Fig. 2.

In vivo visualization and characterization of EVs from cancer tissue in rat mammary tumors. (A) In vivo imaging of control rats and tumor-bearing rats by label-free multiphoton microscopy. (B) FAD/[FAD+NAD(P)H] values of EVs from control (n = 5 animals) and tumor-bearing rats (n = 5 animals). (C) Scatter plot of individual EVs with intensities from the 3 channels. (D) Combined histogram of vesicle optical signatures from cancer and normal tissue. The blue dashed line indicates the computed threshold value (0.65) for identifying the subpopulation of NAD(P)H-rich EVs by using Ostu’s method. (E) Concentration and (F) percentage of NAD(P)H-rich EVs in relation to their cancer status (2-tailed Student’s t test, ***P < 0.001, and ****P < 0.0001; 26 imaging sites from the control group and 82 imaging sites from the cancer group). (G) Receiver operating characteristic (ROC) curve of cancer prediction by logistic regression using different features. The feature of EV density yields an area under the ROC curve (AUC) of 0.786, the NAD(P)H-rich EV density yields an AUC of 0.849, and the percentage of NAD(P)H-rich EVs yields an AUC of 0.868 (Scale bar: 100 µm.).