Media Of Cancer Cell Lines Research Articles

MiR-3940-5p/miR-8069 ratio in urine exosomes is a novel diagnostic biomarker for pancreatic ductal adenocarcinoma.

Despite the development of several therapeutic options, the prognosis of pancreatic cancer remains poor. One reason for this is the difficulty of diagnosing the disease at an early stage. For example, carbohydrate antigen (CA) 19-9, which is the most widely used biomarker for pancreatic cancer, cannot be used to detect the disease at early stages. Some studies have attempted to find novel biomarkers for pancreatic cancer. The aim of the present study was to find a novel diagnostic biomarker for pancreatic ductal adenocarcinoma (PDAC) in urine exosomes. Exosomes were isolated from urine and serum samples of patients with PDAC and control subjects, or culture media of cancer cell lines. MicroRNAs (miRNAs) were purified from exosomes. Novel biomarker candidates for PDCA were identisfied from urine exosome miRNA using expression profiling, and validated in a larger number of samples using 3D digital PCR. The results of a preliminary analysis of nine PDAC and seven control subjects revealed that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in the patients with PDAC. Experiments using cultured cancer cell lines revealed that the elevation of the miR-3940-5p/miR-8069 ratio was specific for PDAC. Furthermore, the elevation of the miR-3940-5p/miR-8069 ratio in exosomes tended to be higher in the urine than in the serum of patients with PDAC. Validation experiments on 43 PDAC, 12 chronic pancreatitis and 25 control subjects demonstrated that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in PDAC at a relatively early stage of the disease. When this ratio was used in combination with CA19-9 for the diagnosis of PDAC, the sensitivity and positive predictive value improved to 93.0 and 78.4%, respectively, when either of them was positive. Additionally, the positive predictive value reached 100% when both were positive. The negative predictive value also improved to 89.7% when both were negative. The miR-3940-5p/miR-8069 ratio in urine exosomes may be useful as a tool for the diagnosis of PDAC, particularly when used in combination with CA19-9.

Characterization of Gene Expression Patterns among Artificially Developed Cancer Stem Cells Using Spherical Self-Organizing Map.

We performed gene expression microarray analysis coupled with spherical self-organizing map (sSOM) for artificially developed cancer stem cells (CSCs). The CSCs were developed from human induced pluripotent stem cells (hiPSCs) with the conditioned media of cancer cell lines, whereas the CSCs were induced from primary cell culture of human cancer tissues with defined factors (OCT3/4, SOX2, and KLF4). These cells commonly expressed human embryonic stem cell (hESC)/hiPSC-specific genes (POU5F1, SOX2, NANOG, LIN28, and SALL4) at a level equivalent to those of control hiPSC 201B7. The sSOM with unsupervised method demonstrated that the CSCs could be divided into three groups based on their culture conditions and original cancer tissues. Furthermore, with supervised method, sSOM nominated TMED9, RNASE1, NGFR, ST3GAL1, TNS4, BTG2, SLC16A3, CD177, CES1, GDF15, STMN2, FAM20A, NPPB, CD99, MYL7, PRSS23, AHNAK, and LOC152573 genes commonly upregulating among the CSCs compared to hiPSC, suggesting the gene signature of the CSCs.

Abstract 5219: Circulating hTERT (human telomerase) mRNA: mechanism of action and potential use for early diagnosis of malignancy

Abstract In contrast to current impressive achievements in the biological knowledge and therapy of cancer, the field of early diagnosis lags behind and presents an unmet need. We hereby present the human telomerase as a potential tool for early diagnosis of cancer. Telomerase activation is a prerequisite for the perpetuation of the malignant clone during cancer progression as it elongates telomeres in each cell division. hTERT mRNA is shed into the serum and remains stable in vesicular forms named “Exosomes”. Exosomes are secreted by most cells containing nucleic acids, proteins and lipids. We established a method for the detection and quantification of hTERT mRNA products in exosomes derived from growth media of cancer cell lines and human sera for the future use as a diagnostic tool for the early detection of cancer. In four cancer cell lines we detected exosomal hTERT RNA expression, which also correlated with its telomerase activity and intracellular hTERT mRNA expression, compared to a non-telomerase expressing cell line. We studied the crosstalk between T cell leukemia derived exosomes and primary human fibroblast cells and revealed that primary cells can uptake exosomes derived from a different type of cell. The secreted exosomes affected the recipient cells by increasing the hTERT mRNA 6 hours post exposure and increasing telomerase levels and expression 24 hours post exposure, while the levels of the hTERT core promoter transcription factors did not change. For the future goal of establishing a method of early diagnosis of malignancies we screened 63 patients with different malignancies and compared their exosomal hTERT mRNA expression in the sera to that of 20 healthy volunteers. The results have shown that the expression of hTERT is variable among malignancies and between different patients with the same cancer type. To exclude some variables, such as drugs, that can influence exosome secretion from cells, we examined the influence of three chronically administered drugs on our chosen experimental cell lines. The drugs tested were Aspirin, Simvastatin and Captopril. While Simvastatin significantly decreased hTERT mRNA in exosomes derived from one cell line, neither aspirin nor Captopril effected the secretion of exosomes, indicated by a similar hTERT expression in the relevant treated cells. Furthermore, no change in the intracellular hTERT expression and telomerase activity after drug exposure was evident. In the light of these results we concluded that exosomes derived from tumor cells can affect the surrounding microenvironment by exploiting the recipient cell mechanism and promoting the activation of telomerase in those recipient cells. Understanding of these mechanisms may have a strong impact on deciphering metastases formation. Hopefully, promoting hTERT mRNA as a novel marker for malignancies will enable us to develop a new diagnostic tool for future use in the early diagnosis of cancer. Citation Format: Orit Uziel, Anna Gutkin, Einat Beery, Jardena Nordenberg, Hadar Goldvaser, Yair Zloof, Steven Henick, Meir Lahav. Circulating hTERT (human telomerase) mRNA: mechanism of action and potential use for early diagnosis of malignancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5219. doi:10.1158/1538-7445.AM2015-5219

Extracellular Vesicles - Powerful Markers of Cancer EVolution.

OPINION article Front. Immunol., 12 January 2015Sec. Inflammation Volume 5 - 2014 | https://doi.org/10.3389/fimmu.2014.00685

Expression, Tissue Distribution and Function of miR-21 in Esophageal Squamous Cell Carcinoma

ObjectiveMiR-21 is an oncomir expressed by malignant cells and/or tumor microenvironment components. In this study we focused on understanding the effects of stromal miR-21 on esophageal malignant cells.DesignMiR-21 expression was evaluated in formalin-fixed paraffin-embedded samples from patients with esophageal squamous-cell carcinoma (SCC) by quantitative RT-PCR. MiR-21 tissue distribution was visualized with in situ hybridization. A co-culture system of normal fibroblasts and esophageal cancer cells was used to determine the effects of fibroblasts on miR-21 expression levels, and on SCC cell migration and invasion.ResultsMiR-21 was overexpressed in SCCs, when compared to the adjacent non-tumor tissues (P = 0.0007), and was mainly localized in the cytoplasm of stromal cells adjacent to malignant cells. Accordingly, miR-21 expression was increased in tumors with high versus low stromal content (P = 0.04). When co-cultured with normal fibroblasts, miR-21 expression was elevated in SCC cells (KYSE-30), while its expression was restricted to fibroblasts when co-cultured with adenocarcinoma cells (OE-33 and FLO-1). MiR-21 was detected in conditioned media of cancer cell lines, illustrating the release of this miRNA into the environment. Co-culturing with normal fibroblasts or addition of fibroblast conditioned media caused a significant increase in cell migration and invasion potency of KYSE-30 cells (P<0.0001). In addition, co-culturing cancer cells with fibroblasts and expression of miR-21 induced the expression of the cancer associated fibroblast (CAF) marker S100A4.ConclusionsMiR-21 expression is mostly confined to the SCC stroma and its release from fibroblasts influences the migration and invasion capacity of SCC cells. Moreover, miR-21 may be an important factor in “activating” fibroblasts to CAFs. These findings provide new insights into the role of CAFs and the extracellular matrix in tumor microenvironment formation and in tumor cell maintenance, and suggest miR-21 may contribute to cellular crosstalk in the tumor microenvironment.

Secretomic analysis of large cell lung cancer cell lines using two-dimensional gel electrophoresis coupled to mass spectrometry

The secretome of cancer cells is a valuable source of biomarkers that can ultimately reach the serum or other body fluids. Cancer biomarkers can facilitate early diagnosis and monitoring of the disease, contribute to our understanding of tumor biology, and support the development of more efficient therapies. Recently, high-throughput proteomic analysis of the conditioned media of cancer cell lines has shown potential to identify novel biomarkers in cancer. We used two-dimensional gel electrophoresis coupled to liquid chromatography tandem mass spectrometry to identify the secretome of the large cell lung cancer cell lines QU-DB and Mehr-80, which they were established from a Canadian and a Persian patient, respectively. A total of 130 distinct protein species were identified. Of these, 124 were previously found in serum or other body fluids, the membrane compartment or conditioned media of other cancer cell lines. Some of the proteins that we identified, e.g. IL-6, triosephosphate isomerase, PGP9.5, α-enolase, Dickkopf-1, and peroxiredoxin-1 are known putative serum markers for lung cancer, whereas others might be candidate markers for further validation in lung cancer body fluids such as IL-25, stathmin, vimentin, peptidyl-prolyl cis-trans isomerase A, transgelin-2, and chloride intracellular channel protein 4.

Secretomic analysis of large cell lung cancer cell lines using two-dimensional gel electrophoresis coupled to mass spectrometry

The secretome of cancer cells is a valuable source of biomarkers that can ultimately reach the serum or other body fluids. Cancer biomarkers can facilitate early diagnosis and monitoring of the disease, contribute to our understanding of tumor biology, and support the development of more efficient therapies. Recently, high-throughput proteomic analysis of the conditioned media of cancer cell lines has shown potential to identify novel biomarkers in cancer. We used two-dimensional gel electrophoresis coupled to liquid chromatography tandem mass spectrometry to identify the secretome of the large cell lung cancer cell lines QU-DB and Mehr-80, which they were established from a Canadian and a Persian patient, respectively. A total of 130 distinct protein species were identified. Of these, 124 were previously found in serum or other body fluids, the membrane compartment or conditioned media of other cancer cell lines. Some of the proteins that we identified, e.g. IL-6, triosephosphate isomerase, PGP9.5, α-enolase, Dickkopf-1, and peroxiredoxin-1 are known putative serum markers for lung cancer, whereas others might be candidate markers for further validation in lung cancer body fluids such as IL-25, stathmin, vimentin, peptidyl-prolyl cis-trans isomerase A, transgelin-2, and chloride intracellular channel protein 4.