TY  - GEN
Y1  - 2018///
TI  - Dissecting tumor cell heterogeneity in 3D cell culture systems by combining imaging and next generation sequencing technologies
N2  - Three-dimensional (3D) in vitro cell culture systems have advanced the modeling of cellular processes in health and disease by reflecting physiological characteristics and architectural features of in vivo tissues. As a result, representative patient-derived 3D culture systems are emerging as advanced pre-clinical tumor models to support individualized therapy decisions. Beside the additional progress that has been achieved in molecular and pathological analyses towards personalized treatments, a remaining problem in both primary lesions and in vitro cultures is our limited understanding of functional tumor cell heterogeneity. This phenomenon is increasingly recognized as key driver of tumor progression and treatment resistance. Recent technological advances in next generation sequencing (NGS) have enabled unbiased identification of gene expression in low-input samples and single cells (scRNA-seq), thereby providing the basis to reveal cellular subtypes and drivers of cell state transitions. However, these methods generally require dissociation of tissues into single cell suspensions, which consequently leads to the loss of multicellular context. Thus, a direct or indirect combination of gene expression profiling with in situ microscopy is necessary for single cell analyses to precisely understand the association between complex cellular phenotypes and their underlying genetic programs.
In this thesis, I will present two complementing strategies based on combinations of NGS and microscopy to dissect tumor cell heterogeneity in 3D culture systems. First, I will describe the development and application of the new method ?pheno-seq? for integrated high-throughput imaging and transcriptomic profiling of clonal tumor spheroids derived from models of breast and colorectal cancer (CRC). By this approach, we revealed characteristic gene expression that is associated with heterogeneous invasive and proliferative behavior, identified transcriptional regulators that are missed by scRNA-seq, linked visual phenotypes and associated transcriptional signatures to inhibitor response and inferred single-cell regulatory states by deconvolution. Second, by applying scRNA-seq to 12 patient-derived CRC spheroid cultures, we identified shared expression programs that relate to intestinal lineages and revealed metabolic signatures that are linked to cancer cell differentiation. In addition, we validated and complemented sequencing results by quantitative microscopy using live-dyes and multiplexed RNA fluorescence in situ hybridization, thereby revealing metabolic compartmentalization and potential cell-cell interactions. 
Taken together, we believe that our approaches provide a framework for translational research to dissect heterogeneous transcriptional programs in 3D cell culture systems which will pave the way for a deeper understanding of functional tumor cell heterogeneity.
AV  - public
A1  - Tirier, Stephan Marius
ID  - heidok25591
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/25591/
ER  -