Real-time Live Cell Imaging Research Articles

Telmisartan attenuates human glioblastoma cells proliferation and oncogenicity by inducing the lipid oxidation.

BackgroundGlioblastoma (GBM) is one of the most common primary brain tumors, which accounts up to 80% of malignant brain tumors and the 5‐year relative survival rate is below 5%. Recent studies showed that the lipid metabolism played an essential role in GBM development. As a peroxisome proliferators‐activated receptors γ (PPAR‐γ) agonist, telmisartan improves the lipid metabolism and has been used to treat hypertension for long time. It has also been shown to have anticancer function, such as in lung cancer and melanoma.MethodsIncucyte real‐time live cell imaging system was used to assess the effect of telmisartan on glioma cell lines U87 and U251 proliferation. Transwell assay and colony formation assay were conducted to detect the effect of telmisartan on oncogenicity of GBM cell lines. Western blot and immunofluorescence analysis were used to detect the effect of telmisartan on the expression of PPAR‐γ and hydroxyacyl‐coenzyme A dehydrogenase alpha subunit (HADHA).ResultsWe demonstrate that telmisartan inhibits two glioma cell lines U87 and U251 proliferation in a time‐ and dose‐dependent manner, and arrests the cell cycle at S phase. We further show that telmisartan decreases the oncogenicity of GBM cell lines. Our data show that telmisartan treatment significantly increases the PPAR‐γ expression level, enhances the lipid oxidation, and upregulates the level of fatty acid oxidation key enzyme HADHA.ConclusionsTelmisartan inhibits the proliferation and oncogenicity while it also increases the lipid oxidation of human GBM cells.

CAMP Imaging at Ryanodine Receptors Reveals β2-Adrenoceptor Driven Arrhythmias.

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Accelerating 3D single-molecule localization microscopy using blind sparse inpainting.

.Significance: Single-molecule localization-based super-resolution microscopy has enabled the imaging of microscopic objects beyond the diffraction limit. However, this technique is limited by the requirements of imaging an extremely large number of frames of biological samples to generate a super-resolution image, thus requiring a longer acquisition time. Additionally, the processing of such a large image sequence leads to longer data processing time. Therefore, accelerating image acquisition and processing in single-molecule localization microscopy (SMLM) has been of perennial interest.Aim: To accelerate three-dimensional (3D) SMLM imaging by leveraging a computational approach without compromising the resolution.Approach: We used blind sparse inpainting to reconstruct high-density 3D images from low-density ones. The low-density images are generated using much fewer frames than usually needed, thus requiring a shorter acquisition and processing time. Therefore, our technique will accelerate 3D SMLM without changing the existing standard SMLM hardware system and labeling protocol.Results: The performance of the blind sparse inpainting was evaluated on both simulation and experimental datasets. Superior reconstruction results of 3D SMLM images using up to 10-fold fewer frames in simulation and up to 50-fold fewer frames in experimental data were achieved.Conclusions: We demonstrate the feasibility of fast 3D SMLM imaging leveraging a computational approach to reduce the number of acquired frames. We anticipate our technique will enable future real-time live-cell 3D imaging to investigate complex nanoscopic biological structures and their functions.

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism.

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism.

Sucrose is the main saccharide used for long‐distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real‐time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne‐tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real‐time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

Endothelial Nitric Oxide Synthase Knockdown in Human Stem Cells Impacts Mitochondrial Biogenesis and Adipogenesis: Live-Cell Real-Time Fluorescence Imaging.

We carried out live-cell real-time fluorescence imaging to follow the effects of genetic (siRNA) knockdown (KD) of endothelial nitric oxide synthase (eNOS) on mitochondrial biogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). We report here that eNOS KD in hMSCs blocks mitochondrial biogenesis and adipogenesis. The transfer of mitochondria from normal hMSCs to eNOS-deficient hMSCs restores adipogenesis. Furthermore, cell-free mitochondria purified from normal hMSCs also restores adipogenesis in eNOS-deficient cells. Thus, eNOS and NO signaling are essential for mitochondrial biogenesis, and mitochondrial activity is indispensable for adipogenesis in hMSC differentiation. We mapped the path and identified the mechanisms of mitochondrial transfer. We captured real-time images of differentiated mature adipocytes in mitosis and replication. These results reveal that human stem cell-differentiated fat cells are capable of replication. This new finding offers novel insights into our understanding of fat cell expansion and the development of obesity. Real-time imaging in live cells allows synchronized investigation of mitochondrial biogenesis and adipogenesis in stem cell differentiation without reducing living cells to nonliving samples for functional analysis. Live-cell real-time imaging can thus be a faithful and immediate tool for molecular diagnostic medicine. Furthermore, our results suggest that mitochondrial remodeling can be a useful approach in treating adiposity, diabetes, and abnormalities in energy metabolism and vascular signaling.

Exosomes derived from stem cells from apical papilla promote craniofacial soft tissue regeneration by enhancing Cdc42-mediated vascularization

BackgroundReconstruction of complex critical-size defects (CSD) in the craniofacial region is a major challenge, and soft tissue regeneration is crucial in determining the therapeutic outcomes of craniofacial CSD. Stem cells from apical papilla (SCAP) are neural crest-derived mesenchymal stem cells (MSCs) that are homologous to cells in craniofacial tissue and represent a promising source for craniofacial tissue regeneration. Exosomes, which contain compound bioactive compounds, are the key factors in stem cell paracrine action. However, the roles of exosomes derived from SCAP (SCAP-Exo) in tissue regeneration are not fully understood. Here, we explored the effects and underlying mechanisms of SCAP-Exo on CSD in maxillofacial soft tissue.MethodsSCAP-Exo were isolated and identified by transmission electron microscopy and nanoparticle tracking analysis. The effects of SCAP-Exo on wound healing and vascularization were detected by measuring the wound area and performing histological and immunofluorescence analysis on the palatal gingival CSD of mice. Real-time live-cell imaging and functional assays were used to assess the effects of SCAP-Exo on the biological functions of endothelial cells (ECs). Furthermore, the molecular mechanisms of SCAP-Exo-mediated EC angiogenesis in vitro were tested by immunofluorescence staining, Western blot, and pull-down assays. Finally, in vivo experiments were carried out to verify whether SCAP-Exo could affect vascularization and wound healing through cell division cycle 42 (Cdc42).ResultsWe found that SCAP-Exo promoted tissue regeneration of palatal gingival CSD by enhancing vascularization in the early phase in vivo and that SCAP-Exo improved the angiogenic capacity of ECs in vitro. Mechanistically, SCAP-Exo elevated cell migration by improving cytoskeletal reorganization of ECs via Cdc42 signalling. Furthermore, we revealed that SCAP-Exo transferred Cdc42 into the cytoplasm of ECs and that the Cdc42 protein could be reused directly by recipient ECs, which resulted in the activation of Cdc42-dependent filopodium formation and elevation in cell migration of ECs.ConclusionThis study demonstrated that SCAP-Exo had a superior effect on angiogenesis and effectively promoted craniofacial soft tissue regeneration. These data provide a new option for SCAP-Exo to be used in a cell-free approach to optimize tissue regeneration in the clinic.

Synthesis and characterization of novel protein nanodots as drug delivery carriers with an enhanced biological efficacy of melatonin in breast cancer cells.

Melatonin is a potent antioxidant, chemotherapeutic and chemo preventive agent against breast cancer. However, its short half-life is one of the major limitations in its application as a therapeutic drug. To overcome this issue, the green-emitting protein nanodot (PND) was synthesized by a one-step hydrothermal method for loading melatonin. The synthesized pH-7 and pH-2 PND showed a quantum yield of 22.1% and 14.0%, respectively. The physicochemical characterization of both PNDs showed similar morphological and functional activities. Furthermore, the biological efficacy of melatonin-loaded PND (MPND) was evaluated in a breast cancer cell line (MDA-MB-231) for live-cell imaging and enhanced nano-drug delivery efficacy. Interestingly, the permeability of neutral pH PND in both cell cytoplasm and nucleus nullifies the limitations of real-time live-cell imaging, and ensures nuclear drug delivery efficacy. Neutral pH PND showed better cell viability and cytotoxicity as a fluorescence bioimaging probe compared to acidic PND. The bioavailability and cell cytotoxicity effect of MPND on MDA-MB-231 breast cancer cells were studied through confocal and migration assay. Results showed that MPND causes enhanced bioavailability, better cellular uptake, and inhibition of the migration of breast cancer cells as compared to the drug alone. Besides, the synthesized MPND showed no sign of fluorescence quenching even at a high concentration of melatonin, making it an ideal nanocarrier for bioimaging and drug delivery.

Testing Cellular Proliferation Responses to Oxidative, Glucocorticoid and Metabolic Challenges in the Baboon

Aging is associated with progressive loss of cellular homeostasis which results from intrinsic and extrinsic challenges. Testing how such challenges relate to the aging process is often limited by the available model systems. We use primary cells (fibroblasts) isolated from baboons as a non-human primate cellular model to address how stressful challenges affect resilience. Using a real-time live-cell imaging system, we characterized a protocol for testing the effects of pro-oxidant compounds (e.g hydrogen peroxide (H2O2), paraquat and thapsigargin), dexamethasone and low glucose environment on cellular proliferation in fibroblasts derived from baboons across the life-course. The inhibitory effect of H2O2 (50 and 100µM), an oxidative stress, on cell proliferation was dose-dependent, with a higher impact in old males (age 14.52-14.80 years; average life span 21 years) compared to young males (6.35-6.39 years). Exposure to a different oxidative stress, paraquat (100 and 200µM) tended to reduce cell proliferation rate with age in males but not females. Inhibitory effects of thapsigargin, an endoplasmic reticulum stress inducer, on cell proliferation were dependent on challenge duration (2 vs 24h), concentration (0.1 and 1µM) and donor age, with greater resilience in young males than young females (4.33-6.70 years). Dexamethasone (100 and 500µM), a glucocorticoid, reduced cell proliferation dose-dependently, with older males exhibiting more resilience than females. In response to low glucose (1mM), cell proliferation reduced with age. Donor’s chronological age and sex are important variables in cellular response to challenge compounds faced during aging, which will guide our on-going studies on the cellular transcriptome and proteome.

The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic β-cells

Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in β-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic β-cells. β-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic β-cells.

Metabolic co-dependence of the oocyte and cumulus cells: essential role in determining oocyte developmental competence.

Within the antral follicle, the oocyte is reliant on metabolic support from its surrounding somatic cells. Metabolism plays a critical role in oocyte developmental competence (oocyte quality). In the last decade, there has been significant progress in understanding the metabolism of the cumulus-oocyte complex (COC) during its final stages of growth and maturation in the follicle. Certain metabolic conditions (e.g. obesity) or ART (e.g. IVM) perturb COC metabolism, providing insights into metabolic regulation of oocyte quality. This review provides an update on the progress made in our understanding of COC metabolism, and the metabolic conditions that influence both meiotic and developmental competence of the oocyte. The PubMed database was used to search for peer-reviewed original and review articles. Searches were performed adopting the main terms 'oocyte metabolism', 'cumulus cell metabolism', 'oocyte maturation', 'oocyte mitochondria', 'oocyte metabolism', 'oocyte developmental competence' and 'oocyte IVM'. Metabolism is a major determinant of oocyte quality. Glucose is an essential requirement for both meiotic and cytoplasmic maturation of the COC. Glucose is the driver of cumulus cell metabolism and is essential for energy production, extracellular matrix formation and supply of pyruvate to the oocyte for ATP production. Mitochondria are the primary source of ATP production within the oocyte. Recent advances in real-time live cell imaging reveal dynamic fluctuations in ATP demand throughout oocyte maturation. Cumulus cells have been shown to play a central role in maintaining adequate oocyte ATP levels by providing metabolic support through gap junctional communication. New insights have highlighted the importance of oocyte lipid metabolism for oocyte oxidative phosphorylation for ATP production, meiotic progression and developmental competence. Within the last decade, several new strategies for improving the developmental competence of oocytes undergoing IVM have emerged, including modulation of cyclic nucleotides, the addition of precursors for the antioxidant glutathione or endogenous maturation mediators such as epidermal growth factor-like peptides and growth differentiation factor 9/bone morphogenetic protein 15. These IVM additives positively alter COC metabolic endpoints commonly associated with oocyte competence. There remain significant challenges in the study of COC metabolism. Owing to the paucity in non-invasive or in situ techniques to assess metabolism, most work to date has used in vitro or ex vivo models. Additionally, the difficulty of measuring oocyte and cumulus cell metabolism separately while still in a complex has led to the frequent use of denuded oocytes, the results from which should be interpreted with caution since the oocyte and cumulus cell compartments are metabolically interdependent, and oocytes do not naturally exist in a naked state until after fertilization. There are emerging tools, including live fluorescence imaging and photonics probes, which may provide ways to measure the dynamic nature of metabolism in a single oocyte, potentially while in situ. There is an association between oocyte metabolism and oocyte developmental competence. Advancing our understanding of basic cellular and biochemical mechanisms regulating oocyte metabolism may identify new avenues to augment oocyte quality and assess developmental potential in assisted reproduction.

Tagging Transferrin Receptor with a Disulfide FRET Probe To Gauge the Redox State in Endosomal Compartments.

Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.

A multiparametric CAR T cell potency assay that employs real-time cellular impedance and live cell imaging

Background & Aim Chimeric antigen receptors (CARs) are engineered proteins that consist of an extracellular antigen binding domain that is linked, via a hinge region and transmembrane domain, to intracellular activation domains that drive T cell activation, proliferation, and target cell killing. A few decades of CAR engineering efforts have made it clear that merely linking these protein domains together is insufficient for generating an effective CAR; subtle differences in how the domains are linked to each other can greatly impact functionality. During CAR optimization biochemical assays can be used to assess critical quality attributes such as potency. Although these reductionistic approaches are useful, their results don't necessarily correlate with cancer killing efficacy within a more physiologically relevant context. To rigorously compare different CAR constructs and identify unwanted behavior such as tonic signaling, it is widely acknowledged that CAR function should be evaluated within the complex milieu of the immune synapse, using bona fide cancer cell killing as the readout. For this purpose, release assays (51Cr, LDH) have historically been the gold standard, but suffer from the fact that they require significant “hands on” time and only provide end point data. Requiring just target cancer cell seeding and a subsequent CAR T cell addition step, herein we use the Agilent xCELLigence RTCA eSight to quantitatively evaluate CAR T cell-mediated killing of cancer cells in real-time over multiple days and at physiologically relevant low E:T ratios. Methods, Results & Conclusion Providing a direct and objective assessment of target cancer cell number, cell size, and cell-substrate attachment strength, impedance biosensors embedded within the base of eSight microplates are shown to quantitatively track the continuum of target cell killing, spanning from early (reduced cell-substrate adhesion strength) to late (lysis) events. Concurrently, eSight is shown to capture live cell images in brightfield and three fluorescence channels, providing an orthogonal readout of the killing process – including real-time assessment of T cell activation and exhaustion markers such as CD69, CD107a, CTLA4, and LAG-3. By combining the strengths of real-time impedance monitoring (simplicity, analytical sensitivity, and objectivity) with that of live cell imaging (specificity of the readout), eSight is shown to increase the information richness of the CAR T cell killing assay without increasing the workload.

Real-time monitoring of cAMP in brown adipocytes reveals differential compartmentation of β1 and β3-adrenoceptor signalling

Objective3′,5′-Cyclic adenosine monophosphate (cAMP) is a central second messenger governing brown adipocyte differentiation and function. β-adrenergic receptors (β-ARs) stimulate adenylate cyclases which produce cAMP. Moreover, cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDEs), which can generate subcellular microdomains of cAMP. Since the spatio-temporal organisation of the cAMP signalling pathway in adipocytes is still unclear, we sought to monitor real-time cAMP dynamics by live cell imaging in pre-mature and mature brown adipocytes. MethodsWe measured the real-time dynamics of cAMP in murine pre-mature and mature brown adipocytes during stimulation of individual β-AR subtypes, as well as its regulation by PDEs using a Förster Resonance Energy Transfer based biosensor and pharmacological tools. We also correlated these data with β-AR stimulated lipolysis and analysed the expression of β-ARs and PDEs in brown adipocytes using qPCR and immunoblotting. Furthermore, subcellular distribution of PDEs was studied using cell fractionation and immunoblots. ResultsUsing pre-mature and mature brown adipocytes isolated from transgenic mice expressing a highly sensitive cytosolic biosensor Epac1-camps, we established real-time measurements of cAMP responses. PDE4 turned out to be the major PDE regulating cytosolic cAMP in brown preadipocytes. Upon maturation, PDE3 gets upregulated and contributes with PDE4 to control β1-AR-induced cAMP. Unexpectedly, β3-AR initiated cAMP is resistant to increased PDE3 protein levels and simultaneously, the control of this microdomain by PDE4 is reduced upon brown adipocyte maturation. Therefore we postulate the existence of distinct cAMP pools in brown adipocytes. One cAMP pool is formed by β1-AR associated with PDE3 and PDE4, while another pool is centred around β3-AR and is much less controlled by these PDEs. Functionally, lower control of β3-AR initiated cAMP by PDE3 and PDE4 facilitates brown adipocyte lipolysis, while lipolysis activated by β1-AR and is under tight control of PDE3 and PDE4. ConclusionsWe have established a real-time live cell imaging approach to analyse brown adipocyte cAMP dynamics in real-time using a cAMP biosensor. We showed that during the differentiation from pre-mature to mature murine brown adipocytes, there was a change in PDE-dependent compartmentation of β1-and β3-AR-initiated cAMP responses by PDE3 and PDE4 regulating lipolysis.

Polyphyllin VI Induces Caspase-1-Mediated Pyroptosis via the Induction of ROS/NF-κB/NLRP3/GSDMD Signal Axis in Non-Small Cell Lung Cancer.

Trillium tschonoskii Maxim (TTM), a traditional Chinese medicine, has been demonstrated to have a potent anti-tumor effect. Recently, polyphyllin VI (PPVI), a main saponin isolated from TTM, was reported by us to significantly suppress the proliferation of non-small cell lung cancer (NSCLC) via the induction of apoptosis and autophagy in vitro and in vivo. In this study, we further found that the NLRP3 inflammasome was activated in PPVI administrated A549-bearing athymic nude mice. As is known to us, pyroptosis is an inflammatory form of caspase-1-dependent programmed cell death that plays an important role in cancer. By using A549 and H1299 cells, the in vitro effect and action mechanism by which PPVI induces activation of the NLRP3 inflammasome in NSCLC were investigated. The anti-proliferative effect of PPVI in A549 and H1299 cells was firstly measured and validated by MTT assay. The activation of the NLRP3 inflammasome was detected by using Hoechst33324/PI staining, flow cytometry analysis and real-time live cell imaging methods. We found that PPVI significantly increased the percentage of cells with PI signal in A549 and H1299, and the dynamic change in cell morphology and the process of cell death of A549 cells indicated that PPVI induced an apoptosis-to-pyroptosis switch, and, ultimately, lytic cell death. In addition, belnacasan (VX-765), an inhibitor of caspase-1, could remarkably decrease the pyroptotic cell death of PPVI-treated A549 and H1299 cells. Moreover, by detecting the expression of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD in A549 and h1299 cells using Western blotting, immunofluorescence imaging and flow cytometric analysis, measuring the caspase-1 activity using colorimetric assay, and quantifying the cytokines level of IL-1β and IL-18 using ELISA, the NLRP3 inflammasome was found to be activated in a dose manner, while VX-765 and necrosulfonamide (NSA), an inhibitor of GSDMD, could inhibit PPVI-induced activation of the NLRP3 inflammasome. Furthermore, the mechanism study found that PPVI could activate the NF-κB signaling pathway via increasing reactive oxygen species (ROS) levels in A549 and H1299 cells, and N-acetyl-L-cysteine (NAC), a scavenger of ROS, remarkably inhibited the cell death, and the activation of NF-κB and the NLRP3 inflammasome in PPVI-treated A549 and H1299 cells. Taken together, these data suggested that PPVI-induced, caspase-1-mediated pyroptosis via the induction of the ROS/NF-κB/NLRP3/GSDMD signal axis in NSCLC, which further clarified the mechanism of PPVI in the inhibition of NSCLC, and thereby provided a possibility for PPVI to serve as a novel therapeutic agent for NSCLC in the future.

BODIPY based realtime, reversible and targeted fluorescent probes for biothiol imaging in living cells.

Real-time live cell imaging and quantification of biothiol dynamics are important for understanding pathophysiological processes. However, the design and synthesis of rational probes that have reversible and real-time capabilities is still challenging. In this work, we have prepared boron-dipyrrolemethene (BODIPY) based fluorescent molecules as ratiometric probes that allow the real-time biothiol dynamics to be observed in living cells. The Michael reaction between α-formyl-BODIPY (BOD-JQ) and GSH exhibited a reversible fluorogenic mechanism with fluorescent emission shifting from 592 nm to 544 nm with t1/2 = 16 ms. In particular, we showed that the probes with targeting agents are capable of detecting biothiols in mitochondria and the endoplasmic reticulum (ER) with high temporal resolution.

Live Cell Imaging with Holotomography and Fluorescence

Abstract

Abstract B086: Cellular context-dependent regulation of cancer cell invasion and migration in pancreatic tumor spheroids co-cultured with pancreatic stellate cells

Abstract Purpose Invasion of cancer cells is the initial step of metastatic progression. Elucidation of the cellular and molecular mechanisms of invasion may provide novel strategies to modulate metastasis and survival. The plasticity of cancer cells and surrounding stroma induces cell migration of which three types have been reported: collective, mesenchymal, or amoeboid type of movement. Here we investigated differential invasion phenotype and underlying mechanisms using two pancreatic cancer cells, BxPC-3 (wild-type KRAS, epithelial type) and PANC-1 (mutated KRAS, intermediate type) under 3D culture conditions. Methods Multicellular tumor spheroids (TS) were cultured in 3D collagen gel matrix either as mono-culture or under pancreatic stellate cell (PSC) co-culture condition using micro-channel chips. TS formation, cell migration within and outside of collagen gel, ECM remodeling, and expression of proteins involved in epithelial-mesenchymal transition (EMT) as well as degradation of matrix proteins were evaluated using immunofluorescence confocal microscopy, real-time live cell imaging and subsequent image analysis. Results BxPC-3 cells formed highly compact TS with strong expression of E-cadherin compared to PANC-1 cells. BxPC-3 cells showed collective migration under mono-culture condition and showed 50% phenotypic conversion to amoeboid single cell migration under PSC co-culture condition. Lamellipodia formation with actin spikes were observed in both types of cell migration in BxPC-3 cells. PANC-1 cells showed mostly mesenchymal invasion with single cells migrating out of TS using invadopodia. PANC-1 cells showed significantly higher level of vimentin and TGF-β as compared to BxPC-3 cells. Extensive ECM remodeling with collagen densification and fiber alignment was shown with BxPC-3 cells, compared to that of PANC-1 cells, under both mono- and PSC co-culture conditions. Similar levels of collagen degradation and expression of MT1-MMP, integrin β1 and pFAK between the two cell lines strongly suggest additional mechanisms of mechanical deformation of ECM facilitated by traction force of BxPC-3 cells, which warrants further studies. When cancer cells and PSCs migrated out of collagen matrix channels to encounter, fibronectin-mediated cell-cell interaction leading to co-migration was observed in both cell lines. Conclusions We demonstrated cellular context-dependent regulation of cancer cell invasion and its association with cancer cell-ECM and cancer cell-PSC interaction in pancreatic TS co-cultured with PSC. Our 3D TS-PSC co-culture recapitulates the differential modes of cancer cell invasion; hence, it may serve as an efficient and in vivo mimic model to study cellular and molecular mechanisms of invasion. Citation Format: Seul-ki Kim, Min-Suk Oh, Hyo-Jeong Kuh. Cellular context-dependent regulation of cancer cell invasion and migration in pancreatic tumor spheroids co-cultured with pancreatic stellate cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B086. doi:10.1158/1535-7163.TARG-19-B086

Live Simultaneous Monitoring of Mineral Deposition and Lipid Accumulation in Differentiating Stem Cells.

Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media, modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells.

Real-Time Live-Cell Imaging Technology Enables High-Throughput Screening to Verify in Vitro Biocompatibility of 3D Printed Materials.

With growing advances in three-dimensional (3D) printing technology, the availability and diversity of printing materials has rapidly increased over the last years. 3D printing has quickly become a useful tool for biomedical and various laboratory applications, offering a tremendous potential for efficiently fabricating complex devices in a short period of time. However, there still remains a lack of information regarding the impact of printing materials and post-processing techniques on cell behavior. This study introduces real-time live-cell imaging technology as a fast, user-friendly, and high-throughput screening strategy to verify the in vitro biocompatibility of 3D printed materials. Polyacrylate-based photopolymer material was printed using high-resolution 3D printing techniques, post-processed using three different procedures, and then analyzed with respect to its effects on cell viability, apoptosis, and necrosis of adipogenic mesenchymal stem cells (MSCs). When using ethanol for the post-processing procedure and disinfection, no significant effects on MSCs could be detected. For the analyses a novel image-based live-cell analysis system was compared against a biochemical-based standard plate reader assay and traditional flow cytometry. This comparison illustrates the superiority of using image-based detection of in vitro biocompatibility with respect to analysis time, usability, and scientific outcome.