ABSTRACT
Molecular profiling of central nervous system lymphomas in cerebrospinal fluid (CSF) samples can be challenging due to the paucicellular and limited nature of the samples. Presented herein is a microfluidic platform for complete CSF lymphoid cell analysis, including single cell capture in sub-nanoliter traps, and molecular and chemotherapeutic response profiling via on-chip imaging, all in less than one hour. The system can detect scant lymphoma cells and quantitate their kappa/lambda immunoglobulin light chain restriction patterns. The approach can be further customized for measurement of additional biomarkers, such as those for differential diagnosis of lymphoma subtypes or for prognosis, as well as for imaging exposure to experimental drugs.
Subject(s)
Central Nervous System Neoplasms/diagnosis , Cerebrospinal Fluid/cytology , Drug Screening Assays, Antitumor/methods , Lymphoma/diagnosis , Microfluidics/methods , Central Nervous System Neoplasms/pathology , Humans , Lymphoma/pathologyABSTRACT
A number of Bruton's tyrosine kinase (BTK) inhibitors are currently in development, yet it has been difficult to visualize BTK expression and pharmacological inhibition in vivo in real time. We synthesized a fluorescent, irreversible BTK binder based on the drug Ibrutinib and characterized its behavior in cells and in vivo. We show a 200â nM affinity of the imaging agent, high selectivity, and irreversible binding to its target following initial washout, resulting in surprisingly high target-to-background ratios. In vivo, the imaging agent rapidly distributed to BTK expressing tumor cells, but also to BTK-positive tumor-associated host cells.
Subject(s)
Molecular Imaging , Protein Kinase Inhibitors/metabolism , Protein-Tyrosine Kinases/metabolism , Single-Cell Analysis , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Animals , Binding Sites , Cell Line, Tumor , Gene Expression , Genes, Reporter , Heterografts , Humans , Lymphoma/diagnosis , Lymphoma/metabolism , Mice , Models, Molecular , Molecular Conformation , Piperidines , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/metabolism , Pyrimidines/pharmacologySubject(s)
Enzyme Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors , Enzyme Inhibitors/chemical synthesis , Fluorine Radioisotopes/chemistry , Humans , Isotope Labeling , Phthalazines/chemistry , Piperazines/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Positron-Emission TomographySubject(s)
Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/methods , Phthalazines/chemistry , Piperazines/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , Green Fluorescent Proteins/genetics , Humans , Kinetics , Ligands , Molecular Structure , Phthalazines/pharmacology , Piperazines/pharmacology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , Protein Transport/drug effects , Transfection , Xanthenes/chemistryABSTRACT
'RNA bandages' are composed of two 6-12-mer 2'-OMe RNA strands complementary to a mRNA target that are joined by a photocleavable linker. These tandem oligonucleotides typically exhibit much higher affinity for the mRNA than the individual strands. An RNA bandage with binding arms of different lengths and a 4-base gap blocked translation in vitro of GFP mRNA; subsequent near-UV irradiation restored translation. This provides a general method of photomodulating hybridization for a variety of oligonucleotide-based technologies.
Subject(s)
Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/radiation effects , RNA, Messenger/chemistry , RNA/chemistry , Nucleic Acid Hybridization/radiation effects , Oligonucleotides, Antisense/chemistry , Protein Biosynthesis/drug effects , RNA/genetics , RNA, Messenger/genetics , Ribosomes/drug effects , Sequence Homology, Nucleic AcidABSTRACT
Previous studies have shown that B cell development is blocked at the pre-B cell stage in IFN regulatory factor (IRF)4 (pip) and IRF8 (IFN consensus sequence binding protein) double mutant mice (IRF4,8(-/-)). In this study, the molecular mechanism by which IRF4,8 regulate pre-B cell development was further investigated. We show that IRF4,8 function in a B cell intrinsic manner to control pre-B cell development. IRF4,8(-/-) mice expressing a Bcl-2 transgene fail to rescue pre-B cell development, suggesting that the defect in B cell development in IRF4,8(-/-) mice is not due to a lack of survival signal. IRF4,8(-/-) pre-B cells display a high proliferation index that may indirectly inhibit the L chain rearrangement. However, forced cell cycle exit induced by IL-7 withdrawal fails to rescue the development of IRF4,8(-/-) pre-B cells, suggesting that cell cycle exit by itself is not sufficient to rescue the development of IRF4,8(-/-) pre-B cells and that IRF4,8 may directly regulate the activation of L chain loci. Using retroviral mediated gene transduction, we show that IRF4 and IRF8 function redundantly to promote pre-B cell maturation and the generation of IgM(+) B cells. Molecular analysis indicates that IRF4, when expressed in IRF4,8(-/-) pre-B cells, induces kappa germline transcription, enhances V(D)J rearrangement activity at the kappa locus, and promotes L chain rearrangement and transcription. Chromatin immunoprecipitation assay further reveals that IRF4 expression leads to histone modifications and enhanced chromatin accessibility at the kappa locus. Thus, IRF4,8 control pre-B cell development, at least in part, by promoting the activation of the kappa locus.
