Identifying somatic changes in drug transporters using whole genome and transcriptome sequencing data of advanced tumors

Drug resistance is a perpetual problem in cancer therapy with many underlying mechanisms. Alterations in drug transport over the cancer cell membrane can severely alter intratumoral drug exposure, contributing to resistance. Here, we present the somatic mutational landscape of 48 ATP-binding cassette and 416 solute carrier transporter genes in a cohort (CPCT-02; NCT01855477) of 3290 patients with different types of advanced and metastasized cancer through analysis of whole genome and transcriptome sequencing. In order to identify potential stressor mechanisms, we stratified patients based on previous systemic therapies and subsequently investigated the enrichment of mutations and copy-number alterations of transporter genes. In tumors from patients pretreated with protein kinase inhibitors (PKIs), genes encoding for specific copper (SLC31A1 and SLC31A2, χ2-test adjusted p-values: 6.9e-09 and 2.5e-09) and nucleoside transporters (SLC28A2 and SLC28A3, χ2-test adjusted p-values: 3.5e-06 and 6.8e-07) were deleted significantly more frequently than in patients pretreated with chemotherapy. Moreover, we detected 16 transporters that were differentially expressed at RNA level between these treatment groups. These findings contradict mechanisms of selective pressure, as they would be expected to originate during treatment with chemotherapy rather than with PKIs. Hence, they might constitute primary drug resistance mechanisms and, therefore, warrant further study.</p

Integrated 3D Acid Fracturing Model for Carbonate Reservoir Stimulation

Acid fracturing is one of the stimulation methods used in carbonate formations and has been proved effective and economical. Because of the stochastic nature of acidizing in carbonate formation, designing and optimizing acid fracture treatment today still remain challenging. In the past, a simple acid fracture conductivity correlation was usually considered sufficient to estimate the overall average fracture conductivity in the formation, leading to the computation of the productivity index for fractured well performance. However, the nature of heterogeneity could not be included in the modeling. Understanding the important role of heterogeneity to stimulation performance becomes a crucial step in design and optimization of acid fracture jobs. In order to study the effect of this stochastic nature on acid fracturing, a fully 3D acid reaction model was developed based on the geostatistical parameters of the formation. It is possible to describe local conductivity distribution related to acid transport and reaction process. In this study, we have developed a new interactive workflow allowing the model of the fracture propagation process, the acid etching process and the well production interactively. This thesis presents the novel approach in integrating fracture propagation, acid transport and dissolution, and well performance models in a seamless fashion for acid fracturing design. In this new approach, the fracture geometry data of a hydraulic fracture is first obtained from commercial models of hydraulic fracture propagation, and then the 3D acid fracture model simulates acid etching and transport from the fracture propagation model using the width distribution as the initial condition. We then calculate the fracture conductivity distribution along the created fracture considering the geostatistical parameters such as permeability correlation length and standard deviation in permeability of the formation. The final step of the approach is to predict well performance after stimulation with a reservoir flow simulator. The significant improvements of the new approach are two folds: (1) capturing the geostatistical effect of the formation; and (2) modeling the acid etching and transport more accurately. The thesis explains the methodology and illustrates the application of the approach with examples. The results from this study show that the new model can successfully design and optimize acid fracturing treatments

Development of an In Vitro Airway Epithelial-Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS‐CoV‐2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial–endothelial cell culture model based on Calu‐3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu‐3 cells, cultured for two weeks at an air–liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO‐1). LMVECs cultured submerged for three weeks were CD31‐positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu‐3 cell cultures and the co‐cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocya-nate‐dextran (FITC‐dextran) permeability assays. Importantly, the Calu‐3/LMVEC co‐cultures showed better cell viability and barrier function than mono‐cultures. Moreover, there was no evidence for epithelial‐ and endothelial‐to‐mesenchymal transition (EMT and EndoMT, respec-tively) based on staining for the mesenchymal markers vimentin and α‐SMA, respectively. These results indicate the potential of this new airway epithelial–endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial–endothelial and alveolar–endothelial models with primary lung epithelial cells

Breathing on chip: Dynamic flow and stretch accelerate mucociliary maturation of airway epithelium in vitro

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying the effects of dynamic medium and airflow as well as stretch on human primary airway epithelial cell differentiation and maturation, including mucociliary clearance, using an organ-on-chip device. Perfused epithelial cell cultures displayed accelerated maturation and polarization of mucociliary clearance, and changes in specific cell-types when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in an increase in polarization of mucociliary clearance towards the applied flow, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and maturation and that their fine-tuned application could generate models of specific epithelial pathologies, including mucociliary (dys)function

Identifying somatic changes in drug transporters using whole genome and transcriptome sequencing data of advanced tumors

Drug resistance is a perpetual problem in cancer therapy with many underlying mechanisms. Alterations in drug transport over the cancer cell membrane can severely alter intratumoral drug exposure, contributing to resistance. Here, we present the somatic mutational landscape of 48 ATP-binding cassette and 416 solute carrier transporter genes in a cohort (CPCT-02; NCT01855477) of 3290 patients with different types of advanced and metastasized cancer through analysis of whole genome and transcriptome sequencing. In order to identify potential stressor mechanisms, we stratified patients based on previous systemic therapies and subsequently investigated the enrichment of mutations and copy-number alterations of transporter genes. In tumors from patients pretreated with protein kinase inhibitors (PKIs), genes encoding for specific copper (SLC31A1 and SLC31A2, χ2-test adjusted p-values: 6.9e-09 and 2.5e-09) and nucleoside transporters (SLC28A2 and SLC28A3, χ2-test adjusted p-values: 3.5e-06 and 6.8e-07) were deleted significantly more frequently than in patients pretreated with chemotherapy. Moreover, we detected 16 transporters that were differentially expressed at RNA level between these treatment groups. These findings contradict mechanisms of selective pressure, as they would be expected to originate during treatment with chemotherapy rather than with PKIs. Hence, they might constitute primary drug resistance mechanisms and, therefore, warrant further study

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future

Identifying somatic changes in drug transporters using whole genome and transcriptome sequencing data of advanced tumors

Drug resistance is a perpetual problem in cancer therapy with many underlying mechanisms. Alterations in drug transport over the cancer cell membrane can severely alter intratumoral drug exposure, contributing to resistance. Here, we present the somatic mutational landscape of 48 ATP-binding cassette and 416 solute carrier transporter genes in a cohort (CPCT-02; NCT01855477) of 3290 patients with different types of advanced and metastasized cancer through analysis of whole genome and transcriptome sequencing. In order to identify potential stressor mechanisms, we stratified patients based on previous systemic therapies and subsequently investigated the enrichment of mutations and copy-number alterations of transporter genes. In tumors from patients pretreated with protein kinase inhibitors (PKIs), genes encoding for specific copper (SLC31A1 and SLC31A2, χ2-test adjusted p-values: 6.9e-09 and 2.5e-09) and nucleoside transporters (SLC28A2 and SLC28A3, χ2-test adjusted p-values: 3.5e-06 and 6.8e-07) were deleted significantly more frequently than in patients pretreated with chemotherapy. Moreover, we detected 16 transporters that were differentially expressed at RNA level between these treatment groups. These findings contradict mechanisms of selective pressure, as they would be expected to originate during treatment with chemotherapy rather than with PKIs. Hence, they might constitute primary drug resistance mechanisms and, therefore, warrant further study