Biobanking of Exosomes in the Era of Precision Medicine: Are We There Yet?

The emerge of personalized medicine demands high-quality human biospecimens with appropriate clinical annotation, especially in complex diseases such as cancer, neurodegenerative, cardiovascular, and metabolic alterations in which specimen heterogeneity and individual responses often complicate the development of precision therapeutic programs. In the growing field of extracellular vesicles (EVs) research, exosomes (EXOs)--a particular type of EVs--have been proposed as an advantageous diagnostic tool, as effective delivery vehicles and as therapeutic targets. However, the lack of consensus on isolation methods and rigorous criteria to characterize them puts the term EXO into question at the time that might explain some of the controversial results found in the literature. A lack of response in the biobank network to warrant standard optimized procedures for the isolation, characterization, and storage of EXOs will undoubtedly lead to a waste of resources and failure. This review is aimed at highlighting the increasing importance of EXOs for the clinic, especially in the cancer field, and at summarizing the initiatives taken to improve current isolation procedures, classification criteria, and storage conditions of EXOs as an effort to identify technological demands that biobank platforms face for the incorporation of EXOs and other extracellular vesicle fractions as valuable biospecimens for research.

Clinicopathological factors associated to HER-2 status in a hospital-based sample of breast cancer patients in Puerto Rico.

Breast cancer is the most common female malignancy in Puerto Rico. Cases with human epidermal growth factor receptor 2 (HER-2) oncoprotein overamplification are associated with aggressive clinical behavior. Given the limited availability of information for Puerto Rico, we aimed to evaluate the prevalence and clinical correlates of HER-2 gene overexpression among a hospital-based female population of breast cancer cases. We analyzed data from 1,049 female patients with invasive breast cancer (diagnosed 2000-2005) at the I. González Martínez Oncologic Hospital and the Auxilio Mutuo Hospital. HER-2 status and other clinical characteristics were retrieved from the hospitals' cancer registries, from the Puerto Rico Central Cancer Registry, and from a review of medical and pathological records. Prevalence odds ratios were estimated with 95% confidence intervals, using logistic regression models to quantify the association between HER-2 status and different clinicophatological factors. The overall prevalence of positive HER-2 expression was 22.5%. In the multivariate logistic regression model, factors significantly associated with HER-2 positivity included a diagnosis age of < 50 years, having a tumor with negative progesterone receptor (PR) status, and having regional disease (p < 0.05). No significant differences in HER-2 positivity were observed by tumor histology or estrogen receptor (ER) status (p > 0.05). This is the most comprehensive epidemiological study to date on HER-2 status in Puerto Rico. The prevalence and correlates of HER-2 overexpression in this study are comparable to those observed in US populations. Study results will aid in the development of breast cancer control strategies in Puerto Rico.

A Recurrent BRCA2 Mutation Explains the Majority of Hereditary Breast and Ovarian Cancer Syndrome Cases in Puerto Rico.

Breast cancer is the most common cause of cancer diagnosis in women and is responsible for considerable mortality among the women of Puerto Rico. However, there are few studies in Puerto Rico on the genetic factors influencing risk. To determine the contribution of pathogenic mutations in BRCA1 and BRCA2, we sequenced these genes in 302 cases from two separate medical centers, who were not selected for age of onset or family history. We identified nine cases that are carriers of pathogenic germline mutation. This represents 2.9% of unselected cases and 5.6% of women meeting National Comprehensive Cancer Network (NCCN) criteria for BRCA testing. All of the identified pathogenic mutations were in the BRCA2 gene and the most common mutation is the p.Glu1308Ter (E1308X) mutation in BRCA2 found in eight out of nine cases, representing 89% of the pathogenic carriers. The E1308X mutation has been identified in breast and ovarian cancer families in Spain, and analysis of flanking DNA polymorphisms shows that all E1308X carriers occur on the same haplotype. This is consistent with BRCA2 E1308X being a founder mutation for the Puerto Rican population. These results will contribute to better inform genetic screening and counseling of breast and ovarian cancer cases in Puerto Rico and Puerto Rican populations in mainland United States.

Differential Effects of EGFL6 on Tumor versus Wound Angiogenesis.

Angiogenesis inhibitors are important for cancer therapy, but clinically approved anti-angiogenic agents have shown only modest efficacy and can compromise wound healing. This necessitates the development of novel anti-angiogenesis therapies. Here, we show significantly increased EGFL6 expression in tumor versus wound or normal endothelial cells. Using a series of in vitro and in vivo studies with orthotopic and genetically engineered mouse models, we demonstrate the mechanisms by which EGFL6 stimulates tumor angiogenesis. In contrast to its antagonistic effects on tumor angiogenesis, EGFL6 blockage did not affect normal wound healing. These findings have significant implications for development of anti-angiogenesis therapies.

Orthomolecular oncology review: ascorbic acid and cancer 25 years later.

The effect of ascorbic acid on cancer has been a subject of great controversy. This is a follow-up review of the 1979 article by Cameron, Pauling, and Leibovitz published in Cancer Research. In this updated version, the authors address general aspects of ascorbic acid and cancer that have been presented before, while reviewing, analyzing, and updating new existing literature on the subject. In addition, they present and discuss their own mechanistic hypothesis on the effect of ascorbic acid on the cancer cell. The objective of this review is to provide an updated scientific basis for the use of ascorbic acid, especially intravenously as adjuvant treatment in pharmacological nutritional oncology.

Erythropoietin Stimulates Tumor Growth via EphB4.

While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo's effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin.

Cell membrane fatty acid composition differs between normal and malignant cell lines.

Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids.

Inhibition of human breast carcinoma cell proliferation by ascorbate and copper.

We tested the effect of different concentrations of ascorbic acid (AA), 50, 100, 250 mg/500 mg/dL) with copper sulfate (CS), 10 mg/dL) on human breast carcinoma (MDA-MB231) cell proliferation in vitro. Cell proliferation was measured using a colori-metric assay (Cell proliferation kit II (XTT), Boehringer, NJ). The results of the mean absorbance of the tissue culture at different AA concentrations and a constant CS concentration were as follow: 0.82 +/- 0.03 (control, mean +/- SE), 0.64 +/- 0.02 (CS above); 0.48 +/- 0.03 (50 mg/dL) AA), 0.21 +/- 0.02 (100 mg/dL), 0.08 +/- 0.01 (250 mg/dL) AA, 0.60 +/- 0.05 (500 mg/dL). These results show that a combination of AA and CS inhibits human breast carcinoma cell proliferation in vitro. This cell proliferation inhibitory effect is directly proportional to the AA concentration with the exception of the 500 mg/dL AA dose. This chemotherapeutic effect was optimally enhanced when AA was added at a concentration of 250 mg/dL. The AA concentrations of 500 mg/dL had a biphasic effect on tumor cell proliferation probably due to back and forth redox reactions between AA and dehydroascorbic acid in a closed system. This study provides preliminary evidence that AA and SC can be used as biological response modifiers (BRM) for tumor growth inhibition.

Intravenous vitamin C as a chemotherapy agent: a report on clinical cases.

A series of seven cases are presented in which intravenous vitamin C has been used as antineoplastic agent in the treatment of different types of cancers. The cancers cases reviewed are the following: Renal cell carcinoma (2), Colorectal cancer (1), Pancreatic cancer (1), Non-Hodgkin's lymphoma (2) and breast cancer (1). Toxic reactions were not observed at these high doses of intravenous Vitamin C. All patients were prescreened for Glucose 6--phosphate dehydrogenase deficiency before administering intravenous Vitamin C in order to prevent hemolysis.

Orthomolecular oncology: a mechanistic view of intravenous ascorbate's chemotherapeutic activity.

The effect of vitamin C in cancer has been a subject of great controversy; mainly because of the inconsistent results obtained by oral intakes of ascorbate when used as an anticancer agent. We believe the intravenous application of ascorbate will provide more consistent results in cancer patients since Vitamin C blood levels attained are substantially higher in a range proven cytotoxic to malignant cells. In this article we will present and discuss our proposed mechanism on the chemotherapeutic activity exhibited by ascorbate.

Intravenous ascorbic acid: protocol for its application and use.

High dose intravenous(i.v.) ascorbic acid (AA) has been used as therapy for infectious disease from bacterial and viral origin and adjuvant therapy for cancer. In this publication we describe a clinical protocol that has been developed over the past twenty years utilizing high dose i.v. AA as therapy for cancer. This includes principles of treatment, rationale, baseline workup, infusion protocol, precautions and side effects.

Proceedings of the 1st Puerto Rico Biobanking Workshop.

The 1st Puerto Rico Biobanking Workshop took place on August 20st, 2014 in the Auditorium of the Comprehensive Cancer Center of the University of Puerto Rico, Medical Sciences Campus in San Juan Puerto Rico. The program for this 1-day, live workshop included lectures by three biobanking experts, followed by presentations from existing biobanks in Puerto Rico and audience discussion. The need for increasing biobanking expertise in Puerto Rico stems from the fact that Hispanics in general are underrepresented in the biobanks in existence in the US, which limits the research conducted specifically to understand the molecular differences in cancer cells compared to other better studied populations. In turn, this lack of information impairs the development of better diagnostic and therapeutic approaches for our population. Dr. James Robb, M.D., F.C.A.P., consulting pathologist to the National Cancer Institute (NCI) and the Office of Biorepositories and Biospecimen Research (OBBR), opened the workshop with a discussion on the basic aspects of the science of biobanking (e.g., what is a biobank; its goals and objectives; protocols and procedures) in his talk addressing the importance of banking tissues for advancing biomedical research. Next, Dr. Gustavo Stefanoff, from the Cancer Institutes Network of Latin America (RINC by its name in Spanish), explained the mission, objectives, and structure of the Network of Latin-American and Caribbean Biobanks (REBLAC by its name in Spanish), which despite limited resources and many challenges, currently accrue high quality human tissue specimens and data to support cancer research in the region. Dr. Robert Hunter-Mellado, Professor of Internal Medicine, Universidad Central del Caribe, followed with an examination of the ethical and regulatory aspects of biobanking tissues for future research, including informed consent of subjects; protection of human subjects rights; and balancing risks and benefit ratios. In the afternoon, the directors of existing biobanks in Puerto Rico (the Puerto Rico Biobank, the Comprehensive Cancer Center biobank, and an HIV-focused biobank at Universidad Central del Caribe) presented their experiences and challenges with establishing biobanks for research in Puerto Rico. In sum, this workshop presented opportunities to share knowledge in the science of biobanking, for further training, and of networking among the participants (34 from 4 different institutions), which will strengthen the collaborative links between investigators studying cancer in Latin America, the Caribbean, and the US.

Synthesis, Stability, Cellular Uptake, and Blood Circulation Time of Carboxymethyl-Inulin Coated Magnetic Nanoparticles.

Iron oxide nanoparticles were coated with the biocompatible, biodegradable, non-immunogenic polysaccharide inulin by introduction of carboxyl groups into the inulin structure and conjugation with amine groups on the surface of iron oxide nanoparticles grafted with 3-aminopropyltriethoxysilane. The resulting nanoparticles were characterized by FT-IR spectroscopy, transmission electron microscopy, dynamic light scattering, zeta potential, SQUID magnetometry, and with respect to their energy dissipation rate in applied alternating magnetic fields. The nanoparticles had a hydrodynamic diameter in the range of 70 ± 10 nm and were superparamagnetic, with energy dissipation rates in the range of 58-175 W/g for an applied field frequency of 233 kHz and an applied field amplitude in the range of 20-48 kA/m. The nanoparticles were stable in a range of pH, at temperatures between 23°C and 53°C, and in short term storage in water, PBS, and culture media. The particles were non-cytotoxic to the immortalized human cancer cell lines Hey A8 FDR, A2780, MDA 468, MCF-7 and Caco-2. The nanoparticles were readily taken up by Caco-2 cells in a time and concentration dependent fashion, and were found to have a pharmacokinetic time constant of 47 ± 3 min. The small size, non-cytotoxicity, and efficient energy dissipation of the particles could make them useful for biomedical applications such as magnetic fluid hyperthermia.

Breast cancer molecular subtypes and survival in a hospital-based sample in Puerto Rico.

Information on the impact of hormone receptor status subtypes in breast cancer (BC) prognosis is still limited for Hispanics. We aimed to evaluate the association of BC molecular subtypes and other clinical factors with survival in a hospital-based female population of BC cases in Puerto Rico. We analyzed 663 cases of invasive BC diagnosed between 2002 and 2005. Information on HER-2/neu (HER-2) overexpression, estrogen (ER), and progesterone (PR) receptor status and clinical characteristics were retrieved from hospitals cancer registries and record review. Survival probabilities by covariates of interest were described using the Kaplan-Meier estimators. Cox proportional hazards models were employed to assess factors associated with risk of BC death. Overall, 17.3% of BC cases were triple-negative (TN), 61.8% were Luminal-A, 13.3% were Luminal-B, and 7.5% were HER-2 overexpressed. In the multivariate Cox model, among patients with localized stage, women with TN BC had higher risk of death (adjusted hazard ratio [HR]: 2.57, 95% confidence interval [CI]: 1.29-5.12) as compared to those with Luminal-A status, after adjusting for age at diagnosis. In addition, among women with regional/distant stage at diagnosis, those with TN BC (HR: 5.48, 95% CI: 2.63-11.47) and those HER-2+, including HER-2 overexpressed and Luminal-B, (HR: 2.73, 95% CI:1.30-5.75) had a higher mortality. This is the most comprehensive epidemiological study to date on the impact of hormone receptor expression subtypes in BC survival in Puerto Rico. Consistent to results in other populations, the TN subtype and HER-2+ tumors were associated with decreased survival.

Biological significance of HORMA domain containing protein 1 (HORMAD1) in epithelial ovarian carcinoma.

The present study was undertaken to determine the expression and biological significance of HORMAD1 in human epithelial ovarian carcinoma. We found that a substantial proportion of human epithelial ovarian cancers expressed HORMAD1. In vitro, HORMAD1 siRNA enhanced docetaxel induced apoptosis and substantially reduced the invasive and migratory potential of ovarian cancer cells (2774). In vivo, HORMAD1 siRNA-DOPC treatment resulted in reduced tumor weight, which was further enhanced in combination with cisplatin. HORMAD1 gene silencing resulted in significantly reduced VEGF protein levels and microvessel density compared to controls. Our data suggest that HORMAD1 may be an important therapeutic target.

Chitosan hydrogel for localized gene silencing.

OBJECTIVE: To achieve effective delivery of siRNA into target cells in vivo, we have developed a novel approach of siRNA delivery by using local drug delivery systems. RESULTS: The chitosan hydrogel (CH-HG) displayed a liquid-solid phase transition in a temperature-dependent manner and formed an endothermic hydrogel in tumor tissue after intra-tumoral injection. Additionally, we tested the extent of in vivo delivery following a single intra-tumoral injection of Alexa555 siRNA/CH-HG into A375SM-bearing mice. The Alexa555 siRNA demonstrated higher localization into tumor cells compared to control. The Alexa555 siRNA delivery extends to tumor cells outside of CH-HG and some tumor cells also infiltrated into CH-HG. For therapeutic proof-of-concept studies, CH-HG including TG2-targeted siRNA significantly inhibited tumor growth in melanoma (A375SM) and breast (MDA-MB231) tumor models compared to control (A375SM: 72% reduction and MDA-MB231: 92% reduction, p < 0.001). EXPERIMENTAL DESIGN: we prepared a CH-HG system loaded with siRNA to enhance localized therapeutic efficacy without risk for systemic side effects. Delivery of siRNA into CH-HG was confirmed by fluorescence microscopy. Antitumor efficacy was examined in mouse models of melanoma (A375SM) and breast (MDA-MD231) cancer. CONCLUSIONS: This study developed a novel local delivery method for siRNA therapy using the CH-HG system. This approach could have broad applications for multiple localized diseases.

Targeted delivery of small interfering RNA using reconstituted high-density lipoprotein nanoparticles.

RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches.

Biological roles of the Delta family Notch ligand Dll4 in tumor and endothelial cells in ovarian cancer.

Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important new targets for antiangiogenesis approaches. In this study, we investigated the clinical and biological significance of Dll4 in ovarian cancer. Dll4 was overexpressed in 72% of tumors examined in which it was an independent predictor of poor survival. Patients with tumors responding to anti-VEGF therapy had lower levels of Dll4 than patients with stable or progressive disease. Under hypoxic conditions, VEGF increased Dll4 expression in the tumor vasculature. Immobilized Dll4 also downregulated VEGFR2 expression in endothelial cells directly through methylation of the VEGFR2 promoter. RNAi-mediated silencing of Dll4 in ovarian tumor cells and tumor-associated endothelial cells inhibited cell growth and angiogenesis, accompanied by induction of hypoxia in the tumor microenvironment. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with control or treatment with bevacizumab alone. Together, our findings establish that Dll4 plays a functionally important role in both the tumor and endothelial compartments of ovarian cancer and that targeting Dll4 in combination with anti-VEGF treatment might improve outcomes of ovarian cancer treatment.

Targeted gene silencing using RGD-labeled chitosan nanoparticles.

PURPOSE: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA). EXPERIMENTAL DESIGN: RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with alphanubeta3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma. RESULTS: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the alphanubeta3 integrin-positive tumor endothelial cells in the A2780 tumor-bearing mice. This approach resulted in significant inhibition of tumor growth compared with controls. CONCLUSIONS: This study shows that RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential for broad applications in human disease.