Protein Complexes in Urine Interfere with Extracellular Vesicle Biomarker Studies

Urine exosomes (extracellular vesicles; EVs) contain (micro)RNA (miRNA) and protein biomarkers that are useful for the non-invasive diagnosis of various urological diseases. However, the urinary Tamm-Horsfall protein (THP) complex, which forms at reduced temperatures, may affect EV isolation and may also lead to contamination by other molecules including microRNAs (miRNAs). Therefore, we compared the levels of three miRNAs within the purified EV fraction and THP- protein-network. Urine was collected from healthy donors and EVs were isolated by ultracentrifugation (UC), two commercial kits or sepharose size-exclusion chromatography (SEC). SEC enables the separation of EVs from protein-complexes in urine. After UC, the isolation of EV-miRNA was compared with two commercial kits. The EV isolation efficiency was evaluated by measuring the EV protein markers, Alix and TSG101, CD63 by Western blotting, or miR-375, miR-204 and miR-21 by RT-qPCR. By using commercial kits, EV isolation resulted in either low yields or dissimilar miRNA levels. Via SEC, the EVs were separated from the protein-complex fraction. Importantly, a different ratio was observed between the three miRNAs in the protein fraction compared to the EV fraction. Thus, protein-complexes within urine may influence EV-biomarker studies. Therefore, the characterization of the isolated EV fraction is important to obtain reproducible results

Models for cancer skeletal metastasis: A reappraisal of Batson's plexus

While skeletal metastasis in prostate cancer is a major and frequent complication, literature data on the mechanisms involved are quite confusing. Recent efforts, however, to establish appropriate animal models for skeletal metastasis have finally yielded positive results which may provide clarity in this discussion. Models involving both human prostate cancer cell transplantation in nude mice as well as syngeneic rat models have contributed to the accumulated evidence in favor of the hypothesis of Batson. According to this hypothesis, prostate tumor cells reach the vertebral venous plexus of the spine especially under transient conditions of increased intra-abdominal pressure and lead to metastatic tumor deposits in the vertebrae. Animal models displaying skeletal metastasis in conjunction with analysis of pathological findings have been instrumental in validating this concept. It is to be expected that such animal models will contribute to the development and optimization of new treatment approaches for prostate cancer bone metastasis

Radiosensitizing effect of cisplatin in prostate cancer cell lines

The radiosensitizing effect of platinum compounds has been demonstrated in a number of tumors. In prostate cancer, clinical and preclinical data concerning an eventual efficacy of the concept of radiosensitization are lacking. In the present study cisplatin and carboplatin have been used as a model to explore radiosensitization in in vitro prostate cancer cell lines. Human (DU-145) and rat (R3327-MATLyLu) prostate tumor cells were irradiated with doses ranging from 0 to 8 Gy in the presence of various concentrations of either cisplatin or carboplatin. For the evaluation of the combined effect of the two treatment modalities, a simple model is presented. Supra-additive treatment effects of combinations of platinum drugs with radiotherapy, both at clinically achievable doses, were shown on the basis of surviving fractions of tumor cells and proved to be significant. These data strongly suggest that radiotherapy may be effectively combined with radiosensitizers such as platinum drugs in prostate cancer therapy, to yield synergism in treatment efficacy

Abstract LB-14: Low connexin-26 predicts the development of metastasis after radical prostatectomy

Abstract Prostate cancer is the second most common cause of death in males. Not all tumors will become metastatic within a patients life time, but when metastasized no curative therapy is available. It is therefore important to identify molecules that predict whether the tumor will become metastatic. Connexin-26 (Cx26) is a gap-junction protein that is involved in cell-cell contacts and in cell adhesion. Cx26 is involved as a tumor suppressor in various tumor types. The aim of the present study was to determine Cx26 expression in localized prostate cancer and relate the expression to the development of metastasis. Cx26 was stained by immunohistochemical staining in radical prostatectomy tissues of 43 patients. The expression levels were scored by a pathologist. Cx26 expression was observed in 95% of the patients. No relation was found to Cx26 expression in the tumor and the development of metastasis (p=0.2). Interestingly, a clear relation was found between low Cx26 expression in benign epithelial cells within the same sections and the development of metastasis (p&amp;lt;0.03). These metastases developed in about 5–40 months after radical prostatectomy. Kaplan Meier analysis showed a relation between a low Cx26 (score &amp;lt;75) expression in benign epithelial cells and time to PSA progression (p=0.01). Time to progression was associated with PSA and Cx26 expression in benign epithelial cells (Multivariate Cox proportional hazards analysis, p=0.05). In conclusion, Cx26 expression in the benign epithelial cells was associated with the development of metastasis. Cx26 expression could therefore be used as a predictor of outcome after radical prostatectomy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-14. doi:10.1158/1538-7445.AM2011-LB-14</jats:p

Amifostine protects against chemotherapy-induced neurotoxicity: An in vitro investigation

Background: Peripheral neurotoxicity is a dose-limiting side-effect of a number of effective chemotherapeutic agents. Neuroprotective agents may help to reduce neurotoxicity, thus allowing the intensification of cytostatic therapy in patients. Materials and Methods: In this in vitro study, using the rat pheochromocytoma cell line PC-12 neurite-outgrowth assay, the potential of amifostine to protect against cisplatin-, paclitaxel- and vincristine-induced neurotoxicity was investigated. Amifostine is described as selectively protecting normal tissue and not tumour tissue. The effect of amifostine on tumour cell kill was investigated using the XTT and colony forming assay. Results: Paclitaxel and vincristine both caused a significant reduction in the percentage of cells expressing neurites. Co-incubation with amifostine significantly increased this percentage of neurites in paclitaxel-induced neurotoxicity, but not in vincristine-induced neurotoxicity. Post-incubation of amifostine also proved to partly reverse already existing cisplatin-induced neurotoxicity, but not paclitaxel-, or vincristine-induced neurotoxicity. Amifostine did not protect tumour cells against cisplatin- and paclitaxel-induced tumour cytotoxicity, using the XTT assay. However, a stimulation of clonogenic capacity was observed when amifostine was co-incubated with cisplatin. Conclusion: Amifostine protects against paclitaxel-induced neurotoxicity, but not against vincristine-induced neurotoxicity in this in vitro model. Furthermore, amifostine has potential to reverse already existing cisplatin-induced neurotoxicity. The tole of amifostine in the proliferative potential of tumour cells in vitro needs further investigation

The metastatic potential of rat prostate tumor variant R3327-MatLyLu is correlated with an increased activity of N-acetylglucosaminyl transferase III and V

AbstractEnzyme activities of N-acetylglucosaminyltransferase (GlcNAc-Tase) I–V involved in N-linked complex-type carbohydrate synthesis were determined in a non-metastatic hormone-dependent rat prostate tumor (R3327-H) and a related, hormone-independent variant metastasizing to lymph nodes and lungs (R3327-MatLyLu). In the metastasizing variant a significantly increased activity of both GlcNAc-Tase III and GlcNAc-Tase V was observed, whereas the activities of GlcNAc-Tase I and II were essentially unchanged. The increase in activity of GlcNAc-Tase III is particularly noteworthy since it indicates that elevated expression of this enzyme cannot be considered as an exclusive marker of hepatic malignancy

Abstract 5217: MMP9 is a prognostic biomarker for metastatic colorectal cancer

Abstract Background: Worldwide almost 700000 patients are diagnosed with colorectal cancer (CRC) each year. Of these patients more than 45% die as a consequence of metastases, of which the majority is located in the liver. Although patient selection for hepatectomy by 18FDG PET has significantly reduced futile surgery (Ruers, J Nucl Med 2009), 70% of all operated patients still die of metastases within 5 years after hepatic resection. To improve prediction of patient outcome, novel PET tracers with better prognostic value are needed. Matrix metalloproteinases, frequently associated with reduced survival in several types of cancer, might serve as prognostic biomarkers that can be used in PET imaging. Aim: To investigate the prognostic value of MMP9 expression in colon cancer liver metastases. Methods: Formalin-fixed paraffin-embedded colon cancer liver metastases, collected from 85 patients having had curative hepatectomy between 1990 and 2009, were incorporated into tissue microarrays (TMAs). MMP9 expression in both epithelial and stromal cells was assessed by immunohistochemistry and expression was correlated to patient survival data. Survival analysis was performed using a log rank test. Results: Mean age of the colon cancer patients (62.1% males, 37.9% females) was 62.0 (standard deviation 10.5). Chemotherapy was received by 45.9% (15.3% neoadjuvant, 14.1% adjuvant, 16.5% palliative) and radiofrequency ablation (RFA) was performed in 10.8%. Median overall survival was 35.0 months (standard deviation 5.7). High expression of MMP9 by epithelial colon cancer cells within the liver metastases was associated with increased survival rates (p=0.007 for disease-free survival and p=0.056 for overall survival). MMP9 expression in stromal cells was not associated with patient survival. Discussion and conclusion: Our results suggest that elevated levels of MMP9 in metastatic epithelial colon cancer cells are indicative of a good prognosis. Whether MMP9 qualifies as a target with prognostic relevance for imaging of lesions in patients with metastatic colorectal cancer remains to be determined. This research was supported by the Center for Translational Molecular Medicine (DeCoDe project). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2011-5217</jats:p

Exosomal ITGA3 interferes with non-cancerous prostate cell functions and is increased in urine exosomes of metastatic prostate cancer patients

Background: Cancer cells are able to change the protein expression and behavior of non-cancerous surrounding cells. Exosomes, secreted by prostate cancer (PCa) cells, may have a functional role in cancer metastasis and present a promising source for protein biomarkers. The aim of the present study was to identify which proteins in exosomes can influence non-cancerous cells, and to determine whether we can use urine exosomal proteins to identify high-risk PCa patients. Method: Exosomes were isolated by ultracentrifugation. Migration and invasion were studied by the transwell (invasion) assay. Proteomics was performed by LC-MS/MS and identified proteins were validated by Western blotting. Cellular uptake of fluorescent labeled PKH67-exosomes was measured by FACS. Results: Based on comparative protein profiling by mass spectrometry-based proteomics of LNCaP- and PC3-exosomes, we selected ITGA3 and ITGB1, involved in migration/invasion, for further analyses. Inhibition of exosomal ITGA3 reduced the migration and invasion of non-cancerous prostate epithelial cells (prEC) almost completely. Cellular uptake of exosomes by prEC was higher with PC3-exosomes compared to LNCaP exosomes. Finally, ITGA3 and ITGB1 were more abundant in urine exosomes of metastatic patients (p<0.05), compared to benign prostate hyperplasia or PCa. Conclusion: These data indicate exosomal ITGA3 and ITGB1 may play a role in manipulating non-cancerous surrounding cells and that measurement of ITGA3 and ITGB1 in urine exosomes has the potential to identify patients with metastatic PCa in a non-invasive manner