Figure 2. CDEs increase proliferation of prostate cancer cells but significantly downregulate their mitochondrial function.

(A) Effect of CAFs-derived exosomes on viability of prostate cancer cells, 48h culture period (PC3) (n≥9). (B) Prostate cancer cells show reduced basal mitochondrial oxygen consumption rate (OCR) when cultured with range of concentrations of CDEs for 24 hr. Basal OCR is a measure of OXPHOS activity. The OCR was normalized with protein content inside cells. PC3 cells were cultured with patient-1 derived CAFs’ exosomes (n≥9). (C) Basal OCR was measured for PC3, DU145, 22RV1, E006AA prostate cancer cell lines cultured with patient derived CDEs and control conditions. Six patient-derived CAFs were used for exosomes isolation. (n≥9). (D) OCR of prostate cancer cells were measured after 24 hr culture with and without CDEs. Cytochalasin D (CytoD), an inhibitor of exosomes uptake through actin depolymerization, rescues reduced OCR in prostate cancer cells when cultured with CAFs exosomes. CytoD disturbs actin filament inside cells, thus inhibit phagocytosis. CytoD concentration of 1.5 μg/ml was used. (n≥5). (E) Maximal and reserve mitochondrial capacities were measured using FCCP and antimycin. Maximal OCR is maximal capacity of mitochondrial OCR. (n≥9). (F) Role of CAFs secreted exosomes in regulating mitochondrial membrane potential (MMP) of prostate cancer cells. MMP is an important indicator of mitochondrial functions. (n≥5). (G) Reduced OXPHOS genes expression in cancer cells cultured with exogenous CDES. (H) qPCR results show that mitochondrial OXPHOS genes of prostate cancer cells were downregulated when cultured with CDEs. (n=3). (I) Most abundant miRNAs targeting OXPHOS genes were abundant in CAFs exosomes. (n=4). (J) miRNAs in CAFs exosomes targeting specific OXPHOS genes. Nanostring was used to measure miRNA expression levels in stromal exosomes. (n=4). (K) OCR of PC3 were measured after transfection of targeted miRNAs together into cells. (n=5). miRNAs were transfected into cells according to the manufacturer’s protocol (Lipofectamine 2000 Transfection Reagent, Thermofisher). Cells were seeded in 6-well plate for 24 hr. Transfection was performed followed by incubation for 48 hr. Cells were then reseeded onto Seahorse plates for OCR measurements after the cells were attached. Data information: data in (A), (B), (C), (F), (H) are expressed as mean ± SD, data in (D), (E), (K) are expressed as mean ± SEM;*p<0.05, **p<0.01, ***p<0.001. Figure 2—figure supplement 1–2.

DOI: http://dx.doi.org/10.7554/eLife.10250.004

Figure 2—figure supplement 1. Specificity of CDEs in regulating mitochondrial respiration of cancer cells is demonstrated.

(A) PC3 secreted exosomes (100 μg/ml) were isolated according to the same procedure as used in Figure 2, and added to PC3 cells. OCR of the PC3 cells cultured with PC3 cells-derived exosomes was measured after 24 hr. (B) IMR90 secreted exosomes (100 μg/ml) were isolated and added to PC3 cells. OCR of the PC3 cells cultured with IMR-90-derived exosomes was measured after 24 hr. (C) Fresh CAF culture medium was incubated for 48 hr, and collected to perform the same steps as for isolating exosomes. Next, RPMI medium was used to dissolve any trace amount of chemicals which may be left from exosomes isolation to culture PC3 cells. OCR of the PC3 cells cultured with blank media-derived isolates was measured after 24 hr. No difference in OCR was observed between experimental and control conditions, while OCR of PC3 showed more than 50% decrease when cultured with CDEs. Data information: data in (A–C) are expressed as mean ± SD, *p<0.05, **p<0.01, ***p<0.001. (n=3).

DOI: http://dx.doi.org/10.7554/eLife.10250.005

Figure 2—figure supplement 2. Effects of synthetic liposomes in regulating PC3 metabolism and viability.

(A,B) Synthetic liposomes were uptaken by PC3 cells. (C,D) Synthetic liposomes did not enhance PC3 cells viability in complete medium or in deprivation condition. (E-G) Synthetic liposomes did not downregulate OCR of BxPC3, MiaPaCa-2, PC3 cells. (H-J) Synthetic liposomes did not upregulate ECAR of BxPC3, MiaPaCa-2, PC3 cells. Data information: data in (C–J) are expressed as mean ± SD, *p<0.05, **p<0.01, ***p<0.001. (n≥3).

DOI: http://dx.doi.org/10.7554/eLife.10250.006