Using genetically encoded fluorescent biosensors to interrogate ovarian cancer metabolism

Abstract Background Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments. Materials and methods Plasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin. Results Exposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients. Conclusions Our study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC

Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells - Fig 6

<p>MALAT1 expression is increased in human primary RPE cells incubated TGF-β1 (10 ng/ml) (A) or PVR vitreous (B) as detected by RT-PCR.</p

Downregulation of MALAT1 inhibited TGF-β1 induced migration and proliferation in RPE cells.

<p>A. ARPE-19 cells were transfected with MALAT1 SiRNA (Si-MALAT1) or negative control SiRNA (Si-NC) and were treated with or without TGF-β1 (10ng/ml) for 48 h. Cells were then subjected to transwell migration assay. B. The number of migrated cells was quantified by counting 5 random vision fields in a microscope (magnification: ×200). C. ARPE-19 cells were transfected with Si-MALAT1 or Si-NC. A scratch was then made to the cell monolayer and TGF-β1 (10ng/ml) was applied. Photographs were taken at indicated times. D. The ratios of remaining of gap at 48 h were calculated. E. After siRNA transfection and TGF-β1 incubation, cell proliferation was assessed using an MTT cell proliferation assay kit.</p

Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Background/Aims: Proliferative vitreoretinopathy (PVR) is a severe blinding complication of rhegmatogenous retinal detachment. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is thought to play a pivotal role in the pathogenesis of PVR. Fucoidan, a marine extract, reportedly has many benefits effects in a variety of tissues and organs such as anti-inflammation, anti-oxidative stress, and anti-carcinogenesis. In this study, we investigated the potential role of fucoidan on EMT in RPE cells and its effect on the development of PVR. Methods: MTS, Transwell, and collagen gel contraction assays were employed to measure the viability, migration, and contraction of RPE cells, respectively. mRNA and protein expression were evaluated via real-time quantitative PCR and western blot analysis, respectively. In vivo, a pigmented rabbit model of PVR was established to examine the anti-PVR effect of fucoidan. Results: Fucoidan reversed the transforming growth factor (TGF)-β1-induced EMT of RPE cells, including the increased expression of α-smooth muscle actin (α-SMA) and fibronectin and down-regulation of E-cadherin in human primary RPE cells. Moreover, the upregulation of phosphorylated Smad2/3 induced by TGF-β1 was suppressed by fucoidan. Fucoidan also inhibited the migration and contraction of RPE cells induced by TGF-β1. In vivo, fucoidan inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that fucoidan suppressed the formation of α-SMA-positive epiretinal membranes. Conclusion: Our findings regarding the protective effects of fucoidan on the EMT of RPE cells and experimental PVR suggest the potential clinical application of fucoidan as an anti-PVR agent

Knocking-down of MALAT1 reduced the TGF-β1-induced up-regulation of Snail, SLUG, and ZEB1 in RPE cells.

<p><b>A-D</b> ARPE-19 cells were transfected with MALAT1 SiRNA (Si-MALAT1) or negative control SiRNA (Si-NC) and were treated with or without TGF-β1 (10ng/ml) for 48 h. The expression level of EMT-related transcription factors (Snail, SLUG, and ZEB1) were detected and quantified by RT-PCR (A), Western blot (B-C) and immunofluorescence (D).</p

Downregulating MALAT1 inhibits the phosphorylation of Smad2/3.

<p>ARPE-19 cells transfected with Si-MALAT1 or Si-NC were treated with TGF-β1 (10 ng/ml) for 1 h. The expression of p-Smad2/3 and Smad2/3 (A), as well as p-38 and p38 (C) were examined by western blot. The relative protein expressions were quantified by normalizing to the GAPDH expression (B and D).</p

Knockdown of MALAT1 attenuates the TGF-β1 induced EMT in RPE cells.

<p>ARPE-19 cells were transfected with MALAT1 SiRNA (Si-MALAT1) or negative control SiRNA (Si-NC) and were treated with or without TGF-β1 (10ng/ml) for 48 h. A. The expression levels of MALAT1 were detected by RT-PCR. B-C. The expression level of EMT-related markers (E-Cadherin, ZO-1, α-SMA, and Fibronection) were detected by RT-PCR and Western blot. D, Quantification of the relative protein expression (normalized to β-actin) in C. E. The morphologic appearances of the cells were captured at 100× magnification. F. The expression of E-Cadherin, ZO-1, α-SMA, and Fibronection werer detected by immunofluorescence.</p

TGF-β1 induces EMT and MALAT1 expression in ARPE-19 cells.

<p>APRE-19 cells were incubated with TGF-β1 (10 ng/ml) for 48h. A. TGF-β1 induces the morphological change of APRE-19 cells were captured at 100× magnification. B-D. The expression level of EMT-related markers (E-Cadherin, ZO-1, α-SMA, and Fibronection) were detected by immunofluorescence, RT-PCR, and Western blot. E. Quantification of the relative protein expression (normalized to β-actin) in western blot. F. The expression of MALAT1 was detected by RT-PCR in indicated time points.</p

Rapid tissue prototyping with micro-organospheres

In vitro tissue models hold great promise for modeling diseases and drug responses. Here, we used emulsion microfluidics to form micro-organospheres (MOSs), which are droplet-encapsulated miniature three-dimensional (3D) tissue models that can be established rapidly from patient tissues or cells. MOSs retain key biological features and responses to chemo-, targeted, and radiation therapies compared with organoids. The small size and large surface-to-volume ratio of MOSs enable various applications including quantitative assessment of nutrient dependence, pathogen-host interaction for anti-viral drug screening, and a rapid potency assay for chimeric antigen receptor (CAR)-T therapy. An automated MOS imaging pipeline combined with machine learning overcomes plating variation, distinguishes tumorspheres from stroma, differentiates cytostatic versus cytotoxic drug effects, and captures resistant clones and heterogeneity in drug response. This pipeline is capable of robust assessments of drug response at individual-tumorsphere resolution and provides a rapid and high-throughput therapeutic profiling platform for precision medicine