Figure 4: Invasive microvesicles can be isolated from peripheral bodily fluids of patients with diagnosed abdomino-pelvic mass.
From: Regulated delivery of molecular cargo to invasive tumour-derived microvesicles
(a) Unfractionated ascites or isolated TMVs were resuspended in sterile, filtered 1 × PBS and subjected to nanoparticle tracking analysis, using a NanoSight LM10 as per the manufacturer’s protocol. Measurements of concentration (upper panel) and percentage undersized (lower panel) versus particle diameter (nm) shown represent the mean of ten individual acquisitions for each sample type. The curves presented are representative of the patient population studied. (b) Microvesicles from patients with Stage IIB serous adenocarcinoma were fixed and examined by whole-mount transmission electron microscopy as described in Methods. Scale bar, 500 nm. (c) Equal amounts of unfractionated ascites fluid and isolated microvesicles (determined using BCA assay) from patient samples were probed by western blotting as indicated.It is worth noting that the protein is equal within but not between patients. Data shown are from patients later diagnosed with serous cystadenocarcinoma of the ovary (28), high-grade serous ovarian carcinoma (32) and poorly differentiated ovarian carcinoma (33). Unfractionated fluid in parallel with isolated TMVs were resuspended in sterile, filtered 1 × PBS (d) or 1 × PBS +NSC405020 (e) before mixing with TMV-free complete cell culture media. The mixture was overlaid onto FITC-gelatin-coated coverslips and allowed to degrade matrix for a period of 14 h. TMVs were fixed, stained as indicated and subjected to confocal microscopy, to examine levels of matrix degradation. Scale bar, 50 μm. (f) Equal amounts of protein from unfractionated serum and isolated TMVs (determined using BCA assay) were probed by western blotting as indicated. In parallel, microvesicles were mixed with 1 × PBS before addition to TMV-free complete cell culture media and incubation with FITC-conjugated gelatin-coated coverslips for a period of 14 h. Scale bar, 10 μm. Data shown are from a patient later diagnosed with serous ovarian carcinoma.
