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679. Heat Stress Elevate Cell Population That Express Cancer Stem Cell Markers in Different Types of Cancer Cell Line
Cancer-Targeted Gene and Cell Therapy II 678. Neural Stem Cell-Based Gene Therapy for Malignant Brain Tumor by Doubl-Stranded AdenoAssociated Viral Vectors Li-juan Wang, Hsin-I Ma Neurological Surgery, National Defense Medical Center, Taipei, Taiwan
Despite intensive efforts exploring gene therapeutic approaches for human cancers, the clinical results in treating of malignant brain tumors are still poor. We demonstrated that the combined therapeutic strategy by using an adenoviral vector carrying the thymidine kinase (suicidal) gene and an adeno-associated viral vector carrying the angiostatin gene achieved better therapeutic results. However, the malignant brain tumor could not be completely eradicated. Therefore, novel vectors and strategies are urgently needed. Neural stem cells (NSCs) may be used either for cell replacement or for gene delivery vehicles in neurodegenerative diseases. The expression of therapeutic proteins by NSCs can be enhanced by viral-mediated gene transfer, though the effects of several common recombinant viruses on primary progenitor cell populations have not been widely tested. The goal of this study is to identify and develop a safe and effective NSCs-based gene therapy protocol to treat malignant glioma. We examined what seral type of the novel double-stranded recombinant adeno-associated viral (AAV) vector can efficiently deliver anti-angiogenic genes and suicidal gene into the rat NSCs. Meanwhile, we investigated the gene product delivered by the genetically engineered NSCs in the adult normal brain. Then, we injected genetically engineered NSCs into different sites of the animal model of human malignant glioma. Flowcytometry data showed that AAV2 had higher transduction efficiency in neural stem cells. The NSCs were engineered to express Decorin and Angiostatin by AAV2, and then injected into the hemisphere contralateral to tumor cell implantation. We demonstrated that genetically-modified NSCs have the capacity to migrate into brain tumors. The therapeutic efficacy of genetically-modified NSCs is remaining explored.
stem cell markers. To answer this, three different types of cancer cell lines were investigated by incubating in elevated temperature without CO2 supplement. They were rhebdomyosarcoma cell line (RD), cervical carcinoma cell line (HeLa), and mice mammary adenocarcenoma (AMN3). In all experiments cell lines were seeded in 12 well tissue culture plates and incubated at 37°C with 5% CO2 until they reach absolute confluent monolayer, after that subsets of plates incubated either at 40°C or 37°C without CO2 for 24 or 48 hr. Cultivation of cell lines in RPMI1640 at 37°C for 24hr without CO2 served as control untreated experiments. We followed changes in cells viability, phenotypic alterations, cologencity, and genetic markers of heat resistant (HSP90-beta and HIF), genetic markers of cancer stem cells (CD44, ALDH, Oct4, and CD133) by RT- qPCR . Results showed that upon incubation at 40°C for 24 hr or 48 hr without CO2 supplement cells were detached floated and acquired spherical shapes and aggregated. Noticeably the formed aggregation resembled to some extent the cell spheres that induced in cancer stem cells at sphere forming assay (fig.1,A,B). The count of viable floated cells for RD, HeLa, and AMN3 cells were 3.93E ̶ 6, 2.1E ̶ 6, and 5.7E ̶ 6 cell/ml at 40°C for 48 hr respectively, and generally it was temperature and time dependant (Fig 1,C). All viable floated cells from the cell lines investigated were capable to form colonies in colony forming assay, the plating efficiency for RD, HeLa, and AMN3 cells were 48%, 64%, and 57% after incubation at 37°C for 10 days with CO2 supplement respectively (Fig.1,D). Up regulation of heat stress gene HSP90-beta and HIF was detected in floated cells derived from all experiment and time of incubation. This was combined by up regulation of CSC markers CD44, ALDH, Oct4 and CD133 in floated cells derived from all experiment. These results may indicate a shift of non-CSC to CSC during heat stress and the increment of cancer stem cell population in the cell line under investigation which need more careful investigation.
679. Heat Stress Elevate Cell Population That Express Cancer Stem Cell Markers in Different Types of Cancer Cell Line
Amer T. Tawfeeq Al-Saeedi Molecular Biology, Iraqi Center for Cancer and Medical Genetics Research, Baghdad, Iraq Discovery of cancer stem cells (CSC) as driving force behind cancer cells tumorigenesis and heterogeneity in solid tumors led to a paradigm shift. The recognition of non-CSC to CSC transient introduces the cancer cell plasticity concept. This behavior appeared to be brought into existence majorly by the hostility of tumor microenvironment and stress imposed by hypoxia, pH, and nutrient depletion in the tumor niche. This recognition draw attention to the correlation between cancer hallmarks such as metastasis, chemotherapy, radiotherapy resistant and the transition of non-CSC to CSC by determination of CSC markers in cells subpopulations. Different types of cancer cell lines were shown to have a subset of cells expressing CSC markers such as CD44, ALDH, C133, Oct 3/4 with tumorigenicity in vivo. Recently, metabolic stress in a long-term nutrient deprivation showed to induces conversion of non-CSC to CSC like state. Additionally the hypoxic microenvironment was shown to upgraded stem-like properties of gastric cancer cells. Since it was recognized that metabolic alterations in tumors often combined by hyperthermia, we hypothesized that hyperthermia could be one of the stress components imposed by microenvironment that may manipulate non-CSC to CSC status and we asked if this elevation in temperature would affect phenotypic characteristics or up-regulate the genes responsible for stress resistant and the genes of cancer S268
680. TransfeX-Mediated HSV-tk/Ganciclovir Suicide Gene Therapy in HeLa Cervical Carcinoma and HSC-3, FaDu, and H357 Oral Cancer Cells Jennifer G. Cheung, Krystyna Konopka, Nejat Düzgüneş Biomedical Sciences, University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco, CA
INTRODUCTION: Herpes Simplex Virus thymidine kinase/ ganciclovir (HSV-tk/GCV)-induced suicide gene therapy has been used to successfully treat various cancers. TransfeX-mediated Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Despite intensive efforts exploring gene therapeutic approaches for human cancers, the clinical results in treating of malignant brain tumors are still poor. We demonstrated that the combined therapeutic strategy by using an adenoviral vector carrying the thymidine kinase (suicidal) gene and an adeno-associated viral vector carrying the angiostatin gene achieved better therapeutic results. However, the malignant brain tumor could not be completely eradicated. Therefore, novel vectors and strategies are urgently needed. Neural stem cells (NSCs) may be used either for cell replacement or for gene delivery vehicles in neurodegenerative diseases. The expression of therapeutic proteins by NSCs can be enhanced by viral-mediated gene transfer, though the effects of several common recombinant viruses on primary progenitor cell populations have not been widely tested. The goal of this study is to identify and develop a safe and effective NSCs-based gene therapy protocol to treat malignant glioma. We examined what seral type of the novel double-stranded recombinant adeno-associated viral (AAV) vector can efficiently deliver anti-angiogenic genes and suicidal gene into the rat NSCs. Meanwhile, we investigated the gene product delivered by the genetically engineered NSCs in the adult normal brain. Then, we injected genetically engineered NSCs into different sites of the animal model of human malignant glioma. Flowcytometry data showed that AAV2 had higher transduction efficiency in neural stem cells. The NSCs were engineered to express Decorin and Angiostatin by AAV2, and then injected into the hemisphere contralateral to tumor cell implantation. We demonstrated that genetically-modified NSCs have the capacity to migrate into brain tumors. The therapeutic efficacy of genetically-modified NSCs is remaining explored.
stem cell markers. To answer this, three different types of cancer cell lines were investigated by incubating in elevated temperature without CO2 supplement. They were rhebdomyosarcoma cell line (RD), cervical carcinoma cell line (HeLa), and mice mammary adenocarcenoma (AMN3). In all experiments cell lines were seeded in 12 well tissue culture plates and incubated at 37°C with 5% CO2 until they reach absolute confluent monolayer, after that subsets of plates incubated either at 40°C or 37°C without CO2 for 24 or 48 hr. Cultivation of cell lines in RPMI1640 at 37°C for 24hr without CO2 served as control untreated experiments. We followed changes in cells viability, phenotypic alterations, cologencity, and genetic markers of heat resistant (HSP90-beta and HIF), genetic markers of cancer stem cells (CD44, ALDH, Oct4, and CD133) by RT- qPCR . Results showed that upon incubation at 40°C for 24 hr or 48 hr without CO2 supplement cells were detached floated and acquired spherical shapes and aggregated. Noticeably the formed aggregation resembled to some extent the cell spheres that induced in cancer stem cells at sphere forming assay (fig.1,A,B). The count of viable floated cells for RD, HeLa, and AMN3 cells were 3.93E ̶ 6, 2.1E ̶ 6, and 5.7E ̶ 6 cell/ml at 40°C for 48 hr respectively, and generally it was temperature and time dependant (Fig 1,C). All viable floated cells from the cell lines investigated were capable to form colonies in colony forming assay, the plating efficiency for RD, HeLa, and AMN3 cells were 48%, 64%, and 57% after incubation at 37°C for 10 days with CO2 supplement respectively (Fig.1,D). Up regulation of heat stress gene HSP90-beta and HIF was detected in floated cells derived from all experiment and time of incubation. This was combined by up regulation of CSC markers CD44, ALDH, Oct4 and CD133 in floated cells derived from all experiment. These results may indicate a shift of non-CSC to CSC during heat stress and the increment of cancer stem cell population in the cell line under investigation which need more careful investigation.
679. Heat Stress Elevate Cell Population That Express Cancer Stem Cell Markers in Different Types of Cancer Cell Line
Amer T. Tawfeeq Al-Saeedi Molecular Biology, Iraqi Center for Cancer and Medical Genetics Research, Baghdad, Iraq Discovery of cancer stem cells (CSC) as driving force behind cancer cells tumorigenesis and heterogeneity in solid tumors led to a paradigm shift. The recognition of non-CSC to CSC transient introduces the cancer cell plasticity concept. This behavior appeared to be brought into existence majorly by the hostility of tumor microenvironment and stress imposed by hypoxia, pH, and nutrient depletion in the tumor niche. This recognition draw attention to the correlation between cancer hallmarks such as metastasis, chemotherapy, radiotherapy resistant and the transition of non-CSC to CSC by determination of CSC markers in cells subpopulations. Different types of cancer cell lines were shown to have a subset of cells expressing CSC markers such as CD44, ALDH, C133, Oct 3/4 with tumorigenicity in vivo. Recently, metabolic stress in a long-term nutrient deprivation showed to induces conversion of non-CSC to CSC like state. Additionally the hypoxic microenvironment was shown to upgraded stem-like properties of gastric cancer cells. Since it was recognized that metabolic alterations in tumors often combined by hyperthermia, we hypothesized that hyperthermia could be one of the stress components imposed by microenvironment that may manipulate non-CSC to CSC status and we asked if this elevation in temperature would affect phenotypic characteristics or up-regulate the genes responsible for stress resistant and the genes of cancer S268
680. TransfeX-Mediated HSV-tk/Ganciclovir Suicide Gene Therapy in HeLa Cervical Carcinoma and HSC-3, FaDu, and H357 Oral Cancer Cells Jennifer G. Cheung, Krystyna Konopka, Nejat Düzgüneş Biomedical Sciences, University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco, CA
INTRODUCTION: Herpes Simplex Virus thymidine kinase/ ganciclovir (HSV-tk/GCV)-induced suicide gene therapy has been used to successfully treat various cancers. TransfeX-mediated Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy