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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

Integrative medicine: a paradigm shift in medical education and practice

The use of alternative/complementary medicine has been increasing considerably. Conventional medicine must begin to address issues related to the use, safety, regulation, research and education of alternative/complementary medicine. Integrative medicine combines conventional medicine and alternative complementary practices. Integrative medicine is an innovative approach to medicine and medical education. It involves the understanding of the interaction of the mind, body and spirit and how to interpret this relationship in the dynamics of health and disease. Integrative medicine shifts the orientation of the medical practice from disease based approach to a healing based approach. It does not reject conventional medicine nor uncritically accepts unconventional practices. Integrative medicine is an effective, more fulfilling human approach to medicine based on the benefit of the patient by following good medicine practices in a scientific manner.