ABSTRACT
High-grade serous ovarian carcinoma (HGSOC) is genetically unstable and characterised by the presence of subclones with distinct genotypes. Intratumoural heterogeneity is linked to recurrence, chemotherapy resistance, and poor prognosis. Here, we use spatial transcriptomics to identify HGSOC subclones and study their association with infiltrating cell populations. Visium spatial transcriptomics reveals multiple tumour subclones with different copy number alterations present within individual tumour sections. These subclones differentially express various ligands and receptors and are predicted to differentially associate with different stromal and immune cell populations. In one sample, CosMx single molecule imaging reveals subclones differentially associating with immune cell populations, fibroblasts, and endothelial cells. Cell-to-cell communication analysis identifies subclone-specific signalling to stromal and immune cells and multiple subclone-specific autocrine loops. Our study highlights the high degree of subclonal heterogeneity in HGSOC and suggests that subclone-specific ligand and receptor expression patterns likely modulate how HGSOC cells interact with their local microenvironment.
Subject(s)
Ovarian Neoplasms , Tumor Microenvironment , Humans , Female , Tumor Microenvironment/genetics , Endothelial Cells/metabolism , Ovarian Neoplasms/pathology , Gene Expression Profiling , DNA Copy Number VariationsABSTRACT
There are an estimated > 400 million people living with a rare disease globally, with genetic variants the cause of approximately 80% of cases. Next Generation Sequencing (NGS) rapidly identifies genetic variants however they are often of unknown significance. Low throughput functional validation in specialist laboratories is the current ad hoc approach for functional validation of genetic variants, which creating major bottlenecks in patient diagnosis. This study investigates the application of CRISPR gene editing followed by genome wide transcriptomic profiling to facilitate patient diagnosis. As proof-of-concept, we introduced a variant in the Euchromatin histone methyl transferase (EHMT1) gene into HEK293T cells. We identified changes in the regulation of the cell cycle, neural gene expression and suppression of gene expression changes on chromosome 19 and chromosome X, that are in keeping with Kleefstra syndrome clinical phenotype and/or provide insight into disease mechanism. This study demonstrates the utility of genome editing followed by functional readouts to rapidly and systematically validating the function of variants of unknown significance in patients suffering from rare diseases.
Subject(s)
Craniofacial Abnormalities/diagnosis , Gene Editing/methods , Gene Expression Profiling/methods , Gene Regulatory Networks , Heart Defects, Congenital/diagnosis , Histone-Lysine N-Methyltransferase/genetics , Intellectual Disability/diagnosis , CRISPR-Cas Systems , Chromosome Deletion , Chromosomes, Human, Pair 19/genetics , Chromosomes, Human, Pair 9/genetics , Chromosomes, Human, X/genetics , Craniofacial Abnormalities/genetics , Early Diagnosis , Gene Expression Regulation , Genetic Variation , HEK293 Cells , Heart Defects, Congenital/genetics , Humans , Intellectual Disability/genetics , Proof of Concept Study , Sequence Analysis, RNAABSTRACT
Since immunoglobulins are conformationally dynamic molecules in solution, we studied the effect of stabilizing and destabilizing excipients on the conformational stability and dynamics of two IgG1 monoclonal antibodies (mAbs; mAb-A and mAb-B) using a variety of biophysical approaches. Even though the two mAbs are of the same IgG1 subtype, the unfolding patterns, aggregation behavior, and pretransition dynamics of these two antibodies were strikingly different in response to external perturbations such as pH, temperature, and presence of excipients. Sucrose and arginine were identified as stabilizers and destabilizers, respectively, on the basis of their influence on conformational stability for both the IgG1 mAbs. The two excipients, however, had distinct effective concentrations and different effects on the conformational stability and pretransition dynamics of the two mAbs as measured by a combination of differential scanning calorimetry, high-resolution ultrasonic spectroscopy, and red-edge excitation shift fluorescence studies. Stabilizing concentrations of sucrose were found to decrease the internal motions of mAb-B, whereas arginine marginally increased its adiabatic compressibility in the pretransition region. Both sucrose and arginine did not influence the pretransition dynamics of mAb-A. The potential reasons for such differences in excipient effects between two IgG1 mAbs are discussed.
