fig7

Figure 7. Effects of EV-PSMA^scFv-mediated ADAM9 silencing on sphere formation stemness-associated programs in prostate cancer. (A and B) Sphere formation assays in LNCaP (A) and CWR22Rv1 (B) cells following treatment with EV-PSMA^scFv loaded with siADAM9 variants (#1 and #2), non-targeting siRNA (siNC), or unloaded EVs. Representative images of tumor spheres are shown alongside quantitative analysis of sphere number and sphere size distribution. Scale bars: 500 μm; (C and D) RT-qPCR analysis of stemness-associated gene expression in LNCaP (C) and CWR22Rv1 (D) sphere cultures following treatment with siADAM9-loaded EV-PSMA^scFv. Expression levels of SOX2, OCT4, NANOG, BMI1, and ADAM9 were evaluated; (E and F) Immunoblot analysis of stemness-associated proteins in LNCaP (E) and CWR22Rv1 (F) sphere cultures following treatment with siADAM9-loaded EV-PSMA^scFv. Expression levels of SOX2, OCT4, NANOG, BMI1, ADAM9, and β-actin were analyzed. Relative protein expression levels normalized to β-actin are indicated below the blots. Results were obtained from three independent biological experiments (n = 3), each using an independently prepared EV batch, with analyses performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Šídák’s multiple-comparisons test for the indicated comparisons. ns; ^*P < 0.05; ^***P < 0.001. EVs: Extracellular vesicles; PSMA: prostate-specific membrane antigen; siADAM9: ADAM9-targeting small interfering RNA; siNC: non-targeting small interfering RNA control; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SOX2: SRY-box transcription factor 2; OCT4: octamer-binding transcription factor 4; NANOG: Nanog homeobox; BMI1: BMI1 proto-oncogene, polycomb ring finger; ADAM9: a disintegrin and metalloproteinase 9; SD: standard deviation; ANOVA: analysis of variance; ns: not significant; β-actin: beta-actin; EV-PSMA^scFv: extracellular vesicles displaying PSMA-targeting single-chain variable fragments.