Although prostatic cancer (PCa) is an extremely common cancer and a frequent cause of cancer deaths in Western males, most cases never metastasize. Currently, diagnostic tests such as serum prostate-specific antigen levels are not sensitive enough to determine whether or not a PCa is aggressive and likely to metastasize.
Cancer cells are known to influence the behavior and protein expression of the normal cells surrounding them.1 This might play a role in metastasis and aggressive invasion into normal tissue. There is strong evidence to suggest this influence involves exosomes,2 small membrane vesicles of around 100 nm in size released by cells into their surroundings and carrying potential signaling molecules such as RNA and proteins. In the case of prostatic cancer, these exosomes are excreted in urine, a relatively easy biosample to obtain non-invasively.
Bijnsdorp et al. (2013) used proteomics techniques to discover whether a more sensitive and accurate protein biomarker for PCa exists, looking in particular at exosomes released by PCa cells into the surrounding tissues and excreted in urine.3 Using cell culture, they also found that these exosomes influence cell behavior and identify the protein involved before showing its presence in urine samples from patients with metastatic PCa.
The researchers cultured two PCa cell lines, LNCaP (a hormone-dependent, less aggressive phenotype) and PC3 (hormone-independent, highly aggressive), and harvested exosomes secreted into the medium. Prior to proteomic analysis using an LTQ FT hybrid mass spectrometer (Thermo Scientific), they separated the proteins using polyacrylamide gel electrophoresis followed by in-gel trypsin digestion.
Referring to the human IPI database 3.59, the researchers identified 1,299 LNCaP and 1,398 PC3 exosome proteins. Of these proteins, 101 were more abundant in PC3 exosomes (p<0.05), with 57 being expressed by both PCa cell lines. The researchers used DAVID (Database for Annotation, Visualization and Integrated Discovery) functional analysis and referral to the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database, identifying six proteins involved in control of cell migration and invasion. From this group, they chose integrins ITGA3 and ITGB1, talin 1 (TLN1) and vinculin (VCL) for further investigation. Using Western ligand blotting, the researchers found that only the integrins, ITGA3 and ITGB1, were present in the PCa exosomes.
Bijnsdorp and co-workers then treated the non-cancerous prostatic cell line, prEC, with media conditioned (CM) by the two PCa cell lines, measuring the effects of specific blocking antibodies on migration and invasion. Antibodies against both integrins blocked invasion and migration induced by CM. However, only the anti-ITGA3 antibody blocked invasion induced when harvested PCa exosomes were added to the culture. Exosome uptake by the prEC cells was confirmed using FACS analysis, showing that PC3 exosomes were internalized much more than the LNCaP exosomes.
As a final step, the researchers isolated urinary exosomes from patients with benign hyperplasia (n=5), PCa (n=5) and metastatic PCa (n=3). Using Western ligand blotting to immunologically identify the proteins, they found that levels of both ITGA3 and ITGB1 were higher in patients with metastatic disease than in those with non-metastasized PCa or benign hyperplasia (p<0.05). The authors suggest that these two proteins may offer important information to clinicians as adjuncts to traditional diagnostic tests, presenting a prognostically useful, non-invasive marker for PCa metastasis in patient assessment.
References
1. Bijnsdorp, I.V., et al. (2012) “A predictive role for noncancerous prostate cells: Low connexin-26 expression in radical prostatectomy tissues predicts metastasis,” British Journal of Cancer, 107 (pp.1963–8).
2. Peinado, H., et al. (2012) “Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET,” Nature Medicine, 18 (pp. 883–91).
3. Bijnsdorp, I.V., et al. (2013) “Exosomal ITGA3 interferes with non-cancerous prostate cell functions and is increased in urine exosomes of metastatic prostate cancer patients,” Journal of Extracellular Vesicles, 2 (pp. 22097), doi: 10.3402/jev.v2i0.22097.
Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer.
A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.
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