Figure 5.

Tspan8 supports metastases and mediates a significant increase in EV number in the circulation in vivo. (A) Orthotopic injection of rats was performed using 1 × 10⁶ MTPa or MTPa‐Tspan8 cells per animal; cells were injected into the mammary fat pad (five animals per group). At day 18, animals were sacrificed and organs isolated. Representative images of primary tumours, liver, and spleen are shown. Pictures were taken and metastases counted using ImageJ. (B) Immunofluorescence was performed on frozen sections of primary tumours and liver and spleen specimens from both the animals harbouring MTPa‐ and those harbouring MTPa‐Tspan8 tumours. (C) EVs were collected from rat blood and measured by NTA. Significantly higher numbers of EV were detected in the blood of rats injected with MTPa‐Tspan8 cells. (D) Transmission electron microscopy of EV preparations revealed vesicular structures typical for EVs isolated from blood. (E) EVs were lysed and equal amounts of proteins were loaded for WB analysis and tested with CD9, Alix, and Tspan8 antibodies. No differences in CD9 and Alix protein amounts were observed in control animals and in animals harbouring MTPa or MTPa‐Tspan8 tumours. (F) Flow cytometry analysis of EVs. Tspan8, CD9, and CD81 were tested and the percentage of positive EVs and the MFI value were counted. Strong differences in the MFI values between the origin of EVs from control animals (green line) and the origin of EVs from MTPa (blue line) and MTPs‐Tspan8 (red line) animals were noticed, showing a strong increase in the MFI of Tspan8 and CD81 in EVs derived from MTPa‐Tspan8 animals. Flow cytometry was performed twice and representative images are shown.