Cancer genomics studies have identified thousands of putative cancer driver genes
. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. ...Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif
from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.
Despite its prolific track record of research into primary tumor dynamics, the field of cancer biology has yet to develop a reliable methodology for interrogating the genetic profiles of recurrent ...tumors. CRISPR/Cas9 gene editing has proven to be foundational for recent advancements in oncogenetic screening, due in part to its high degree of specificity in achieving on‐target gene knockout while minimizing off‐target effects. The primary limitation to its application in screening pro‐recurrence factors in cancer, however, is the constitutive nature of canonical Cas9 activity, which may not be remotely regulated after the point of Cas9 translation. To address this problem, we cultivated and transduced populations of a resected invasive mammary adenocarcinoma cell type with a selection of four recombinant Cas9 transgenes and characterized the relative inducibility of each system. These Cas9's are fused to peptides whose tertiary structure renders the endonuclease inactive, restoring catalytic activity only in the presence of a fusion‐specific ligand. Populations of EGFP‐expressing adenocarcinoma cells were transduced with a modified estrogen receptor‐Cas9 fusion (ERT2‐Cas9) and assayed for target knockout efficiency upon addition of small guide GFP (sgGFP) and a Cas9‐enabling estrogen analog. After three days exposure to the analogue, flow cytometry revealed GFP expression to be markedly reduced in cells expressing ERT2‐Cas9 and minimal change in those not exposed. Once the timing and treatment of this model are refined, ERT2‐Cas9‐expressing adenocarcinoma cells will be orthotopically‐injected into mice for an in vivo screening assay. By equipping neoplastic cells with the inducible‐Cas9 machinery before the point of tumor outgrowth, it becomes possible to systematically screen pro‐survival factors at late time points in cancer progression without drastically disturbing the microenvironment of the tumor.
Support or Funding Information
This work was supported by ah ASPET summer research award in partnership with SURPH at Duke University.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
In situations of oxidative stress, the antioxidant processes of any eukaryotic organism can eventually become overwhelmed. Exposure to ambient particulate matter 2.5 (PM
2.5
), composed ...of transition metals and organic compounds 2.5μm or less in diameter, has been shown to cause oxidative stress in alveolar macrophages (AMs). This can lead to various adverse health effects, including cardiovascular and pulmonary diseases. In this study, we examine the capacity of ambient PM
2.5
samples collected during periods of peak air pollution in Fresno and Claremont, CA to trigger ROS production in AM cells. These PM
2.5
samples were characterized for their chemical content using chromatography and mass spectrometry. A DCF‐fluorescence based microplate assay was used to estimate the potential of the PM
2.5
samples to trigger ROS production in NR8383, rat AM cells. Two types of quinones (1,2‐Naphtaquinone and 1,4‐Naphtaquinone) and two transition metals (Cu
2+
and Fe
2+
) were associated with samples that had a high capacity to induce ROS generation in NR8383. While establishing a cytotoxic link between PM
2.5
constituents and ROS, we also seek to understand the underlying biological mechanisms through which PM
2.5
interacts with AM cells to produce excessive ROS. Previous observations suggest that the upregulation of NADPH Oxidase (NOX1), an enzyme that enhances ROS production, may contribute to cellular oxidative stress in macrophages. In this study, NOX1’s potential role in a PM
2.5
‐ induced oxidative stress pathway is investigated in NR8383 cells incubated with concentrations of iron and copper relevant with those found in the collected PM
2.5
samples. The change in NOX1 expression was quantified using fluorescence‐based western blotting, with zymosan, a well‐known inducer of oxidative stress in macrophages, used as positive control. Our preliminary results indicate that 4‐hour treatments with increasing relevant iron concentrations triggered both an increase of ROS and NOX1 protein levels in NR8383. Our current experiments consist of testing the potential of relevant concentrations of other chemical constituents of PM
2.5
suspected of triggering increased ROS production and NOX1 expression (these include naphtaquinones, phenanthraquinones, and copper). Understanding which chemical components of PM
2.5
are able to induce significant amounts of ROS will allow us to understand how pollution is pernicious and advocate for the global reduction of PM
2.5
emission.
Support or Funding Information
This work is supported by Fresno State ASI, the Division of Research and Graduate Studies, and Fresno State’s CSU‐LSAMP program funded by NSF under grant #HRD‐1826490.
When oxidative stress exceeds normal levels, the antioxidant system of any eukaryotic organism can become overwhelmed. This may lead to various adverse health effects, including cardiovascular and ...pulmonary diseases. The risk for these conditions is heightened by increased exposure to particulate matter 2.5 (PM2.5), composed of transition metals and organic compounds 2.5μm or less in diameter. These particles reach the alveolar macrophages (AMs) in the respiratory tract, where they can induce an inflammatory response by producing excessive reactive oxygen species (ROS). During 2012 and 2013, samples of ambient PM2.5 samples were collected during periods of peak air pollution from Fresno and Claremont, CA. These samples were tested for their potential to induce a ROS response in the rat NR8383 AM cell line using the intracellular ROS‐sensitive fluorescent probe 2′‐7′‐Dichlorodihydrofluorescein diacetate. Interestingly, the specific cellular ROS response was dependent on the sample origin; strongly suggesting that the chemical composition of PM2.5 is a major determinant of its effect on AM ROS metabolism. The PM2.5 samples were characterized for their chemical content using chromatography and mass spectrometry based techniques. Two types of quinones (1,2‐Naphtaquinone, and 1,4‐Naphtaquinone) and one transition metal (Cu2+) were associated with samples that had a high capacity to create ROS in NR8383. Currently, each of the above components' potential to induce a cellular ROS response is being investigated using mock samples with concentration ranges similar to those of the PM2.5 samples. While establishing a cytotoxic link between PM2.5 and ROS, we also seek to understand the underlying biological mechanisms through which PM2.5 interacts with AM cells to produce excessive ROS. Previous studies have suggested the upregulation of heme oxygenase‐1 (HO‐1) during oxidative stress plays an essential role in intracellular ROS regulation. Our study consists of optimizing a gene knockdown protocol for HO‐1 in NR8383 to investigate the potential involvement of HO‐1 in a PM2.5‐induced ROS response. Having established an optimal protein concentration of 50μg to see distinct HO‐1 bands in western blots, a range of siRNA concentrations are being used in conjunction with our positive control, zymosan, to understand their cytotoxicity effects. Zymosan, extracted from the cell wall of saccharomyces cerevisiae, is a known inflammatory agent which stimulates ROS production in NR8383. Repeated trials of western blotting confirm that 4 hour zymosan treatments increase the HO‐1 protein levels in NR8383. The HO‐1 knockdown will be confirmed through quantitative reverse transcription polymerase chain reaction (qrt‐PCR) and western blotting. Cells with the HO‐1 knockdown will then be subjected to a fluorescence‐based microplate assay to monitor their PM2.5‐induced ROS response. We hypothesize enhanced ROS production in PM2.5 treated HO1‐knockdown cells due to the lack of the protective enzyme, HO‐1. Understanding the specific chemical components which have proved noxious to NR8383 and the biological mechanisms through which PM2.5 is able to induce ROS will help us understand exactly how pollution is pernicious and advocate for global reduction of PM2.5.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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