Browsing by Subject "Three-dimensional culture"

Now showing 1 - 3 of 3
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    Applying Small Molecule Signal Transducer and Activator of Transcription-3 (STAT3) Protein Inhibitors as Pancreatic Cancer Therapeutics
    (American Association for Cancer Research, 2016-05) Arpin, Carolynn C.; Mac, Stephen; Jiang, Yanlin; Cheng, Huiwen; Grimard, Michelle; Page, Brent D. G.; Kamocka, Malgorzata M.; Haftchenary, Sina; Su, Han; Ball, Daniel; Rosa, David A.; Lai, Ping-Shan; Gómez-Biagi, Rodolfo F.; Ali, Ahmed M.; Rana, Rahul; Hanenberg, Helmut; Kerman, Kagan; McElyea, Kyle C.; Sandusky, George E.; Gunning, Patrick T.; Fishel, Melissa L.; Pediatrics, School of Medicine
    Constitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells.
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    Directed Differentiation of Mouse Embryonic Stem Cells Into Inner Ear Sensory Epithelia in 3D Culture
    (Springer Nature, 2017) Nie, Jing; Koehler, Karl R.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
    The inner ear sensory epithelium harbors mechanosensory hair cells responsible for detecting sound and maintaining balance. This protocol describes a three-dimensional (3D) culture system that efficiently generates inner ear sensory epithelia from aggregates of mouse embryonic stem (mES) cells. By mimicking the activations and repressions of key signaling pathways during in vivo inner ear development, mES cell aggregates are sequentially treated with recombinant proteins and small molecule inhibitors for activating or inhibiting the Bmp, TGFβ, Fgf, and Wnt signaling pathways. These stepwise treatments promote mES cells to sequentially differentiate into epithelia representing the non-neural ectoderm, preplacodal ectoderm, otic placodal ectoderm, and ultimately, the hair cell-containing sensory epithelia. The derived hair cells are surrounded by a layer of supporting cells and are innervated by sensory neurons. This in vitro inner ear organoid culture system may serve as a valuable tool in developmental and physiological research, disease modeling, drug testing, and potential cell-based therapies.
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    Modeling cancer predisposition: Profiling Li-Fraumeni syndrome patient-derived cell lines using bioinformatics and three-dimensional culture models
    (2015-10-07) Phatak, Amruta Rajendra; Herbert, Brittney-Shea; Liu, Yunlong; Mendonca, Marc S.; Wells, Clark D.
    Although rare, classification of over 200 hereditary cancer susceptibility syndromes accounting for ~5-10% of cancer incidence has enabled the discovery and understanding of cancer predisposition genes that are also frequently mutated in sporadic cancers. The need to prevent or delay invasive cancer can partly be addressed by characterization of cells derived from healthy individuals predisposed to cancer due to inherited "single-hits" in genes in order to develop patient-derived samples as preclinical models for mechanistic in vitro studies. Here, we present microarray-based transcriptome profiling of Li-Fraumeni syndrome (LFS) patient-derived unaffected breast epithelial cells and their phenotypic characterization as in vitro three-dimensional (3D) models to test pharmacological agents. In this study, the epithelial cells derived from the unaffected breast tissue of a LFS patient were cultured and progressed from non-neoplastic to a malignant stage by successive immortalization and transformation steps followed by growth in athymic mice. These cell lines exhibited distinct transcriptomic profiles and were readily distinguishable based upon their gene expression patterns, growth characteristics in monolayer and in vitro 3D cultures. Transcriptional changes in the epithelial-to-mesenchymal transition gene signature contributed to the unique phenotypes observed in 3D culture for each cell line of the progression series; the fully transformed LFS cells exhibited invasive processes in 3D culture with disorganized morphologies due to cell-cell miscommunication, as seen in breast cancer. Bioinformatics analysis of the deregulated genes and pathways showed inherent differences between these cell lines and targets for pharmacological agents. After treatment with small molecule APR-246 that restores normal function to mutant p53, we observed that the neoplastic LFS cells had reduced malignant invasive structure formation from 73% to 9%, as well as an observance of an increase in formation of well-organized structures in 3D culture (from 27% to 91%) by stereomicroscopy and confocal microscopy. Therefore, the use of well-characterized and physiologically relevant preclinical models in conjunction with transcriptomic profiling of high-risk patient derived samples as a renewable laboratory resource can potentially guide the development of safer and more effective chemopreventive approaches.