Project description:Cancer-derived exosomes can deliver nucleic acids, proteins, and lipids to neighboring or distant cells and subsequently modulate recipient cells. Recently, high levels of microRNAs (miRNAs) have been identified in cancer-derived exosomes, which provide a favorable microenvironment that contribute to tumorigenesis, tumor metastasis, angiogenesis, chemoresistance, and immune escape. Howerer, the mechanism of the cancer-derived exosomes regulating CD45+EpCAM+ cell apoptosis remains unclear.
Project description:To further development of our gene expression approach to biodosimetry, we have employed microRNA microarray expression profiling to identify genes with the potential to distinguish liver metastasis related microRNA. Colorectal cancer patients were administered anesthesia and 20 mL BM was taken from the right and left anterior iliac crests before surgery. Mononucleated cells were collected using a standard Ficoll-Hypaque gradient technique. To enrich for EpCAM+ cells, CD14+ cells were removed from the whole bone marrow using auto MACSTM pro (Milteny Biotec, Bergisch Gladbach, Germany) with anti-CD14 immunomagnetic beads (clone; TÜK4, Milteny Biotec). Next, CD45+ cells were removed by treatment with anti-CD45 immunomagnetic beads (clone; 5B1; Milteny Biotec). The residual CD14?CD45? cells were then incubated with FcR blocking reagent (Milteny Biotec), followed by incubation with anti-EpCAM immunomagnetic beads (clone; HEA-125, Milteny Biotec), and the CD14?CD45?EpCAM+ cells were taken up. Total RNA of these cells we analyzed the microRNA levels of CD14?CD45?EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7). Ten-microRNA consensus signature was identified that distinguished between CD14?CD45?EpCAM+ cells from liver metastasis patients and CD14?CD45?EpCAM+ cells from non-liver metastasis patients. MicroRNA expression of CD14-CD45-EpCAM+ cells in human bone marrow was measured. RNA of these cells we analyzed the microRNA levels of CD14?CD45?EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7).
Project description:To further development of our gene expression approach to biodosimetry, we have employed microRNA microarray expression profiling to identify genes with the potential to distinguish liver metastasis related microRNA. Colorectal cancer patients were administered anesthesia and 20 mL BM was taken from the right and left anterior iliac crests before surgery. Mononucleated cells were collected using a standard Ficoll-Hypaque gradient technique. To enrich for EpCAM+ cells, CD14+ cells were removed from the whole bone marrow using auto MACSTM pro (Milteny Biotec, Bergisch Gladbach, Germany) with anti-CD14 immunomagnetic beads (clone; TÜK4, Milteny Biotec). Next, CD45+ cells were removed by treatment with anti-CD45 immunomagnetic beads (clone; 5B1; Milteny Biotec). The residual CD14−CD45− cells were then incubated with FcR blocking reagent (Milteny Biotec), followed by incubation with anti-EpCAM immunomagnetic beads (clone; HEA-125, Milteny Biotec), and the CD14−CD45−EpCAM+ cells were taken up. Total RNA of these cells we analyzed the microRNA levels of CD14−CD45−EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7). Ten-microRNA consensus signature was identified that distinguished between CD14−CD45−EpCAM+ cells from liver metastasis patients and CD14−CD45−EpCAM+ cells from non-liver metastasis patients.
Project description:Comparison of Villin-Cre;Apc min;Lef1 fl/fl (VApcMinL) and ApcMin Epcam- cells and CD45-Epcam- stromal cells from lamina propria at the age of 11 wks. Two male mice per group was used in the experiment.
Project description:Lung cancer has the highest mortality rate among human cancers, and the majority of deaths result from metastatic spread. The tumor microenvironment plays an important role in suppressing the immune surveillance and elimination of tumor cells. A few studies have reported the presence of CD45+EpCAM+ double-positive cells in cancer, but the underlying mechanism remains unclear with respect to how these cells originate and their function in cancer biology. In this study, we analyzed 25 lung tumor samples. We confirmed the presence of CD45+EpCAM+ cells in lung cancer, and these cells exhibited higher apoptosis than CD45+EpCAM- cells. Using co-culture of lung cancer cell-derived exosomes with healthy donor peripheral blood mononuclear cells, we recapitulated CD45+EpCAM+ cell formation and increased apoptosis that occurs in patients with primary lung cancer. Further analysis suggested that microRNAs in lung cancer cell-derived exosomes may alter the gene expression profile of CD45+EpCAM+ cells, resulting in elevated TP53 expression and increased apoptosis. To our knowledge, this is the first report of cancer cell-derived exosomes that can inhibit the immune system by promoting immune cell apoptosis.
Project description:Tumour tissue was immediately obtained post-surgery along with paired healthy tissue near (<2cm from tumour margin) and far (>10cm) from the tumour site. Each tissue was dissociated and stained with flow cytometry antibodies and sorted by FACS into Live/Dead-negative EpCAM-positive CD3-negative populations. An additional sample was passed through the FACS however no sorting was applied (control for the effect of sorting). Total RNA was purified and tissues compared for gene expression to identify potential hits that were assessed for protein expression and immunogenicity in later experiments.
Project description:siRNAs have played a major role in cancer drug discovery, but their potential is hampered due to off-target effects. Thus, delivery systems like RNA aptamers have been used to enhance the specific delivery of these siRNAs to cancer stem cells. We report the efficacy of three different EpCAM aptamer siRNA chimeras, which were investigated both in vitro and in vivo for their ability to reduce cancer cell progression. Using these chimeras, we demonstrated specific gene knockdown in EpCAM positive cells which ultimately led to the apoptosis. To study the efficacy of these aptamer chimeras in vivo, retinoblastoma xenografts bearing NCC Rb C 51 cells were created for the first time. Systemic administration of these aptamer chimeras reduced tumour growth to about 50%. We further investigated the central Role of PLK1 in Cancer Progression and demonstrated the anti-cancer effects of targeted EpCAM siPLK1 approach. Using SILAC-Mass spectrometry analysis, we showed that silencing PLK 1 gene can lead to p53 mediated cell cycle arrest. Thus, we establish EpCAM-siRNA chimeras as potential markers for targeted anti-cancer applications, which paves a platform for efficient second line of therapies in addition to existing chemotherapy options.
Project description:Exploration of proteome differences between CD45+ and CD45- cell types in renal cell carcinoma tumors and normal adjacent tissue patient samples.
Project description:We developed a novel approach to isolate tumor cells with high purity from bone marrow which was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays. For non-tumor controls, RNA profiling was performed on matched leukocytes (CD45+) isolated from the same enriched bone marrow samples from 17 of the 30 patients.