 0.01.

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