Project description:Single clones were isolated from the Detroit 562 cell line and implanted into NOD/SCID mice to determine whether clonal populations within one cell line had fuctionally distinct phenotypes, in the form of differing tumour initiating activity. From these data I found that different clonal populations had different tumour initiating activity, which lead to this investigation as to whether these functionally distinct populations were also genetically distinct.
Project description:We report changes in H3K27ac following LPS stimulation in Detroit 562 cells. We were able to identified LPS-increased H3K27ac regions which correlated with RELA binding as well as gene up-regulation. This data set is relevant for airborne bacterial sensing as Detroit 562 cells are nasopharyngeal epithelial cells and LPS is a gram negative bacterial endotoxin.
Project description:A total of 432 genes were found to be differentially expressed in M1SF370 bacterial population internalized in Detroit 562 human pharyngeal cells when compared with the same strain incubated in the absence of Detroit 562 cells. While most of them (349/432 i.e. 80.8%) were up regulated, 83 genes were down regulated contributing to 19.2% of the total differentiated genes. The major contributor of the latter category was phage-related genes (35 genes). Almost ¼ of these genes (106) belonged to a category of Unknown or possible predicted function. Most notably, up-regulated genes belonged to amino acid transport , cell division, cell envelope biogenesis, DNA replication correlated well with up-regulated 67 genes belonged to translation and ribosomal structure. Further, up-regulation of 12/15 virulence-related genes indicated that human host cell internalized bacteria are highly virulent as compared to laboratory grown culture in test-tubes. S. pyogenes strain type M1 SF370 (wild-type) was procured from ATCC (ATCC 700294). Detroit 562 pharyngeal cells were obtained from ATCC and maintained in MEM with 10% FBS in humidified CO2-incubator. Purified cDNA preparations from the Detroit cells-internalized bacteria and that were cultured without Detroit cells, were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using four independently isolated RNA preparations (biological replicates), a total of 8 experiments (incorporating 4 dye swaps) were performed. Accordingly eight hybridization measurements for this mutant were obtained. Exp-1 and -2 (GSM687276, GSM687310-dye swap) are the technical replicates of the biological sample-1, Exp-3 and -4 (GSM687311, GSM687312-dye swap) are technical replicates of biological sample-2, Exp-5 and -6, (GSM687313,GSM687319-dye swap,) are technical replicates of the biological sample-3, and finally Exp-7 and -8, (GSM687320,GSM687321-dye swap) are technical replicates of the biological sample-4. .
Project description:A total of 432 genes were found to be differentially expressed in M1SF370 bacterial population internalized in Detroit 562 human pharyngeal cells when compared with the same strain incubated in the absence of Detroit 562 cells. While most of them (349/432 i.e. 80.8%) were up regulated, 83 genes were down regulated contributing to 19.2% of the total differentiated genes. The major contributor of the latter category was phage-related genes (35 genes). Almost ¼ of these genes (106) belonged to a category of Unknown or possible predicted function. Most notably, up-regulated genes belonged to amino acid transport , cell division, cell envelope biogenesis, DNA replication correlated well with up-regulated 67 genes belonged to translation and ribosomal structure. Further, up-regulation of 12/15 virulence-related genes indicated that human host cell internalized bacteria are highly virulent as compared to laboratory grown culture in test-tubes.
Project description:Divergence of K-562 genomes through in vitro clonal evolution revealed by comparing three sublines. Comparison of three K-562 sublines
Project description:The remarkable evolutionary capacity of cancer poses major challenges to current therapeutic efforts, and results from the vast clonal heterogeneity and ability of individual cancer cells to adapt to diverse selective pressures. More complete mechanistic understanding of the basis of therapeutic resistance has been limited by difficulties in coupling information regarding genetic and epigenetic alterations present within individual cells to their respective transcriptomic and functional outputs. To this end, we developed a novel high-complexity expressed barcode system, ClonMapper, that integrates DNA barcoding with single-cell RNA-sequencing and clonal isolation to characterize thousands of clones within a mixed cancer cell population. In applying this system to the chronic lymphocytic leukemia cell line HG3 in the setting of resistance to fludarabine-based chemotherapy, we discover pretreatment sub-populations with distinct expression profiles (i.e. upregulated Wnt, Notch and CXCR4 signaling) that not only confer distinct treatment survivorship trajectories, but also provide the basis for long-term clonal equilibrium between co-existing clones in the absence of treatment. Characterization of individual clones comprising these sub-populations revealed remarkable genetic heterogeneity and the persistence of unique transcriptomic signatures throughout treatment exposure. These data reveal the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population and highlight the unprecedented resolution that can be achieved using ClonMapper.
Project description:The remarkable evolutionary capacity of cancer poses major challenges to current therapeutic efforts, and results from the vast clonal heterogeneity and ability of individual cancer cells to adapt to diverse selective pressures. More complete mechanistic understanding of the basis of therapeutic resistance has been limited by difficulties in coupling information regarding genetic and epigenetic alterations present within individual cells to their respective transcriptomic and functional outputs. To this end, we developed a novel high-complexity expressed barcode system, ClonMapper, that integrates DNA barcoding with single-cell RNA-sequencing and clonal isolation to characterize thousands of clones within a mixed cancer cell population. In applying this system to the chronic lymphocytic leukemia cell line HG3 in the setting of resistance to fludarabine-based chemotherapy, we discover pretreatment sub-populations with distinct expression profiles (i.e. upregulated Wnt, Notch and CXCR4 signaling) that not only confer distinct treatment survivorship trajectories, but also provide the basis for long-term clonal equilibrium between co-existing clones in the absence of treatment. Characterization of individual clones comprising these sub-populations revealed remarkable genetic heterogeneity and the persistence of unique transcriptomic signatures throughout treatment exposure. These data reveal the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population and highlight the unprecedented resolution that can be achieved using ClonMapper.
Project description:The remarkable evolutionary capacity of cancer poses major challenges to current therapeutic efforts, and results from the vast clonal heterogeneity and ability of individual cancer cells to adapt to diverse selective pressures. More complete mechanistic understanding of the basis of therapeutic resistance has been limited by difficulties in coupling information regarding genetic and epigenetic alterations present within individual cells to their respective transcriptomic and functional outputs. To this end, we developed a novel high-complexity expressed barcode system, ClonMapper, that integrates DNA barcoding with single-cell RNA-sequencing and clonal isolation to characterize thousands of clones within a mixed cancer cell population. In applying this system to the chronic lymphocytic leukemia cell line HG3 in the setting of resistance to fludarabine-based chemotherapy, we discover pretreatment sub-populations with distinct expression profiles (i.e. upregulated Wnt, Notch and CXCR4 signaling) that not only confer distinct treatment survivorship trajectories, but also provide the basis for long-term clonal equilibrium between co-existing clones in the absence of treatment. Characterization of individual clones comprising these sub-populations revealed remarkable genetic heterogeneity and the persistence of unique transcriptomic signatures throughout treatment exposure. These data reveal the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population and highlight the unprecedented resolution that can be achieved using ClonMapper.
Project description:The remarkable evolutionary capacity of cancer poses major challenges to current therapeutic efforts, and results from the vast clonal heterogeneity and ability of individual cancer cells to adapt to diverse selective pressures. More complete mechanistic understanding of the basis of therapeutic resistance has been limited by difficulties in coupling information regarding genetic and epigenetic alterations present within individual cells to their respective transcriptomic and functional outputs. To this end, we developed a novel high-complexity expressed barcode system, ClonMapper, that integrates DNA barcoding with single-cell RNA-sequencing and clonal isolation to characterize thousands of clones within a mixed cancer cell population. In applying this system to the chronic lymphocytic leukemia cell line HG3 in the setting of resistance to fludarabine-based chemotherapy, we discover pretreatment sub-populations with distinct expression profiles (i.e. upregulated Wnt, Notch and CXCR4 signaling) that not only confer distinct treatment survivorship trajectories, but also provide the basis for long-term clonal equilibrium between co-existing clones in the absence of treatment. Characterization of individual clones comprising these sub-populations revealed remarkable genetic heterogeneity and the persistence of unique transcriptomic signatures throughout treatment exposure. These data reveal the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population and highlight the unprecedented resolution that can be achieved using ClonMapper.