Figure 4a, Comparison of the pre-treatment levels of USP8 mRNA in patient-derived tissue samples by qRT-PCR (left panel), enzyme activity of USP8 in patient-derived tissue samples by Ub-VME (middle panel) and TβRII+ crEVs level in the plasma from breast cancer patients by ELISA (right panel) between breast cancer patients with or without clinical response to PTX. R, responders; n = 16; NR, non-responders; n = 14.b, Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient-derived tissue samples with the EV-TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).c, Objective response rate (ORR) for patients as in a (n = 30) with high (>0.95 ng/ml) and low (<0.95 ng/ml) pre-treatment levels of circulating EV-TβRII.d, Pearson correlation of the percentage of IFN-γ+ of CD8+ cells to the TβRII+ crEV level in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).e, TβRII+ crEVs (left panel), the USP8 enzyme activity (midde panel) and USP8 mRNA (right panel) from breast cancer patients with or without clinical response to combined therapy using anti-PD-L1 (Atezolizumab) and paclitaxel. R, responders; n = 10 (left panel), n = 5 (middle and right panels, only 5 patient-derived tissue samples obtained). NR, non-responders; n = 10 (left panel), n = 6 (middle and right panels, only 5 patient-derived tissue samples obtained).Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. Bioluminescent imaging (BLI) of representative mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). BLI images are shown (f (left panel)). BLI signal of each mice (f (middle panel)) and number of bone metastasis (f (right panel)) of every mouseExperimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). the percentage of bone metastasis free mice survival in each experimental group followed in time (g).Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). Fold change of circulating EV-TβRII by ELISA analysis in plasma samples of mice in each experimental group at week 6 (h).nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Normalized BLI signals (left panel) and representative mice (right panel) from each group at the indicated times (j).nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Immunoblot analysis of the circulating EVs from plasma in mice (k).nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times (l).