Docetaxel-resistance in prostate cancer: evaluating associated phenotypic changes and potential for resistance transfer via exosomes

Abstract

Background: Hormone-refractory prostate cancer remains hindered by inevitable progression of resistance to first-line treatment with docetaxel. Recent studies suggest that phenotypic changes associated with cancer may be transferred from cell-to-cell via microvesicles/exosomes. Here we aimed to investigate phenotypic changes associated with docetaxel-resistance in order to help determine the complexity of this problem and to assess the relevance of secreted exosomes in prostate cancer.

Methodology/principal findings: Docetaxel-resistant variants of DU145 and 22Rv1 were established and characterised in terms of cross-resistance, morphology, proliferation, motility, invasion, anoikis, colony formation, exosomes secretion their and functional relevance. Preliminary analysis of exosomes from relevant serum specimens was also performed. Acquired docetaxel-resistance conferred cross-resistance to doxorubicin and induced alterations in motility, invasion, proliferation and anchorage-independent growth. Exosomes expelled from DU145 and 22Rv1 docetaxel-resistant variants (DU145RD and 22Rv1RD) conferred docetaxel-resistance to DU145, 22Rv1 and LNCap cells, which may be partly due to exosomal MDR-1/P-gp transfer. Exosomes from prostate cancer patients' sera induced increased cell proliferation and invasion, compared to exosomes from age-matched controls. Furthermore, exosomes from sera of patients undergoing a course of docetaxel treatment compared to matched exosomes from the same patients prior to commencing docetaxel treatment, when applied to both DU145 and 22Rv1 cells, showed a correlation between cellular response to docetaxel and patients' response to treatment with docetaxel.

Conclusions/significance: Our studies indicate the complex and multifaceted nature of docetaxel-resistance in prostate cancer. Furthermore, our in vitro observations and preliminary clinical studies indicate that exosomes may play an important role in prostate cancer, in cell-cell communication, and thus may offer potential as vehicles containing predictive biomarkers and new therapeutic targets.

Figure 1. Cell Morphology.

Images of sensitive parent and docetaxel-resistant variants of DU145 and 22Rv1 cell lines. (Olympus CKX4, 20X magnification).

Figure 2. Cell motility.

Wound-healing assays were performed to assess cell motility. Monolayers were scratched with a pipette tip and the resulting wounded areas were monitored by phase contrast microscopy for 24 hours (DU145) and 48 hours (22Rv1). Results are displayed as n = 3± SEM, where * = p<0.05 (Student’s t-test).

Figure 3. Cell migration and invasion. A:

Migration assays were performed using 8 µm pore size 24-well transwell chambers; B: For invasion assays the inserts were pre-coated with ECM. Cells were allowed to migrate/invade for 48 hours (DU145) and 72 hours (22Rv1). Results are displayed as n = 3± SEM, where * = p<0.05, ** =  p<0.01 (Student’s t-test).

Figure 4. Anchorage independent growth. A:

For anoikis assays, cell line variants were plated onto Poly (hydroxyethyl methactylic) acid-coated 24 well plates – or 95% ethanol-coated plates, as controls - and were cultured for 24 hours. 100 µl of Alamar blue dye was then added to each well, incubated for 4 hours and absorbance was measured at 570nm; B: Colony formation assays were performed using Cytoselect™ 96-Well Cell Transformation kit. Cells were incubated for 8 days in semisolid agar media before being lysed and detected with CyQuant GR Dye in a fluorescence plate reader. Results are displayed as n = 3± SEM, where * = p<0.05 (Student’s t-test).

Figure 5. Exosome characterisation and assessment of affects on cell motility and invasion.

A: Transmission Electron Microscopy was performed to investigate size and structure of exosomes; B: Western blotting was performed to assess the expression of common exosomes markers in 30 µg (TSG101) and 8 µg (PDC6I/Alix) exosomes isolated from DU145 and 22Rv1 cell line variants; C: DU145 wound-healing assays in the presence of exosomes (5 µg) from DU145 cell line variants; D (i): DU145 invasion assays in the presence of exosomes (15 µg) from DU145 cell line variants; D (ii): 22Rv1 invasion assays in the presence of exosomes (15 µg) from DU145 cell line variants.

Figure 6. Isolation of exosomes and assessment of affects on toxicity assays.

A-C (i): DU145, 22Rv1 and LNCap proliferation in the presence of exosomes (20 µg) from DU145 cell line variants; A–C (ii) Response of DU145, 22Rv1 and LNCap cells to docetaxel in the presence of exosomes (20 µg) from DU145 cell line variants; D–E (i): DU145 and LNCap proliferation in the presence of exosomes (20 µg) from 22Rv1 cell line variants; D–E (ii): Response of DU145 and LNCap cells to docetaxel in the presence of exosomes (20 µg) from 22Rv1 cell line variants; F: Western blotting was performed to assess the expression of MDR-1/P-gp in total cellular protein (50 µg) and corresponding exosomes (30 µg) of DU145 and 22Rv1 cell line variants. Results are displayed as n = 3± SEM, where * = p<0.05, ** =  p<0.01, *** = p<0.001 (Student’s t-test).

Figure 7. Isolation of exosomes from serum and assessment of affects on cells. A:

Western blotting was performed to assess the expression of common exosomes markers in 30 µg exosomes isolated from sera of docetaxel-naïve patients (patient #1–6) and age-matched healthy controls (control #1–6). B: DU145 invasion in the presence of exosomes from docetaxel-naïve patients and aged-matched healthy controls (25 µg), with representative invasion image displayed. C: 22Rv1 proliferation in the presence of exosomes from treatment-naïve patients and age-matched healthy controls (25 µg). Results are displayed as n = 6± SEM, where ** = p<0.01, *** = p<0.001 (Student’s t-test). D: Response, to docetaxel, of 22Rv1 and DU145 cells in the presence of serum-derived exosomes from patients with elevated PSA levels (n = 2; Patients A & B) and in the presence of exosomes from patients with decreased PSA levels (n = 6; Patients C–H).