@article{holden_park_price_floyd_oldham_2022, title={Evaluation of a method to measure fluorescent cell burden in complex culture systems}, volume={8}, url={http://dx.doi.org/10.1088/2057-1976/ac6701}, DOI={10.1088/2057-1976/ac6701}, abstractNote={Abstract Purpose. This work introduces and evaluates a method for accurate in-vitro measurement of fluorescent cell burden in complex 3D-culture conditions. Methods. The Fluorescent Cell Burden (FCB) method was developed to analyze the burden of 4T1 mCherry-expressing cells grown in an organotypic co-culture model of brain metastasis using 400 μm rat brain slices. As a first step, representative simulated image-data accurately reflecting the 4T1 experimental data, but with known ground truth burden, were created. The FCB method was then developed in the CellProfiler software to measure the integrated intensity and area of the colonies in the simulated image data. Parameters in the pipeline were varied to span the experimentally observed range (e.g. of cell colony size) and the result compared with simulation ground truth to evaluate and optimize FCB performance. The optimized CellProfiler pipeline was then applied to the original 4T1 tumor cell images to determine colony growth with time, and re-applied with upper and lower bound parameters to determine uncertainty estimates. Results. The FCB method measured integrated intensity across 10 simulated images with an accuracy of 99.23% ± 0.75%. When colony density was increased by increasing colony number to 450, 600, and 750, the FCB measurement was 98.68%, 100.9%, 97.6% and 113.5% of the true value respectively. For the increasing number of cells plated on the rat brain slices, the integrated intensity increased nearly linearly with cell count except for at high cell counts, where it is hypothesized that shadowing from clumped cells causes a sub-linear relationship. Conclusion. The FCB method accurately measured an integrated fluorescent light intensity to within 5% of ground truth for a wide range of simulated image data spanning the range of observed variability in experimental data. The method is readily customizable to in-vitro studies requiring estimation of fluorescent tumor cell burden.}, number={3}, journal={Biomedical Physics & Engineering Express}, publisher={IOP Publishing}, author={Holden, R and Park, J and Price, A and Floyd, S and Oldham, M}, year={2022}, month={Apr}, pages={037003} } @article{edwards_maganti_tanksley_luo_park_balkanska-sinclair_ling_floyd_2020, title={BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation}, volume={32}, url={http://dx.doi.org/10.1016/j.celrep.2020.108166}, DOI={10.1016/j.celrep.2020.108166}, abstractNote={Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery, causing DNA damage and cell death. BRD4 allows efficient transcriptional elongation by stimulating phosphorylation of RNA polymerase II (RNAPII). We report that bromodomain and extra-terminal domain (BET) protein loss of function (LOF) causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. This persistent RNAPII-dependent pausing leads to an accumulation of RNA:DNA hybrids (R-loops) at sites of BRD4 occupancy, leading to transcription-replication conflicts (TRCs), DNA damage, and cell death. Finally, our data show that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET protein LOF.}, number={12}, journal={Cell Reports}, publisher={Elsevier BV}, author={Edwards, Drake S. and Maganti, Rohin and Tanksley, Jarred P. and Luo, Jie and Park, James J.H. and Balkanska-Sinclair, Elena and Ling, Jinjie and Floyd, Scott R.}, year={2020}, month={Sep}, pages={108166} } @article{tay_park_price_engelward_floyd_2020, title={HTS-Compatible CometChip Enables Genetic Screening for Modulators of Apoptosis and DNA Double-Strand Break Repair}, volume={5}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85085376126&partnerID=MN8TOARS}, DOI={10.1177/2472555220918367}, abstractNote={Dysfunction of apoptosis and DNA damage response pathways often drive cancer, and so a better understanding of these pathways can contribute to new cancer therapeutic strategies. Diverse discovery approaches have identified many apoptosis regulators, DNA damage response, and DNA damage repair proteins; however, many of these approaches rely on indirect detection of DNA damage. Here, we describe a novel discovery platform based on the comet assay that leverages previous technical advances in assay precision by incorporating high-throughput robotics. The high-throughput screening (HTS) CometChip is the first high-throughput-compatible assay that can directly detect physical damage in DNA. We focused on DNA double-strand breaks (DSBs) and utilized our HTS CometChip technology to perform a first-of-its-kind screen using an shRNA library targeting 2564 cancer-relevant genes. Conditions of the assay enable detection of DNA fragmentation from both exogenous (ionizing radiation) and endogenous (apoptosis) sources. Using this approach, we identified LATS2 as a novel DNA repair factor as well as a modulator of apoptosis. We conclude that the HTS CometChip is an effective assay for HTS to identify modulators of physical DNA damage and repair.}, number={8}, journal={SLAS DISCOVERY: Advancing the Science of Drug Discovery}, publisher={SAGE Publications}, author={Tay, Ian J. and Park, James J. H. and Price, Anna L. and Engelward, Bevin P. and Floyd, Scott R.}, year={2020}, month={May}, pages={247255522091836} } @article{nehama_di ianni_musio_du_patané_pollo_finocchiaro_park_dunn_edwards_et al._2019, title={B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres.}, volume={47}, url={http://europepmc.org/articles/PMC6796553}, DOI={10.1016/j.ebiom.2019.08.030}, abstractNote={BackgroundThe dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM.MethodsWe compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models.FindingsB7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines.InterpretationWe demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM.FundAlex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.}, journal={EBioMedicine}, author={Nehama, D. and Di Ianni, N. and Musio, S. and Du, H. and Patané, M. and Pollo, B. and Finocchiaro, G. and Park, J.J.H. and Dunn, D.E. and Edwards, D.S. and et al.}, year={2019}, month={Aug}, pages={33–43} } @article{edwards_maganti_tanksley_luo_park_balkanska-sinclair_ling_floyd_2019, title={BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation}, volume={11}, url={https://doi.org/10.1101/854737}, DOI={10.1101/854737}, abstractNote={ABSTRACTEffective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery causing DNA damage and cell death. BRD4, a BET bromodomain protein and known transcriptional regulator, interacts with P-TEFb to ensure efficient transcriptional elongation by stimulating phosphorylation of RNA Polymerase II (RNAPII). Here we report that disruption of BET bromodomain protein function causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. We find that this persistent, RNAPII-dependent pausing leads to accumulation of RNA:DNA hybrids (R-loops), which are known to lead to transcription-replication conflicts (TRCs), DNA damage, and cell death. Furthermore, we show that resolution of R-loops abrogates BET bromodomain inhibitor-induced DNA damage, and that BET bromodomain inhibition induces both R-loops and DNA damage at sites of BRD4 occupancy. Finally, we see that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET bromodomain inhibition and that oncogenes which promote transcription and replication exacerbate BET bromodomain inhibitor-induced DNA damage. Together, these findings demonstrate that BET bromodomain inhibitors can damage DNA via induction of R-loops and TRCs in highly replicative cells.}, publisher={Cold Spring Harbor Laboratory}, author={Edwards, Drake and Maganti, Rohin and Tanksley, Jarred P. and Luo, Jie and Park, James J.H. and Balkanska-Sinclair, Elena and Ling, Jinjie and Floyd, Scott R.}, year={2019}, month={Nov} }