In this report we demonstrate the effect of a novel electron emission-based cell culture device on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells. Our device has an electron emission element that allows, for the first time, stable emission of electrons into an atmosphere. Atmospheric electrons react with gas molecules to generate radicals and negative ions, which induce a variety of biochemical reactions in the attached cell culture system. In this study, we demonstrated the effect of this new electron emissionbased cell culture device on cell proliferation and differentiation using pre-osteoblastic MC3T3-E1 cells. Electron emission stimulation (EES) was applied directly to culture medium containing plated cells, after which the number of living cells, the mRNA levels of osteogenesis-related genes, and the alkaline phosphatase (ALP) activity were evaluated. The growth rate of EES-exposed cells increased by approximately 20% in comparison with unexposed control cells. We also found the mRNA levels of osteogenic specific genes such as collagen type I α-1, core-binding factor α-1, and osteocalcin to be up-regulated following EES. ALP activity, a marker for osteogenic activity, was significantly enhanced in EEStreated cells. Furthermore, reactive oxygen species generated by EES were measured to determine their effect on MC3T3-E1 cells. These results suggest that our new electron emission- based cell culture device, while providing a relatively weak stimulus in comparison with atmospheric plasma systems, promotes cell proliferation and differentiation. This system is expected to find application in regenerative medicine, specifically in relation to bone regeneration.
FGFR2 gene is frequently amplified in gastric cancer. Recently, targeting FGFR2 has drawn attention as a form of gastric cancer therapy, and FGFR-selective inhibitors have shown promising efficacy in clinical studies. Because overcoming acquired resistance is a common problem with molecular targeting drugs, we investigated a resistant mechanism of FGFR inhibitors using the gastric cancer cell line SNU-16, which harbors FGFR2 amplification. We established single-cell clones of FGFR inhibitor–resistant SNU-16 (AZD-R) by continuous exposure to AZD4547, a selective FGFR inhibitor. To screen the genetic alterations acquired in AZD-R, we ran a comparative genomic hybridization assay and found an amplification of Chr7q34 region. The chromosomal breakpoints were located between the 12th and the 13th exon of jumonji C domain containing histone demethylase 1 homolog D (JHDM1D) and between the 3rd and the 4th exon of BRAF. We sequenced cDNA of the AZD-R clones and found fusion kinase JHDM1D-BRAF, which has previously been identified in primary ovarian cancer. Because JHDM1D–BRAF fusion lacks a RAS-binding domain, the dimerization of JHDM1D–BRAF was enhanced. A cell growth inhibition assay using MEK inhibitors and RAF-dimer inhibitors indicated the dependence of AZD-R clones for growth on the MAPK pathway. Our data provide a clinical rationale for using a MEK or RAF dimer inhibitor to treat FGFR2-amplified gastric cancer patients who have acquired resistance through the JHDN1D–BRAF fusion. Mol Cancer Ther; 17(10); 2217–25. 2018 AACR.
This thesis focuses on the design, realization and application of gravity-driven microfluidic systems to create a physiological culture environment for the experimentation with 3D microtissues. As the pharmaceutical industry is facing a prediction dilemma in substance testing, new testing methods are required to bridge the gap between conventional 2D cell culture methods, animal testing, and human physiology. Advanced 3D cell culture techniques in combination with microfluidic technology bear the potential to increase the predictive power of preclinical testing methods and to better characterize the effects of substances on the human organism. However, such systems have yet to be adapted to industrial requirements in terms of material properties, user-friendliness, and experimental throughput. In this thesis, two gravitydriven chip systems and the respective proof-of-concept applications are presented: (i) A scalable, polystyrene-based microfluidic chip was used for combining multiple tissue types in an in vitro system. Primary human liver microtissues and colon cancer microtissues were cultured together and fluidically connected through cell culture medium. Upon co-administration of anticancer therapeutics and other compounds, liver-related drug-drug interactions were detected. (ii) Dynamically changing dosing curves were generated by applying a specific microfluidic channel layout. Colon cancer microtissues were exposed to physiologically-relevant pharmacokinetic dosing curves, and their time-dependent response was assessed. By relying on standardized 3D microtissues and gravity-driven perfusion, the systems presented in this thesis are scalable, robust, and easy to use so that a transfer to industrial settings is conceivable. The overall goal is to devise in vitro systems that closely mimic human physiology.
Organism size and growth curves are important biological characteristics. Current methods to measure organism size, and in particular growth curves, are often resource intensive because they involve many manual steps. Here we demonstrate a method for automated, high-throughput measurements of size and growth in individual aquatic invertebrates kept in microtiter well-plates. We use a spheroid counter (Cell3iMager, cc-5000) to automatically measure size of seven different freshwater invertebrate species. Further, we generated calibration curves (linear regressions, all p < 0.0001, r2 >=0.9 for Ceriodaphnoa dubia, Asellus aquaticus, Daphnia magna, Daphnia pulex; r2 >=0.8 for Hyalella azteca, Chironomus spec. larvae and Culex spec. larvae) to convert size measured on the spheroid counter to traditional, microscope based, length measurements, which follow the longest orientation of the body. Finally, we demonstrate semi-automated measurement of growth curves of individual daphnids (C. dubia and D. magna) over time and find that the quality of individual growth curves varies, partly due to methodological reasons. Nevertheless, this novel method could be adopted to other species and represents a step change in experimental throughput for measuring organisms’ shape, size and growth curves. It is also a significant qualitative improvement by enabling high-throughput assessment of interindividual variation of growth.
The subcutaneous transplantation of microencapsulated islets has been extensively studied as a therapeutic approach for type I diabetes. However, due to the lower vascular density and strong inflammatory response in the subcutaneous area, there have been few reports of successfully normalized blood glucose levels. To address this issue, we developed mosaic-like aggregates comprised of mesenchymal stem cells (MSCs) and recombinant peptide pieces called MSC CellSaics, which provide a continuous release of angiogenic factors and anti-inflammatory cytokines. Our previous report revealed that the diabetes of immunodeficient diabetic model mice was reversed by the subcutaneous co-transplantation of the MSC CellSaics and rat islets. In this study, we focused on the development of immune-isolating microcapsules to co-encapsulate the MSC CellSaics and rat islets, and their therapeutic eciency via subcutaneous transplantation into immunocompetent diabetic model mice. As blood glucose level was monitored for 28 days following transplantation, the normalization rate of the new immuno-isolating microcapsules was confirmed to be significantly higher than those of the microcapsules without the MSC CellSaics, and the MSC CellSaics transplanted outside the microcapsules (p < 0.01). Furthermore, the number of islets required for the treatment was reduced. In the stained sections, a larger number/area of blood vessels was observed around the new immuno-isolating microcapsules, which suggests that angiogenic factors secreted by the MSC CellSaics through the microcapsules function locally for their enhanced efficacy.
Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes Akt- Ser473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchoragedeficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell—cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy.
Background: Metastasis is a leading cause of breast cancer mortality. The induction of epithelial-to-mesenchymal transition (EMT) and complex oncogenic signaling is a vital step in the evolution of highly metastatic and therapeutically-intractable breast cancer; necessitating novel target discovery or development of therapeutics that target metastatic breast cells (MBCs). Methods: To achieve this, this study employs a combination of in silico bioinformatics analyses, protein and transcript analyses, drug sensitivity assays, functional assays and animal studies. Results: The present study identified CDH11 as an inductor and/or facilitator of metastatic signaling, and biomarker of poor prognosis in MBCs. Furthermore, we showed that in the presence of CDH11-rich cancer-associated fibroblasts (CAFs), MCF7 and MDA-MB-231 MBC cell lines acquired enhanced metastatic phenotype with increased CDH11, β-catenin, vimentin, and fibronectin (FN) expression. We also demonstrated, for the first time to the best of our knowledge that exposure to anti- CDH11 antibody suppresses metastasis, reduces CDH11, FN and β-catenin expression, and abrogate the cancer stem cell (CSC)-like traits of MBC cells. Interestingly, ectopic expression of miR-335 suppressed CDH11, β-catenin and vimentin expression, in concert with attenuated metastatic and CSC potentials of the MBC cells; conversely, inhibition of miR-335 resulted in increased metastatic potential. Finally, corroborating the in silica and in vitro findings, in vivo assays showed that the administration of anti-CDH11 antibody or miR-335 mimic suppressed tumorigenesis and inhibited cancer metastasis. Conclusions: These findings validate our hypotheses that miR-335 mediates anti-CDH11 antibody therapy response and that an enhanced miR-335/CDH11 ratio elicits marked suppression of the MBC CSC-like and metastatic phenotypes, thus revealing a therapeutically-exploitable inverse correlation between CDH11-enhanced CSC-like and metastatic phenotype and miR-335 expression in MBCs. Thus, we highlight the therapeutic promise of humanized anti-CDH11 antibodies or miR-335- mimic, making a case for their clinical application as efficacious therapeutic option in patients with MBC.
An integrated genomic and functional analysis to elucidate DNA damage signaling factors promoting self-renewal of glioma stem cells (GSCs) identified proliferating cell nuclear antigen (PCNA)- associated factor (PAF) up-regulation in glioblastoma. PAF is preferentially overexpressed in GSCs. Its depletion impairs maintenance of self-renewal without promoting differentiation and reduces tumorinitiating cell frequency. Combined transcriptomic and metabolomic analyses revealed that PAF supports GSC maintenance, in part, by influencing DNA replication and pyrimidine metabolism pathways. PAF interacts with PCNA and regulates PCNA-associated DNA translesion synthesis (TLS); consequently, PAF depletion in combination with radiation generated fewer tumorspheres compared with radiation alone. Correspondingly, pharmacological impairment of DNA replication and TLS phenocopied the effect of PAF depletion in compromising GSC self-renewal and radioresistance, providing preclinical proof of principle that combined TLS inhibition and radiation therapy may be a viable therapeutic option in the treatment of glioblastoma multiforme (GBM).
Colorectal cancer represents one of the most prevalent malignancies globally, with an estimated 140,000 new cases in the United States alone in 2019. Despite advancements in interventions, drug resistance occurs in virtually all patients diagnosed with late stages of colon cancer. Amplified epidermal growth factor receptor (EGFR) signaling is one of the most prevalent oncogenic drivers in patients and induces increased Janus kinase (JAK)/signal transduction and activator of transcription (STAT) and -catenin functions, all of which facilitate disease progression. Equally important, cancer-associated fibroblasts (CAFs) transformed by cancer cells within the tumor microenvironment (TME) further facilitate malignancy by secreting interleukin (IL)-6 and augmenting STAT3 signaling in colon cancer cells and promoting the generation of cancer stem-like cells (CSCs). Based on these premises, single-targeted therapeutics have proven ineective for treating malignant colon cancer, and alternative multiple-targeting agents should be explored. Herein, we synthesized a tetracyclic heterocyclic azathioxanthone, MSI-N1014, and demonstrated its therapeutic potential both in vitro and in vivo. First, we used a co-culture system to demonstrate that colon cancer cells co-cultured with CAFs resulted in heightened 5-fluorouracil (5-FU) resistance and tumor sphere-forming ability and increased side populations, accompanied by elevated expression of cluster of dierentiation 44 (CD44), -catenin, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and ATP-binding cassette super-family G member 2 (ABCG2). MSI-N1014 suppressed cell viability, colony formation, and migration in both DLD1 and HCT116 cells. MSI-N1014 treatment led to decreased expressions of oncogenic markers, including mammalian target of rapamycin (mTOR), EGFR, and IL-6 and stemness markers such as CD44, -catenin, and LGR5. More importantly, MSI-N1014 treatment suppressed the transformation of CAFs, and was associated with decreased secretion of IL-6 and vascular endothelial growth factor (VEGF) by CAFs. Furthermore, MSI-N1014 treatment resulted in significantly reduced oncogenic properties, namely the migratory ability, tumor-sphere generation, and resistance against 5-FU. Notably, an increased level of the tumor suppressor, miR-142-3p, whose targets include LGR5, IL-6, and ABCG2, was detected in association with MSI-N1014 treatment. Finally, we demonstrated the therapeutic potential of MSI-N1014 in vivo, where combined treatment with MSI-N1014 and 5-FU led to the lowest tumor growth, followed by MSI-N1014 only, 5-FU, and the vehicle control. Tumor samples from the MSI-N1014 group showed markedly reduced expressions of LGR5, -catenin, IL-6, and mTOR, but increased expression of the tumor suppressor, miR-142-3p, according to qRT-PCR analysis. Collectively, we present preclinical support for the application of MSI-N1014 in treating 5-FU-resistant colon cancer cells. Further investigation is warranted to translate these findings into clinical settings.
Dysregulation of repressor-element 1 silencing transcription factor REST/NRSF is related to several neuropathies, including medulloblastoma, glioblastoma, Huntington’s disease, and neuropathic pain. Inhibitors of the interaction between the N-terminal repressor domain of REST/NRSF and the PAH1 domain of its corepressor mSin3 may ameliorate such neuropathies. In-silico screening based on the complex structure of REST/NRSF and mSin3 PAH1 yielded 52 active compounds, including approved neuropathic drugs. We investigated their binding affinity to PAH1 by NMR, and their inhibitory activity toward medulloblastoma cell growth. Interestingly, three antidepressant and antipsychotic medicines, sertraline, chlorprothixene, and chlorpromazine, were found to strongly bind to PAH1. Multivariate analysis based on NMR chemical shift changes in PAH1 residues induced by ligand binding was used to identify compound characteristics associated with cell growth inhibition. Active compounds showed a new chemo-type for inhibitors of the REST/NRSF-mSin3 interaction, raising the possibility of new therapies for neuropathies caused by dysregulation of REST/NRSF.
roles in the maintenance of homeostasis, and also in the regeneration of the damaged intestinal epithelia. However, whether the inflammatory environment of Crohn’s disease (CD) affects properties of resident small intestinal stem cells remain uncertain. Methods CD patient-derived small intestinal organoids were established from enteroscopic biopsy specimens taken from active lesions (aCD-SIO), or from mucosa under remission (rCD-SIO). Expression of ISC-marker genes in those organoids was examined by immunohistochemistry, and also by microfluid-based single-cell multiplex gene expression analysis. The ISC-specific function of organoid cells was evaluated using a single-cell organoid reformation assay. Results ISC-marker genes, OLFM4 and SLC12A2, were expressed by an increased number of small intestinal epithelial cells in the active lesion of CD. aCD-SIOs, rCDSIOs or those of non-IBD controls (NI-SIOs) were successfully established from 9 patients. Immunohistochemistry showed a comparable level of OLFM4 and SLC12A2 expression in all organoids. Single-cell gene expression data of 12 ISC-markers were acquired from a total of 1215 cells. t-distributed stochastic neighbor embedding analysis identified clusters of candidate ISCs, and also revealed a distinct expression pattern of SMOC2 and LGR5 in ISCcluster classified cells derived from aCD-SIOs. Single-cell organoid reformation assays showed significantly higher reformation efficiency by the cells of the aCD-SIOs compared with that of cells from NI-SIOs. Conclusions aCD-SIOs harbor ISCs with modified marker expression profiles, and also with high organoid reformation ability. Results suggest modification of small intestinal stem cell properties by unidentified factors in the inflammatory environment of CD.
We evaluated the effect of gut bacterial metabolites of polyunsaturated fatty acids on inflammation and found that 10-oxo-cis-6,trans-11-octadecadienoic acid (gKetoC) strikingly suppressed LPS-induced IL-6 release from bone marrow-derived macrophages (BMMs), which was accompanied by reduced mRNA expression of Il6, TNF, and Il1b. gKetoC decreased the cAMP concentration in BMMs, suggesting that gKetoC stimulated G protein-coupled receptors. A Gq agonist significantly suppressed LPS-induced IL-6 expression in BMMs, whereas a Gi inhibitor partially abrogated gKetoC-mediated IL-6 suppression. Cytosolic Ca2þ was markedly increased by gKetoC, which was partly but not fully abrogated by an ion channel inhibitor. Taken together, these data suggest that gKetoC suppresses inflammatory cytokine expression in macrophages primarily through Gq and partially through Gi. gKetoC suppressed osteoclast development and IL-6 expression in synovial fibroblasts from rheumatoid arthritis (RA) patients, suggesting the beneficial effect of gKetoC on the prevention or treatment of RA.
Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. Aggressive surgical resection plus radiotherapy and temozolomide have prolonged patients’ median survival to only 14.6 months. Therefore, there is a critical need to develop novel therapeutic strategies for GBM. In this study, we evaluated the effect of NOTCH signaling intervention by gamma-secretase inhibitors (GSIs) on glioma sphere-forming cells (GSCs). GSI sensitivity exhibited remarkable selectivity among wild-type TP53 (wt-p53) GSCs. GSIs significantly impaired the sphere formation of GSCs harboring wt-p53. We also identified a concurrence between GSI sensitivity, NOTCH1 expression, and wt-p53 activity in GSCs. Through a series of gene editing and drug treatment experiments, we found that wt-p53 did not modulate NOTCH1 pathway, whereas NOTCH1 signaling positively regulated wt-p53 expression and activity in GSCs. Finally, GSIs (targeting NOTCH signaling) synergized with doxorubicin (activating wt-p53) to inhibit proliferation and induce apoptosis in wt-p53 GSCs. Taken together, we identified wt-p53 as a potential marker for GSI sensitivity in GSCs. Combining GSI with doxorubicin synergistically inhibited the proliferation and survival of GSCs harboring wt-p53.