No abstract

Basal forebrain GABAergic and cholinergic circuits regulate the activity of cholinergic projections to the cortex and hippocampus. Because these projections influence cortical development and function, the development of basal forebrain excitatory and inhibitory neurons is critical for overall brain development. We show that the neurotransmitter phenotype of these neurons is developmentally regulated by neurotrophins and the p75 receptor. Neurotrophins (nerve growth factor and brain-derived neurotrophic factor) increased the number of both cholinergic and GABAergic neurons in neonatal basal forebrain neuron cultures from the region of the medial septum. However, the p75 receptor is required only for neurotrophin-dependent expansion of the GABAergic, not the cholinergic, population. Neurotrophin-induced GABAergic development can be rescued in p75 Ϫ/Ϫ cultures by expression of a p75 rescue construct in neighboring cells or by treatment with medium collected from neurotrophin-treated wild-type cultures. Because p75 is not expressed in basal forebrain GABAergic neurons, this defines a new, non-cell-autonomous mechanism of p75 action in which ligand binding results in release of a soluble factor that modifies neurotrophin responses of nearby neurons. p75 is also required for the maintenance of basal forebrain GABAergic neurons in vivo, demonstrating that p75-mediated interactions between cholinergic and GABAergic neurons regulate the balance of excitatory and inhibitory components of basal forebrain circuits.

The purpose of this study was to examine the pathways involved in the induction of immune checkpoint molecules in basal breast and cervical cancer cell lines by measuring classical biomarker expression using AlphaLISA no-wash homogeneous assays. Programmed cell death-ligand 1 (PD-L1) expression increases with tumor severity in basal-like breast cancer and is enhanced in intraepithelial neoplasia and cervical cancers. Basal-breast tumors can adapt to lymphocytic infiltration by responding to heightened concentrations of interferon gamma (IFN-y) secreted by Type 1 helper T cells with upregulation of PD-L1 protein allowing the tumors to evade immune targeting and reduce the immune response. We show here that human peripheral blood mononuclear cells (PBMCs) secrete IFN-y and other cytokines in response to stimulation with CD3/CD38 Dynabeads. We then examined modulation in expression of immune checkpoint molecules (PD-L1 and others) in HCC38 (basal breast cancer-derived) and HeLa (cervical cancer) cells in response to co-culturing with activated PBMCs, treatment with conditioned media collected from activated PBMCs, and direct treatment with recombinant IFN-y. As expected, IFN-y treatment induced dose-dependent upregulation of PD-L1 protein expression in both HCC38 and HeLa cell lines, whereas the effects of conditioned media and direct co-culturing with PBMCs resulted in more complex effects which are discussed. To determine if tumor cytoarchitecture influences responsiveness to the PBMCs, biomarker expression was measured from cultures grown in both traditional adherent monolayers and in 3D spheroid cultures using Ultra-Low Attachment (ULA) microplates. Cellular health and proliferation was assessed by measuring ATP concentration with ATPlite luminescence assays and cellular imaging with viability dyes. The upregulation of PD-L1 expression was observed to be largely independent of cellular proliferation and is further shown to be dependent on signaling through the JAK/STAT pathway by probing with AlphaLISA SureFire phosphorylation assays. These data illustrate and address some of the challenges in developing a biologically relevant culture system for examining the complex mechanisms involved in tumor evasion of the innate immune response. Citation Format: Jeanine M. Hinterneder, Jen Carlstrom, Adam Carlson, Dawn Nida. Rapid, no-wash measurement of immune checkpoint molecules expression induced by interaction with peripheral blood mononuclear cells in breast and cervical cancer cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4671.

The physiological function of the epidermal growth factor receptor (EGFR) is to regulate epithelial tissue development and homeostasis, but its overexpression and activation are strongly linked to oncogenesis and tumor progression in a variety of human cancers. Many EGFR-targeted therapies have been developed which alone have proven to be effective, but resistance often develops, indicating a need for more studies probing the efficacy of combinatorial drug treatments. These studies will further require the discovery and characterization of biomarkers that can predict therapeutic efficacy and resistance. The purpose of this study was to examine the effects of individual and combined treatment with two classical EGFR-targeted therapies with different modes of action on cellular models of human skin cancer and non-small cell lung carcinoma (NSCLC). We characterized the effects of a two-day treatment with varying concentrations of EGF on cellular health and proliferation and on the expression of several key biomarker proteins in A431 and A549 cell lines. We then examined the individual and combined effects of treatment with the tyrosine kinase inhibitor (TKI) gefitinib and the humanized monoclonal antibody cetuximab. Cellular growth, proliferation and health were assessed with ATPlite luminescence assays and well-imaging and cytometry measures using a high-throughput multimodal imager and plate reader. Biomarkers measured included EGFR, immune checkpoint proteins (PD-L1 and CD276/B7-H3), chemokines (IL-8/CXCL8 and GRO-α/CXCL1), a protease inhibitor (TIMP-2), and growth factor (VEGF). All these biomarker proteins were rapidly assessed using AlphaLISA no-wash, mix-and-read assays on 5 μL samples collected from the same wells of the culture plate. We observed that EGF treatment resulted in decreased EGFR expression and upregulation of PD-L1 protein in a dose-dependent manner in both culture models. CD276, VEGF and TIMP-2 expression levels, however, were upregulated with EGF treatment only in the A431 cells. Complex drug treatment effects on biomarker expression are presented. In addition, treatment effects on EGFR phosphorylation and downstream signaling pathways (including MEK/ERK and AKT) were probed using AlphaLISA SureFire Ultra phosphorylation assays and demonstrate the different pathways effected by either drug or a combination of both. These data illustrate the effects of EGFR stimulation on the expression of an array of biomarkers and signaling pathways and in the cellular tolerance for individual and combined treatment with two EGFR-targeted therapies with different mechanisms of action between two different cellular models of human cancer. Citation Format: Jeanine M. Hinterneder, Lauren Berstler, Adam Carlson, Jen Carlstrom. A systems approach to evaluating the effects of two EGFR-targeted therapies on cellular models of human skin and lung cancer using an integrated imaging and analysis platform and the rapid quantification of multiple key biomarkers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5197.

The purpose of this study was to examine the induction of epithelial to mesenchymal transition (EMT) in a prostate cancer cell line by measuring classical biomarker expression in three-dimensional (3D) spheroid cultures compared to traditional 2D monolayers in an effort to develop a more biologically relevant assay. Using Ultra-Low Attachment (ULA) microplates, we grew spheroids from a human prostate cancer cell line (DU 145). Numerous studies have implicated a role for EMT in carcinoma invasion and metastasis. EMT is characterized by rearrangement of the extracellular matrix (ECM) and differential regulation of ECM proteins. We induced EMT using TGF-beta and phorbol-12-myristate-13-acetate (PMA) and compared expression levels of specific biomarkers, such as E-cadherin, fibronectin, and IL-6, using AlphaLISA and LANCE (TR-FRET) assay technologies. We confirmed that treatment of DU 145 cells with TGF-beta is sufficient for inducing changes in both EMT biomarker expression and characteristic cellular morphology in monolayer cultures. However, in 3D spheroid cultures, we observed only a partial EMT response to the same TGF-beta treatment as demonstrated by changes in the expected biomarker expression pattern. Using the small molecule, PMA, we see significant differences in the levels of IL-6 secretion after EMT induction between cells grown in monolayer and those grown in spheroids. Cellular proliferation, growth and vitality were assessed using ATPlite luminescence assays and confocal microscopy of live-stained cells with a high content imaging system. Though we observe increased proliferation in monolayer cultures compared to 3D spheroids, the changes observed in protein expression patterns cannot be sufficiently explained by differences in cell number or viability. These data illustrate the differences in protein expression levels and in cellular tolerance for compound treatment between a human prostate cancer cell line grown in monolayers and those same cells grown in 3D spheroids. Citation Format: Jen Carlstrom, Jeanine M. Hinterneder, Lindsay Nelson, Stephen Hurt. Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5793. doi:10.1158/1538-7445.AM2017-5793