Project description:Glioblastoma (GBM) is among the most aggressive cancers. Despite aggressive radiotherapy and treatment with the alkylating agent temozolomide (TMZ), patients ultimately succumb to the disease. Although much interest has focused on highly tumorigenic GBM stem cells (GSCs), adaption of a concept from microbial research proposes that a minor population of dormant âpersisterâ cells in cancer evade current therapies. To separate dormant and treatment-resistant tumor cells in human GBM tumorspheres, we have refined density gradient protocols previously used for separation of neurosphere-forming neural stem cells (NSCs). We find that a minor cell population in human GBM tumorsphere cultures and patient-derived tumor biopsies display increased cell density. These high-density GBM cells (HDGCs) display dormancy, variable expression of proposed GSC markers, and 10-100 fold higher levels of reprogramming gene expression compared to low-density GBM cells (LDGCs). Transcriptional profiling data confirmed the slow-cycling state of HDGCs. As a result, HDGCs show decreased tumorsphere formation capacity in vitro and reduced tumorigenicity in vivo. Using tumorspheres and xenografts, we demonstrated that HDGCs show increased treatment-resistance to ionizing radiation (IR) and temozolomide treatment compared to LDGCs. Similar to the NSC lineage, our data suggest that dormant HDGCs become increasingly sensitive to anti-proliferative therapies as they become activated and further differentiate. In conclusion, density gradients represents a marker-independent approach to separate dormant and treatment-resistant tumor cells in human GBMs and other solid cancers. 12 samples, no replicates, derived from 5 individual patients
Project description:Glioblastoma (GBM) is among the most aggressive cancers. Despite aggressive radiotherapy and treatment with the alkylating agent temozolomide (TMZ), patients ultimately succumb to the disease. Although much interest has focused on highly tumorigenic GBM stem cells (GSCs), adaption of a concept from microbial research proposes that a minor population of dormant “persister” cells in cancer evade current therapies. To separate dormant and treatment-resistant tumor cells in human GBM tumorspheres, we have refined density gradient protocols previously used for separation of neurosphere-forming neural stem cells (NSCs). We find that a minor cell population in human GBM tumorsphere cultures and patient-derived tumor biopsies display increased cell density. These high-density GBM cells (HDGCs) display dormancy, variable expression of proposed GSC markers, and 10-100 fold higher levels of reprogramming gene expression compared to low-density GBM cells (LDGCs). Transcriptional profiling data confirmed the slow-cycling state of HDGCs. As a result, HDGCs show decreased tumorsphere formation capacity in vitro and reduced tumorigenicity in vivo. Using tumorspheres and xenografts, we demonstrated that HDGCs show increased treatment-resistance to ionizing radiation (IR) and temozolomide treatment compared to LDGCs. Similar to the NSC lineage, our data suggest that dormant HDGCs become increasingly sensitive to anti-proliferative therapies as they become activated and further differentiate. In conclusion, density gradients represents a marker-independent approach to separate dormant and treatment-resistant tumor cells in human GBMs and other solid cancers.
Project description:This experiment investigates the effect of mild cold-shock (32 degrees C, 24 hours) on the translatome of human HEK293 cells. After cold-shock, cells were treated with cycloheximide to block elongation, then sucrose density gradients were used to separate post-nuclear extracts into polysome and subpolysome fractions. The pooled polysomal fraction was labelled with Cy5, the pooled subpolysomal fraction was labelled with Cy3, and both were hybridised to the same array.
Project description:Cellular dormancy and heterogeneous cell cycle lengths provide important explanations for treatment failure following adjuvant therapy with S-phase cytotoxics in colorectal cancer (CRC) yet the molecular control of the dormant versus cycling state remains unknown. In CRCs dormant cells are found to be highly clonogenic and resistant to chemotherapies. We sought to understand the molecular features of dormant CRC cells to facilitate rationale identification of compounds to target both dormant and cycling tumour cells.
Project description:affy_tour_2012-02 - Identification of transcripts that are addressed to traduction in imbibed seeds in relation with dormancy: comparison of the translatome in Dormant versus Non-Dormant seeds -- At harvest seeds are dormant. They stay dormant if they are stored at -20°C (D) and become non-dormant (ND) if they are stored 2 months at +20°C. Polysomal fractions were purified on sucrose gradients from sunflower axis isolated from dormant and non-dormant seeds imbibed at 10°C during 3h, 15h or 24h. - These fractions allow to identify the transcripts addressed to translation (translatome) during the seed imbibition process (3, 15 and 24h) - The translatome of 2 types of seeds are compared: Dormant vs Non-Dormant at the 3 time points. 18 arrays - SUNFLOWER; time course,treated vs untreated comparison
Project description:affy_tour_2012-02 - Identification of transcripts that are addressed to traduction in imbibed seeds in relation with dormancy: comparison of the translatome in Dormant versus Non-Dormant seeds -- At harvest seeds are dormant. They stay dormant if they are stored at -20°C (D) and become non-dormant (ND) if they are stored 2 months at +20°C. Polysomal fractions were purified on sucrose gradients from sunflower axis isolated from dormant and non-dormant seeds imbibed at 10°C during 3h, 15h or 24h. - These fractions allow to identify the transcripts addressed to translation (translatome) during the seed imbibition process (3, 15 and 24h) - The translatome of 2 types of seeds are compared: Dormant vs Non-Dormant at the 3 time points.
Project description:au13-12_polysome - transcriptome and translatome of arabidopsis wt seeds according to dormancy - Identification of transcripts that are differentially abundant (transcriptome) and transcripts that are addressed to translation (translatome) in imbibed Arabidopsis seeds in relation with dormancy. During imbibition of seeds (16h and 24h in darkness at 25°C, dormant and non-dormant seeds), transcriptome analysis is done on total RNA and translatome analysis on polysomal RNA. - At harvest seeds are dormant. They stay dormant if they are stored at -20°C (D) and become non-dormant (ND) if they are stored 3 weeks at +20°C. Arabidopsis dormant seeds do not germinate at 25°C in darkness while non-dormant seeds do. Total RNA and polysomal RNA (polysomal fractions purified on sucrose gradients) were extracted from imbibed seeds for 16h or 24h at 25°C in darkness (3 biological replicates). Transcriptome and translatome are compared for Dormant vs Non-Dormant for 16h and 24 imbibition. In silico comparison will allow to compare transcriptome and translatome for each point and type of seeds and to compare the time points (16 vs 24h) for each type of sample. 12 dye-swap - time course