Tracing Clonal Dynamics Reveals that Two- and Three-dimensional Patient-derived Cell Models Capture Tumor Heterogeneity of Clear Cell Renal Cell Carcinoma.

BACKGROUND: Extensive DNA sequencing has led to an unprecedented view of the diversity of individual genomes and their evolution among patients with clear cell renal cell carcinoma (ccRCC). OBJECTIVE: To understand subclonal architecture and dynamics of patient-derived two-dimensional (2D) and three-dimensional (3D) ccRCC models in vitro, in order to determine whether they mirror ccRCC inter- and intratumor heterogeneity. DESIGN, SETTING, AND PARTICIPANTS: We have established a comprehensive platform of living renal cancer cell models from ccRCC surgical specimens. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We confirmed the concordance of 2D and 3D patient-derived cell (PDC) models with the original tumor tissue in terms of histology, biomarker expression, cancer driver mutations, and copy number alterations. We addressed inter- and intrapatient heterogeneity by analyzing clonal dynamics during serial passaging. RESULTS AND LIMITATIONS: In-depth genetic characterization verified the presence of heterogeneous cell populations, and revealed a high degree of similarity between subclonal compositions of monolayer and organoid cell cultures and the corresponding parental ccRCCs. Clonal dynamics were evident during serial passaging of cells in vitro, suggesting that PDC cultures can offer insights into evolutionary potential and treatment susceptibility of ccRCC subclones in vivo. Proof-of-concept drug profiling using selected ccRCC-targeted therapy agents highlighted patient-specific vulnerabilities in PDC models that could not be anticipated by interrogating commercially available cell lines. CONCLUSIONS: We demonstrate that PDC models mirror inter- and intratumor heterogeneity of ccRCC in vitro. Based on our findings, we envision that the use of these models will advance our understanding of the trajectories that cause genetic diversity and their consequences for treatment on an individual level. PATIENT SUMMARY: In this study, we developed two- and three-dimensional patient-derived models from clear cell renal cell carcinoma (ccRCC) as "mini-tumors in a dish." We show that these cell models retain important features of the human ccRCCs such as the profound tumor heterogeneity, thus highlighting their importance for cancer research and precision medicine.

A Framework for Optimizing High-Content Imaging of 3D Models for Drug Discovery.

Three-dimensional (3D) spheroid models are rapidly gaining favor for drug discovery applications due to their improved morphological characteristics, cellular complexity, long lifespan in culture, and higher physiological relevance relative to two-dimensional (2D) cell culture models. High-content imaging (HCI) of 3D spheroid models has the potential to provide valuable information to help researchers untangle disease pathophysiology and assess novel therapies more effectively. The transition from 2D monolayer models to dense 3D spheroids in HCI applications is not trivial, however, and requires 3D-optimized protocols, instrumentation, and resources. Here, we discuss considerations for moving from 2D to 3D models and present a framework for HCI and analysis of 3D spheroid models in a drug discovery setting. We combined scaffold-free, multicellular spheroid models with scalable, automation-compatible plate technology enabling image-based applications ranging from high-throughput screening to more complex, lower-throughput microphysiological systems of organ networks. We used this framework in three case studies: investigation of lipid droplet accumulation in a human liver nonalcoholic steatohepatitis (NASH) model, real-time immune cell interactions in a multicellular 3D lung cancer model, and a high-throughput screening application using a 3D co-culture model of gastric carcinoma to assess dose-dependent drug efficacy and specificity. The results of these proof-of-concept studies demonstrate the potential for high-resolution image-based analysis of 3D spheroid models for drug discovery applications, and confirm that cell-level and temporal-spatial analyses that fully exploit multicellular features of spheroid models are not only possible but soon will be routine practice in drug discovery workflows.

In vitro cell culture of patient derived malignant pleural and peritoneal effusions for personalised drug screening.

BACKGROUND: Malignant serous effusion (MSE) denotes a manifestation of metastatic disease with typical high concentrations of both cancer and immune cells, making them an ideal resource for in vitro cytologic studies. Hence, the aim of the study was to investigate the features of 2D and 3D MSE culture systems as well as their feasibilities for in vitro drug screening. METHODS: Pleural and peritoneal effusions from 8 patients were collected and processed for 2D monolayer and 3D hanging drop cell culture into GravityPLUS™ plates. Representative markers for cell components, proliferation rate and tumour classification were investigated by immunohistochemistry, followed by absolute quantification using a digitalised image analysis approach. Further, we implemented another 3D cell culture model based on a low attachment method for in vitro drug sensitivity testing of carboplatin, pemetrexed and pembrolizumab for 5 patients. RESULTS: Monolayer cell culture was favourable for the growth of mesothelial cells, while hanging drop culture in GravityPLUS™ plates showed better ability for preserving cancer cells, inducing positive diagnostic markers expression and restraining the growth of mesothelial cells. For in vitro drug testing, MSE from five patients presented various drug sensitivities, and one case showed strong response to PD-1 checkpoint inhibition (pembrolizumab). For some patients, the application of combinatorial drugs had better therapeutic responses compared to monotherapy. CONCLUSIONS: Digitalised quantification of data offers a better understanding of different MSE culture models. More importantly, the proposed platforms are practical and amenable for performing in vitro chemo-/immunotherapeutic drug testing by using routine cytologic MSE in a personalised manner. Next to cell blocks, our work demonstrates the prognostic and predictive value of cytologic effusion samples.

Transcriptional regulation of tenascin-W by TGF-beta signaling in the bone metastatic niche of breast cancer cells.

Tenascin-W is a matricellular protein with a dynamically changing expression pattern in development and disease. In adults, tenascin-W is mostly restricted to stem cell niches, and is also expressed in the stroma of solid cancers. Here, we analyzed its expression in the bone microenvironment of breast cancer metastasis. Osteoblasts were isolated from tumor-free or tumor-bearing bones of mice injected with MDA-MB231-1833 breast cancer cells. We found a fourfold upregulation of tenascin-W in the osteoblast population of tumor-bearing mice compared to healthy mice, indicating that tenascin-W is supplied by the bone metastatic niche. Transwell and co-culture studies showed that human bone marrow stromal cells (BMSCs) express tenascin-W protein after exposure to factors secreted by MDA-MB231-1833 breast cancer cells. To study tenascin-W gene regulation, we identified and analyzed the tenascin-W promoter as well as three evolutionary conserved regions in the first intron. 5'RACE analysis of mRNA from human breast cancer, glioblastoma and bone tissue showed a single tenascin-W transcript with a transcription start site at a noncoding first exon followed by exon 2 containing the ATG translation start. Site-directed mutagenesis of a SMAD4-binding element in proximity of the TATA box strongly impaired promoter activity. TGFß1 induced tenascin-W expression in human BMSCs through activation of the TGFß1 receptor ALK5, while glucocorticoids were inhibitory. Our experiments show that tenascin-W acts as a niche component for breast cancer metastasis to bone by supporting cell migration and cell proliferation of the cancer cells.

Transcriptional regulation of tenascin genes.

Extracellular matrix proteins of the tenascin family resemble each other in their domain structure, and also share functions in modulating cell adhesion and cellular responses to growth factors. Despite these common features, the 4 vertebrate tenascins exhibit vastly different expression patterns. Tenascin-R is specific to the central nervous system. Tenascin-C is an "oncofetal" protein controlled by many stimuli (growth factors, cytokines, mechanical stress), but with restricted occurrence in space and time. In contrast, tenascin-X is a constituitive component of connective tissues, and its level is barely affected by external factors. Finally, the expression of tenascin-W is similar to that of tenascin-C but even more limited. In accordance with their highly regulated expression, the promoters of the tenascin-C and -W genes contain TATA boxes, whereas those of the other 2 tenascins do not. This article summarizes what is currently known about the complex transcriptional regulation of the 4 tenascin genes in development and disease.