12 research outputs found
scPred: accurate supervised method for cell-type classification from single-cell RNA-seq data
Single-cell RNA sequencing has enabled the characterization of highly specific cell types in many tissues, as well as both primary and stem cell-derived cell lines. An important facet of these studies is the ability to identify the transcriptional signatures that define a cell type or state. In theory, this information can be used to classify an individual cell based on its transcriptional profile. Here, we present scPred, a new generalizable method that is able to provide highly accurate classification of single cells, using a combination of unbiased feature selection from a reduced-dimension space, and machine-learning probability-based prediction method. We apply scPred to scRNA-seq data from pancreatic tissue, mononuclear cells, colorectal tumor biopsies, and circulating dendritic cells and show that scPred is able to classify individual cells with high accuracy. The generalized method is available at https://github.com/powellgenomicslab/scPred
Dynamic Immune Modulation Seen By Single Cell RNA-Sequencing of Serial Lymphoma Biopsies in Patients Undergoing in Situ Vaccination
Applying the chicken embryo chorioallantoic membrane assay to study treatment approaches in urothelial carcinoma
Additional file 1 of GITR and TIGIT immunotherapy provokes divergent multicellular responses in the tumor microenvironment of gastrointestinal cancers
Additional file 1. Supplementary tables S1 – 13
Parallel PI3K, AKT and mTOR inhibition is required to control feedback loops that limit tumor therapy
<div><p>Targeting the PI3K pathway has achieved limited success in cancer therapy. One reason for the disappointing activity of drugs that interfere with molecules that are important player in this pathway is the induction of multiple feedback loops that have been only partially understood. To understand these limitations and develop improved treatment strategies, we comprehensively characterized molecular mechanisms of PI3K pathway signaling in bladder cancer cell lines upon using small molecule inhibitors and RNAi technologies against all key molecules and protein complexes within the pathway and analyzed functional and molecular consequences. When targeting either mTORC1, mTOR, AKT or PI3K, only S6K1 phosphorylation was affected in most cell lines examined. Dephosphorylation of 4E-BP1 required combined inhibition of PI3K and mTORC1, independent from AKT, and resulted in a robust reduction in cell viability. Long-term inhibition of PI3K however resulted in a PDK1-dependent, PIP3 and mTORC2 independent rephosphorylation of AKT. AKT rephosphorylation could also be induced by mTOR or PDK1 inhibition. Combining PI3K/mTOR inhibitors with AKT or PDK1 inhibitors suppressed this rephosphorylation, induced apoptosis, decreased colony formation, cell viability and growth of tumor xenografts. Our findings reveal novel molecular mechanisms that explain the requirement for simultaneous targeting of PI3K, AKT and mTORC1 to achieve effective tumor growth inhibition.</p></div
Multiple targeting of the PI3K signaling pathway inhibits 3-dimensional tumor growth.
<p>RT112 cells were grown on the CAM as xenografts and were treated with 1000 nM MK-2206, 2000 nM PIK-90, 5 nM RAD001, 200 nM NVP-BEZ235, their indicated combinations or control (ctrl). (A) Tumor weight shown as percentage of control from 7–21 tumors per condition in two independent experiments. Horizontal line indicates median, upper whisker indicates the difference between maximum and first quartile, and lower whisker indicates the difference between minimum and third quartile. * indicates p < 0.05. (B) Ki-67 positive cells were quantified from three fields from at least three tumors treated with the indicated inhibitors and expressed as a percentage. * indicates p < 0.05 (unpaired Student’s T-test). (C) Representative images of tumor sections treated with respective inhibitors and stained with Ki-67 antibody by immunohistochemistry. Scale bar indicates 20 um.</p
Inhibition of PI3K results in a positive feedback loop on AKT.
<p>RT112 cells were treated with 200 nM NVP-BEZ235 for the indicated duration or with control (ctrl). (A) Cells that were treated for 24 and 48 hours were additionally retreated with the same concentration for 1 hour (indicated by +, no retreatment indicated by -) and immunoblotting was performed with the respective antibodies (B) NVP-BEZ235 or control treatment was combined with 500 nM of GSK2334470 for the indicated duration or with control (ctrl). Immunoblotting was performed with the indicated antibodies. Results are representative of at least three independent experiments. (C) T24 cells were treated with control (ctrl) or 100 nM of NVP-BEZ235 for 1 or 24 hours. PIP3 and PI(4,5)P2 were extracted and the quantity was determined by ELISA. Results represent the mean +/- standard deviation of triplicate wells and indicate the PIP3/PI(4,5)P2 relative to control expressed as percentage. Results are representative of two independent experiments. * indicates p < 0.05 (unpaired Student’s T-test). (D-F) Cells were treated with control (ctrl), 500 nM GSK2334470, 500 nM PIK-90, 25 nM INK128 or 10 nM NVP-BEZ235 for the indicated duration and immunoblotting was performed on lysates with the indicated antibodies. Results are representative of at least three independent experiments.</p
Effect of PI3K and PI3K/mTOR inhibitors on bladder cancer cell lines.
<p>(A-C) Cells were treated with indicated concentrations of PIK-90, BKM-120 or INK128 for 1 hour and immunoblotting was performed on lysates with the denoted antibodies. (D) Cell lysates and immunoblotting were performed with inhibitor or control treated cells for 1 hour after transduction with control (ctrl) or mTOR shRNA for 72 hours. (E) Cells were treated with respective inhibitors using indicated concentrations for 72 hours and cell viability assay was performed. Results indicate the mean +/- standard error of relative cell fluorescence in arbitrary units expressed as a percentage of control. All results are representative of at least three independent experiments.</p
PI3K and mTORC1 regulate phosphorylation of 4E-BP1.
<p>Cells were treated with respective inhibitors at the indicated concentrations for 1 hour and immunoblotting was performed on lysates with the denoted antibodies (a) or cell viability was detected after 72 hours of treatment. Results indicate the mean +/- standard error of relative cell fluorescence in arbitrary units expressed as a percentage of control from three independent experiments. (c) Cells were transduced with control (ctrl) or mTOR shRNA for 72 hours, or (d-f) transfected with control (ctrl), Raptor or Rictor siRNAs for 48 hours. Cells were then treated with control or PIK-90 or MK-2206 at the indicated concentrations for 1 hour and immunoblotting was performed on lysates with the denoted antibodies. Normalized p4E-BP1 Thr 37/46 was calculated as percentage of control. Results are representative of at least three independent experiments.</p
The combination of dual PI3K and AKT inhibitors regulate colony formation and apoptosis.
<p>(A) RT112 cells were treated with control (ctrl), 1000 nM MK-2206, 500 nM GSK2334470, 200 nM NVP-BEZ235 or their combinations for 12 days and colonies were stained with 0.5% crystal violet. (B) Quantification of surviving colonies in duplicate samples relative to control expressed as a percentage and three independent experiments. (C) Cells were lysed and immunoblotting was performed with the indicated antibodies after treating cells with the respective inhibitors or control (ctrl) for the indicated time points. Results are representative of at least three independent experiments. (D) RT112, T24 or 647V cells were treated with 200 nM, 100 nM and 200 nM NVP-BEZ235 respectively, 1000 nM MK-2206, their combination or control (ctrl) for 48 hours and caspase 3/7 assay was performed. Results obtained in arbitrary luminescence units were normalized to the number of living cells per trypan blue staining conducted in parallel and indicate the mean +/- standard error of three independent experiments relative to control expressed as a percentage. * indicates p < 0.05 (unpaired Student’s T-test.</p