scholarly journals An Unbiased Functional Genetics Screen Identifies Rare Activating ERBB4 Mutations

2022 ◽  
Vol 2 (1) ◽  
pp. 10-27
Author(s):  
Deepankar Chakroborty ◽  
Veera K. Ojala ◽  
Anna M. Knittle ◽  
Jasmin Drexler ◽  
Mahlet Z. Tamirat ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Cellular Growth ◽  
Expression Library ◽  
Functional Genetics ◽  
Mutational Hotspots ◽  
Cancer Types

Despite the relatively high frequency of somatic ERBB4 mutations in various cancer types, only a few activating ERBB4 mutations have been characterized, primarily due to lack of mutational hotspots in the ERBB4 gene. Here, we utilized our previously published pipeline, an in vitro screen for activating mutations, to perform an unbiased functional screen to identify potential activating ERBB4 mutations from a randomly mutated ERBB4 expression library. Ten potentially activating ERBB4 mutations were identified and subjected to validation by functional and structural analyses. Two of the 10 ERBB4 mutants, E715K and R687K, demonstrated hyperactivity in all tested cell models and promoted cellular growth under two-dimensional and three-dimensional culture conditions. ERBB4 E715K also promoted tumor growth in in vivo Ba/F3 cell mouse allografts. Importantly, all tested ERBB4 mutants were sensitive to the pan-ERBB tyrosine kinase inhibitors afatinib, neratinib, and dacomitinib. Our data indicate that rare ERBB4 mutations are potential candidates for ERBB4-targeted therapy with pan-ERBB inhibitors. Statement of Significance: ERBB4 is a member of the ERBB family of oncogenes that is frequently mutated in different cancer types but the functional impact of its somatic mutations remains unknown. Here, we have analyzed the function of over 8,000 randomly mutated ERBB4 variants in an unbiased functional genetics screen. The data indicate the presence of rare activating ERBB4 mutations in cancer, with potential to be targeted with clinically approved pan-ERBB inhibitors.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

scholarly journals Bacterial Infection-Mimicking Three-Dimensional Phagocytosis and Chemotaxis in Electrospun Poly(ε-caprolactone) Nanofibrous Membrane

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 569
Author(s):  
Seung-Jun Lee ◽  
Perry Ayn Mayson A Maza ◽  
Gyu-Min Sun ◽  
Petr Slama ◽  
In-Jeong Lee ◽  
...  
Keyword(s):  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Conditions ◽  
Lung Epithelial Cells ◽  
Nanofibrous Membrane ◽  
Infection Model ◽  
Layer System

In this study, we developed a three-dimensional (3D) in vitro infection model to investigate the crosstalk between phagocytes and microbes in inflammation using a nanofibrous membrane (NM). Poly(ε-caprolactone) (PCL)-NMs (PCL-NMs) were generated via electrospinning of PCL in chloroform. Staphylococcus aureus and phagocytes were able to adhere to the nanofibers and phagocytes engulfed S. aureus in the PCL-NM. The migration of phagocytes to S. aureus was evaluated in a two-layer co-culture system using PCL-NM. Neutrophils, macrophages and dendritic cells (DCs) cultured in the upper PCL-NM layer migrated to the lower PCL-NM layer containing bacteria. DCs migrated to neutrophils that cultured with bacteria and then engulfed neutrophils in two-layer system. In addition, phagocytes in the upper PCL-NM layer migrated to bacteria-infected MLE-12 lung epithelial cells in the lower PCL-NM layer. S. aureus-infected MLE-12 cells stimulated the secretion of tumor necrosis factor-α and IL-1α in 3D culture conditions, but not in 2D culture conditions. Therefore, the PCL-NM-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo and is applicable to the biomimetic study of various microbe infections.

scholarly journals Cancer Cell Lines Are Useful Model Systems for Medical Research

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1098 ◽  
Author(s):  
Mirabelli ◽  
Coppola ◽  
Salvatore
Keyword(s):  
Medical Research ◽  
Cell Lines ◽  
Cancer Cell ◽  
Three Dimensional ◽  
Cancer Cell Lines ◽  
Culture Conditions ◽  
The Cancer Genome Atlas ◽  
Model Systems

Cell lines are in vitro model systems that are widely used in different fields of medical research, especially basic cancer research and drug discovery. Their usefulness is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes. Under the right conditions and with appropriate controls, authenticated cancer cell lines retain most of the genetic properties of the cancer of origin. During the last few years, comparing genomic data of most cancer cell lines has corroborated this statement and those that were observed studying the tumoral tissue equivalents included in the The Cancer Genome Atlas (TCGA) database. We are at the disposal of comprehensive open access cell line datasets describing their molecular and cellular alterations at an unprecedented level of accuracy. This aspect, in association with the possibility of setting up accurate culture conditions that mimic the in vivo microenvironment (e.g., three-dimensional (3D) coculture), has strengthened the importance of cancer cell lines for continuing to sustain medical research fields. However, it is important to consider that the appropriate use of cell lines needs to follow established guidelines for guaranteed data reproducibility and quality, and to prevent the occurrence of detrimental events (i.e., those that are linked to cross-contamination and mycoplasma contamination)

scholarly journals Multiscale modelling of drug transport and metabolism in liver spheroids

2020 ◽  
Vol 10 (2) ◽  
pp. 20190041 ◽  
Author(s):  
Joseph A. Leedale ◽  
Jonathan A. Kyffin ◽  
Amy L. Harding ◽  
Helen E. Colley ◽  
Craig Murdoch ◽  
...  
Keyword(s):  
Drug Transport ◽  
Temporal Dynamics ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Isolated Cells ◽  
Modelling Framework ◽  
The Impact ◽  
Cells Cultured

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo . While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

scholarly journals Enrichment of Melanoma Stem-Like Cells via Sphere Assays

2021 ◽  
pp. 185-199
Author(s):  
Nabanita Mukherjee ◽  
Karoline A. Lambert ◽  
David A. Norris ◽  
Yiqun G. Shellman
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Stem Cell ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Low Density ◽  
In Vitro Techniques ◽  
Sphere Culture

AbstractSphere assays are widely used in vitro techniques to enrich and evaluate the stem-like cell behavior of both normal and cancer cells. Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.

scholarly journals Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1650 ◽  
Author(s):  
Christina McKee ◽  
Christina Brown ◽  
G. Rasul Chaudhry
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Extended Period ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Self Assembling

The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine.

scholarly journals Molecular dynamics simulation-guided drug sensitivity prediction for lung cancer with rareEGFRmutations

2019 ◽  
Vol 116 (20) ◽  
pp. 10025-10030 ◽  
Author(s):  
Shinnosuke Ikemura ◽  
Hiroyuki Yasuda ◽  
Shingo Matsumoto ◽  
Mayumi Kamada ◽  
Junko Hamamoto ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Kinase Inhibitors ◽  
Dynamics Simulation ◽  
Cell Lung Carcinoma ◽  
Variants Of Unknown Significance ◽  
Mutational Hotspots

Next generation sequencing (NGS)-based tumor profiling identified an overwhelming number of uncharacterized somatic mutations, also known as variants of unknown significance (VUS). The therapeutic significance ofEGFRmutations outside mutational hotspots, consisting of >50 types, in nonsmall cell lung carcinoma (NSCLC) is largely unknown. In fact, our pan-nation screening of NSCLC without hotspotEGFRmutations (n= 3,779) revealed that the majority (>90%) of cases with rareEGFRmutations, accounting for 5.5% of the cohort subjects, did not receive EGFR-tyrosine kinase inhibitors (TKIs) as a first-line treatment. To tackle this problem, we applied a molecular dynamics simulation-based model to predict the sensitivity of rare EGFR mutants to EGFR-TKIs. The model successfully predicted the diverse in vitro and in vivo sensitivities of exon 20 insertion mutants, including a singleton, to osimertinib, a third-generation EGFR-TKI (R2= 0.72,P= 0.0037). Additionally, our model showed a higher consistency with experimentally obtained sensitivity data than other prediction approaches, indicating its robustness in analyzing complex cancer mutations. Thus, the in silico prediction model will be a powerful tool in precision medicine for NSCLC patients carrying rareEGFRmutations in the clinical setting. Here, we propose an insight to overcome mutation diversity in lung cancer.

In Vitro Reconstitution of Differentiated Human Epithelia

1992 ◽  
Vol 20 (1) ◽  
pp. 95-102
Author(s):  
Graziella Pellegrini ◽  
Roberto Gherzi ◽  
Adriano Tito Franzi ◽  
Fiorella D'Anna ◽  
Michele De Luca ◽  
...  
Keyword(s):  
Donor Site ◽  
Three Dimensional ◽  
Basal Layer ◽  
Human Keratinocytes ◽  
Culture Conditions ◽  
3T3 Fibroblasts ◽  
Stratified Squamous Epithelium ◽  
Original Donor

Human keratinocytes obtained from skin biopsies can be serially cultured in vitro. When plated on lethally irradiated 3T3 fibroblasts, keratinocyte colonies reconstitute a stratified squamous epithelium devoid of stratum corneum. The expression of a mature cornified epidermis, expressing all the morphological and biochemical markers of the in vivo epidermis, can be obtained by the “emerged dermal equivalent” culture system. Melanocytes grown under the same culture conditions, maintain a physiological melanocyte/keratinocyte ratio, are organised in the basal layer of the cultured epidermis, and maintain differentiated functions such as dendritic arborisation, melanin synthesis and melanosome transfer. This allows the reconstitution of an epidermis physiologically populated by functionally active melanocytes. Epithelial cells from different mucosal body sites, namely palate, urethra, conjunctiva, cornea and vagina, can also be cultured and maintain the characteristics of the original donor sites. The in vitro reconstituted human epithelia, permanently transplanted onto patients presenting large epidermal or mucosal defects, retain the characteristics of the original donor site, suggesting an intrinsic site-specific differentiation programme. These three-dimensional human epithelium models could prove useful in standard cytotoxicity assays and could be used as a tool to study the effects of a variety of compounds on normal human epithelia in vitro.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Approach in improving potency and selectivity of kinase inhibitors: Allosteric kinase inhibitors

2020 ◽  
Vol 21 ◽  
Author(s):  
Shangfei Wei ◽  
Tianming Zhao ◽  
Jie Wang ◽  
Xin Zhai
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Binding Modes ◽  
Allosteric Inhibitors ◽  
Wide Range ◽  
Allosteric Sites ◽  
Protein Functions ◽  
Regulate Protein

: Allostery is an efficient and particular regulatory mechanism to regulate protein functions. Different from conserved orthosteric sites, allosteric sites have distinctive functional mechanism to form the complex regulatory network. In drug discovery, kinase inhibitors targeting the allosteric pockets have received extensive attention for the advantages of high selectivity and low toxicity. The approval of trametinib as the first allosteric inhibitor validated that allosteric inhibitors could be used as effective therapeutic drugs for treatment of diseases. To date, a wide range of allosteric inhibitors have been identified. In this perspective, we outline different binding modes and potential advantages of allosteric inhibitors. In the meantime, the research processes of typical and novel allosteric inhibitors are described briefly in terms of structureactivity relationships, ligand-protein interactions and in vitro and in vivo activity. Additionally, challenges as well as opportunities are presented.

Sign in / Sign up

Export Citation Format

Share Document