The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1α). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo-like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.
Label‐free cell‐based assays offer a powerful approach to drug discovery and compound profiling for endogenously expressed receptors in a variety of cell types, including primary and stem cells. Dynamic mass redistribution (DMR) responses in whole cells following receptor stimulation provide phenotypic activity profiles that are readily amenable to evaluation of compound pharmacology. Protocols are provided in this unit to obtain DMR response profiles in adherent and suspension cells, and then to use known tool compounds to delineate the biology of the underlying signaling pathways from the information‐rich kinetic traces that are recorded. Curr. Protoc. Chem. Biol. 6:39‐51 © 2014 by John Wiley & Sons, Inc.
EGFR has been found frequently mutated in human cancers, and more than 10% lung cancer patients associated with EGFR mutation. In addition, several critical mutations within the EGFR have been shown as gain of function mutations or can cause drug resistance to the EGFR inhibitors. A panel of ATCC tumor cell lines has been characterized for EGFR mutation and EGFR signaling pathway. Particularly, lung cell line NCI-H1975, which contains both drug sensitive mutation EGFR p.L858R and drug resistant mutation p.T790M, has displayed a resistance to EGFR inhibitor.Overall, NCI-H1975 cell line can be used as a nice model to study drug resistance, as well as to screen novel EGFR inhibitors that overcome the p.T790M-mediated resistance. Moreover, we have used next generation sequencing to analyze the mutation allelic frequency of both mutations, and observed tumor heterogeneity within this cell line. Impact of cell passage numbers on the mutation allele frequency of the cell line was also examined. Furthermore, a novel single cell western blot assay has been used to analyze the EGFR mutation, EGFR protein expression and downstream signaling within individual cells of three lung cancer cell lines at the single cell level. Collectively, NCI-H1975 cell line represents the acquired drug resistance found in clinical cancer patients, and this study demonstrates the tumor heterogeneity represented in human cell lines. Citation Format: Lysa-Anne Volpe, John Foulke, Michael Jackson, Luping Chen, David H. Randle, Hannah J. Gitschier, Kelly Gardner, Fang Tian. Tumor heterogeneity represented in human cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4919. doi:10.1158/1538-7445.AM2015-4919
Developmental neurotoxicity (DNT) of environmental chemicals has long been identified as a threat to the health of the human population, as the developing nervous system is particularly susceptible to toxicant exposure. The resulting neurological deficits can have long‐term effects on families and society both financially and emotionally. Current DNT testing guidelines involve the use of animal models; primarily rodents. The testing strategy incorporates large numbers of animals, which can be extremely time‐ and cost‐intensive; particularly due to the backlog of chemicals needing to be tested (Lein et al. , 2005). This demand, in addition to current and future proposed regulations on the use of animals for testing makes it imperative that new models be found to reduce animal experimentation while providing a suitable method to test new chemicals. Three‐dimensional cell models, which incorporate human neural stem cells (hNSCs) aggregated into neurospheres, have been proposed as a viable alternative for DNT testing. The in vitro system has the ability to recapitulate the processes of brain development, including proliferation, migration, differentiation and apoptosis (Salma et al ., 2015). Inclusion of human cells, as opposed to murine, also meets recommendations to circumvent the drawback of species differences between in vivo testing and actual exposure effects. Here we will demonstrate the use of a 3D neurosphere model, composed of hNSCs, to conduct toxicity testing of potential neurotoxicants. A spheroid microplate was used to create and maintain cells in the 3D model. 3D neurosphere proliferation, multipotency, along with the continued capacity to differentiate into neurons, astrocytes, and oligodendrocytes was initially validated. Neurotoxicity testing was then performed using neurospheres maintained in the 3D spheroid plate. Detection of induced levels of oxidative stress, apoptotic, and necrotic activity within treated neurospheres, compared to negative control spheres, was evaluated. Monitoring of cell proliferation, differentiation, multipotency and experimental testing was performed using a novel cell imaging multi‐mode reader.
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