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Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term “exosomes” was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use “exosomes” as synonymous with “extracellular vesicles.” While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle’s membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions. [ABSTRACT FROM AUTHOR]

Background: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. Methodology/Principal Findings: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. Conclusions/Significance: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma. [ABSTRACT FROM AUTHOR]

Cancer cells, both in vivo and in vitro, have been demonstrated to release membranous structures, defined as microvesicles or exosomes, consisting of an array of macromolecules derived from the originating cells, including proteins, lipids, and nucleic acids. While only recently have the roles of these vesicular components in intercellular communication become elucidated, significant evidence has demonstrated that tumor exosomes can exert a broad array of detrimental effects on the immune system-ranging from apoptosis of activated cytotoxic T cells to impairment of monocyte differentiation into dendritic cells, to induction of myeloid-suppressive cells and T regulatory cells. Immunosuppressive exosomes of tumor origin can be found within neoplastic lesions and in biologic fluids from cancer patients, implying a potential role of these pathways in in vivo tumor progression and systemic paraneoplastic syndromes. Through the expression of molecules involved in angiogenesis promotion, stromal remodeling, signaling pathway activation through growth factor/receptor transfer, chemoresistance, and genetic intercellular exchange, tumor exosomes could represent a central mediator of the tumor microenvironment. By understanding the nature of these tumor-derived exosomes/microvesicles and their roles in mediating cancer progression and modulating the host immune response will significantly impact therapeutic approaches targeting exosomes. [ABSTRACT FROM AUTHOR]

Tumor cells release membranous structures, defined as microvesicles or exosomes, consisting of an array of macromolecules derived from the originating cells, including proteins, lipids, and RNA. The expression of antigenic molecules recognizable by T cells originally suggested a role for these vesicles as a cell-free antigen source for anti-cancer vaccines; however, evidence demonstrates that tumor exosomes can exert a broad array of detrimental effects on the immune system - ranging from apoptosis of activated cytotoxic T cells to impairment of monocyte differentiation into dendritic cells, to induction of myeloid-suppressive cells. Immunosuppressive exosomes of tumor origin can be found in neoplastic lesions and biologic fluids from cancer patients, implying a potential role of these pathways in in vivo tumor progression and systemic paraneoplastic syndromes. Through the expression of molecules involved in angiogenesis promotion, stromal remodeling, signaling pathway activation through growth factor/receptor transfer, chemoresistance, and intercellular genetic exchange, tumor exosomes could represent a central mediator of the tumor microenvironment. Their release by tumor cells may represent the future for targeting therapeutic interventions and for development of multiplexed diagnostic biomarkers. [ABSTRACT FROM AUTHOR]

Currently, a combination of technologies is typically required to assess the malignancy of cancer cells. These methods often lack the specificity and sensitivity necessary for early, accurate diagnosis. Here we demonstrate using clinical samples the application of laser trapping Raman spectroscopy as a novel approach that provides intrinsic biochemical markers for the noninvasive detection of individual cancer cells. The Raman spectra of live, hematopoietic cells provide reliable molecular fingerprints that reflect their biochemical composition and biology. Populations of normal T and B lymphocytes from four healthy individuals and cells from three leukemia patients were analyzed, and multiple intrinsic Raman markers associated with DNA and protein vibrational modes have been identified that exhibit excellent discriminating power for cancer cell identification. A combination of two multivariate statistical methods, principal component analysis (PCA) and linear discriminant analysis (LDA), was used to confirm the significance of these markers for identifying cancer cells and classifying the data. The results indicate that, on average, 95% of the normal cells and 90% of the patient cells were accurately classified into their respective cell types. We also provide evidence that these markers are unique to cancer cells and not purely a function of differences in their cellular activation. [ABSTRACT FROM AUTHOR]

Abstract: Objective.: Early stage cervical cancer is treated with surgery or radiation with equivalent results. Radiation is used for curative therapy of locally advanced disease and is combined with additional anti-tumor agents to improve control. We determined the potential role of genistein as a radiosensitizer for cervical cancer cells. Methods. : Human cervical cell lines (CaSki and ME180) were used. Sensitivity of cells to genistein, radiation and the combination was determined by colony assays. Western blotting was used to study the expression of cell-response-related gene products. Results.: Genistein results in the dose-dependent inhibition of all cell lines (2.5–40.0 μM). Effect of genistein on the radiosensitivity of the two cervical tumor cells was variable. Me180 cells were more sensitive at 20 and 40 μM of genistein. At 40 μM, less than 5% of Me180 cells survived the radiation (200–800 cGy). Potentiation of the radiation effect in CaSki cells was seen (500–800 cGy). The most significant enhancement of radiosensitivity was seen at 20 and 40 μM genistein at 500 and 800 cGy. G2M arrest was demonstrated only in ME180 cells with genistein. There was significant inhibition of Mcl-1 by genistein that correlated with increase in radiosensitivity in Me180 cells. Activated pAKT (Thr 308) was inhibited with genistein and radiation in CaSki cells. Conclusions.: Genistein inhibits growth of cervical cancer cells. Genistein results in variable and significant enhancement of the radiation effect that may be partially mediated by G2M arrest, Mcl-1 and activation of the AKT gene. [Copyright &y& Elsevier]

A combination of E and progestogens significantly increased matrix metalloproteinase (MMP)-2 in both T47D cells (E2-medroxyprogesteroneacetate [MPA] and E2-P) and MCF-7 cells (E2-MPA, E2-P, and equilin-MPA). All combinations resulted in higher MMP-9 levels in MCF-7 cells, but higher MMP-9 levels resulted only with equilin-norethinderone in T47D cells. [Copyright &y& Elsevier]