Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis.

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

Risk Classification for Progression to Subfoveal Geographic Atrophy in Dry Age-Related Macular Degeneration Using Machine Learning-Enabled Outer Retinal Feature Extraction.

BACKGROUND AND OBJECTIVE: To evaluate the utility of spectral-domain optical coherence tomography biomarkers to predict the development of subfoveal geographic atrophy (sfGA). PATIENTS AND METHODS: This was a retrospective cohort analysis including 137 individuals with dry age-related macular degeneration without sfGA with 5 years of follow-up. Multiple spectral-domain optical coherence tomography quantitative metrics were generated, including ellipsoid zone (EZ) integrity and subretinal pigment epithelium (sub-RPE) compartment features. RESULTS: Reduced mean EZ-RPE central subfield thickness and increased sub-RPE compartment thickness were significantly different between sfGA convertors and nonconvertors at baseline in both 2-year and 5-year sfGA risk assessment. Longitudinal change assessment showed a significantly higher degradation of EZ integrity in sfGA convertors. The predictive performance of a machine learning classification model based on 5-year and 2-year risk conversion to sfGA demonstrated an area under the receiver operating characteristic curve of 0.92 ± 0.06 and 0.96 ± 0.04, respectively. CONCLUSIONS: Quantitative outer retinal and sub-RPE feature assessment using a machine learning-enabled retinal segmentation platform provides multiple parameters that are associated with progression to sfGA. [Ophthalmic Surg Lasers Imaging. 2022;53:31-39.].

Regulation of cytochrome P450 4F11 by nuclear transcription factor-κB.

Although the mechanisms that regulate CYP4F genes have been and are currently being studied in a number of laboratories, the specific mechanisms for the regulation of these genes are not yet fully understood. This study shows that nuclear factor κB of the light-chain-enhancer in activated B cells (NF-κB) can inhibit CYP4F11 expression in human liver carcinoma cell line (HepG2) as summarized below. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, has been shown to activate NF-κB signaling while also activating the c-Jun NH(2)-terminal kinase (JNK) signaling pathway. Other studies have reported that JNK signaling can up-regulate CYP4F11 expression. The results of this study demonstrate that in the presence of TNF-α and the specific NF-κB translocation inhibitor N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354), there is a greater increase in CYP4F11 expression than that elicited by TNF-α alone, indicating that NF-κB plays an inhibitory role. Moreover, NF-κB stimulation by overexpression of mitogen-activated protein kinase kinase kinase inhibited CYP4F11 promoter expression. CYP4F11 promoter inhibition can also be rescued in the presence of TNF-α when p65, a NF-κB protein, is knocked down. Thus, NF-κB signaling pathways negatively regulate the CYP4F11 gene.

Gene regulation of CYP4F11 in human keratinocyte HaCaT cells.

Mechanisms regulating CYP4F genes remain under investigation, although characterization of CYP4F regulatory modalities would facilitate the discovery of new drug targets. This present study shows that all-trans- and 9-cis-retinoic acids can inhibit CYP4F11 expression in human keratinocyte-derived HaCaT cells. Transrepression of many genes by retinoic acids is mediated by interactions between retinoid receptors and the activator protein 1 (AP-1) complex. Proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta, which can activate the AP-1 complex, induce CYP4F11 transcription in HaCaT cells. The c-Jun N-terminal kinase (JNK)-specific inhibitor 1,9-pyrazoloanthrone (SP600125) blocked the induction of CYP4F11 by both cytokines, indicating involvement of the JNK pathway. Furthermore, TNF-alpha failed to induce CYP4F11 transcription when HaCaT cells were preincubated with retinoic acids. Retinoic acids are ligands for the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RXR agonist 6-(1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl) nicotinic acid (LG268) greatly induced CYP4F11 transcription, whereas the RAR agonist 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB) markedly inhibited CYP4F11 transcription, indicating that down-regulation of CYP4F11 transcription by retinoic acid is mediated by RARs and may also be related to ligand competition for RXRs. Thus, the CYP4F11 gene is positively regulated by multiple signaling pathways in HaCaT keratinocytes, including RXR and JNK signaling pathways.