Live Cell Imaging Analysis Research Articles

Yunnan Baiyao exerts anti-glioma activity by inducing autophagy-dependent necroptosis

Ethnopharmacological relevanceYunnan Baiyao (YB), a traditional herbal formulation, has been used for over a century to manage bleeding and enhance blood circulation. Its ingredients are widely recognized for their anti-cancer properties. However, its impact on glioma, the most common primary malignant tumor of the central nervous system, remains unexplored. Aim of the studyThis study aims to investigate the anti-glioma activity of YB in vitro and in vivo, and to elucidate the underlying mechanism of action. MethodsU-87 MG cells were treated with YB and subjected to cell proliferation assay, colony formation assay, and flow cytometry with Annexin V/PI staining to confirm anti-glioma activity. The induction of necroptosis and autophagy was confirmed through live-cell imaging, western blotting, and immunofluorescence analysis. The role of apoptosis, necroptosis, autophagy, and AMPK was validated using specific inhibitors. The in vivo anti-glioma activity of YB was evaluated using subcutaneous and orthotopic xenograft models in nude mice and chemically induced glioma rat models. ResultsYB induced necroptotic rather than apoptotic cell death in glioma U-87 MG cells, as evidenced by increased phosphorylated MLKL levels and plasma membrane disruptions. Rescue experiments further confirmed the role of necroptosis. Importantly, YB-triggered necroptosis was found to be dependent on autophagy induction, which relies on the AMPK signaling pathway. In line with these findings, YB demonstrated significant anti-glioma activity in vivo. ConclusionsOur study reveals that YB exerts potent anti-glioma effects both in vitro and in vivo through the induction of autophagy-dependent necroptosis.

Development of a microfluidic system with a bearing micropump for applying fluid shear force in an intervertebral disc degeneration model involving mechanical stimulation

Background: The intervertebral disc (IVD) functions as a shock absorber with viscoelastic tissues, including the gelatinous nucleus pulposus and the collagenous annulus fibrosus (AF). External mechanical loading influences IVD homeostasis but can cause damage and inflammatory reactions, contributing to disc degeneration. There is a lack of in vitro platforms for modeling IVD disease with mechanical stimulation.Methods: This study aimed to create a mechanical stimulation-based model by subjecting AF cells to shear stress using a micropump system. A micropump platform was used to measure pulsation, flow rate, and shear stress. AF cells were exposed to fluid shear stress of 0.5 and 1 dyne/cm², and morphological changes, cytotoxicity, and cell viability were analyzed through a live/dead assay. Perimeter, area, diameter, and elongation were assessed using live cell imaging and imaging analysis software.Results: The micropump platform exhibited optimal rotor characteristics for uniform pulsation and shear stress. Fluid shear stress at 0.5 dyne/cm² showed no significant difference from the control group, while 1 dyne/cm² reduced cell adhesion and survival. Comparing the 0.5 dyne/cm² shear stress group and interleukin-1β group to the control, significant decreases in perimeter, area, and diameter were observed.Conclusion: The study successfully developed a micro-peristaltic pump platform for applying fluid shear stress to cells, identifying an optimal rotor for uniform stress application. The platform effectively modeled IVD disease, revealing reduced cell adhesion and survival under specific shear stress conditions. This platform has potential promise for discovering biomarkers and exploring cellular responsiveness to external stimuli.

Determination of adipogenesis stages of human umbilical cord-derived mesenchymal stem cells using three-dimensional label-free holotomography

Adipogenesis involves complex changes in gene expression, morphology, and cytoskeletal organization. However, the quantitative analysis of live cell images to identify their stages through morphological markers is limited. Distinct adipogenesis markers on human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were identified through holotomography, a label-free live cell imaging technique. In the MSC-to-preadipocyte transition, the nucleus-to-cytoplasm ratio (0.080 vs. 0.052) and lipid droplet (LD) refractive index variation decreased (0.149 % vs. 0.061 %), whereas the LD number (20 vs. 65) increased. This event was also accompanied by the downregulation and upregulation of THY1 and Preadipocyte Factor-1 (PREF-1), respectively. In the preadipocyte to immature adipocyte shift, cell sphericity (0.20 vs. 0.43) and LD number (65 vs. 200) surged, large LDs (>10 μm3) appeared, and the major axis of the cell was reduced (143.7 μm vs. 83.12 μm). These findings indicate features of preadipocyte and immature adipocyte stages, alongside the downregulation of PREF-1 and upregulation of Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In adipocyte maturation, along with PPARγ and Fatty Acid-Binding Protein 4 upregulation, cell compactness (0.15 vs. 0.29) and sphericity (0.43 vs. 0.59) increased, and larger LDs (>30 μm3) formed, marking immature and mature adipocyte stages. The study highlights the distinct adipogenic morphological biomarkers of adipogenesis stages in UC-MSCs, providing potential applications in biomedical and clinical settings, such as fostering innovative medical strategies for treating metabolic disease.

Metabolic modulation of melanoma enhances the therapeutic potential of immune checkpoint inhibitors.

Lactate is a pivotal molecule with diverse functions in the metabolic reprogramming of cancer cells. Beyond its role in metabolism, lactate exerts a modulatory effect within the tumor microenvironment; it is utilized by stromal cells and has been implicated in the suppression of the immune response against the tumor. Using in vitro assays (including flow cytometry, live-cell imaging and metabolic analyses), the impact of lactate dehydrogenase inhibitors (LDHIs) on melanoma cells were assessed. The therapeutic potential of LDHIs with immune checkpoint inhibitors (ICIs) were tested in vivo in murine models of melanoma tumors. A potent anti-proliferative effect (via both cell cycle alterations and enhanced apoptosis) of LDHIs, Oxamate (Oxa) and methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2), was found upon treatment of melanoma cell lines. Using a combination of Oxa and NHI-2, a synergistic effect to inhibit proliferation, glycolysis, and ATP production was observed. Metabolic analysis revealed significant alteration in glycolysis and oxidative phosphorylation, while metabolite profiling emphasized consequential effects on lactate metabolism and induced energy depletion by LDHIs. Detection of increased RANTES and MCP-1, with Oxa and NHI-2 treatment, prompted the consideration of combining LDHIs with ICIs. In vivo studies using a murine B78 melanoma tumor model revealed a significant improvement in treatment efficacy when LDHIs were combined with ICIs. These findings propose the potential of targeting lactate metabolism to enhance the efficacy of ICI treatments in patients with melanoma.

Uptake of substances into living mammalian cells by microwave induced perturbation of the plasma membrane

Delivering foreign molecules and genetic material into cells is a crucial process in life sciences and biotechnology, resulting in great interest in effective cell transfection methods. Importantly, physical transfection methods allow delivery of molecules of different chemical composition and are, thus, very flexible. Here, we investigated the influence of microwave radiation on the transfection and survival of mammalian cells. We made use of an optimized microwave-poration device and analyzed its performance (frequency and electric field strength) in comparison with simulations. We, then, tested the effect of microwave irradiation on cells and found that 18 GHz had the least impact on cell survival, viability, cell division and genotoxicity while 10 GHz drastically impacted cell physiology. Using live-cell fluorescence microscopy and image analysis, we tested the uptake of small chemical substances, which was most efficient at 18 GHz and correlated with electric field strength and frequency. Finally, we were able to obtain cellular uptake of molecules of very different chemical composition and sizes up to whole immunoglobulin antibodies. In conclusion, microwave-induced poration enables the uptake of widely different substances directly into mammalian cells growing as adherent cultures and with low physiological impact.

Diverse calcium signaling profiles regulate migratory behavior in avascular wound healing and aberrant signal hierarchy occurs early in diabetes.

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.

Rit1-TBC1D10B signaling modulates FcγR-mediated phagosome formation in RAW264 macrophages.

Phagocytosis is an important immune response that protects the host from pathogen invasion. Rit1 GTPase is known to be involved in diverse cellular processes. However, its role in FcγR-mediated phagocytosis remains unclear. Our live-cell imaging analysis revealed that Rit1 was localized to the membranes of F-actin-rich phagocytic cups in RAW264 macrophages. Rit1 knockout and expression of the GDP-locked Rit1 mutant suppressed phagosome formation. We also found that TBC1D10B, a GAP for the Rab family GTPases, colocalizes with Rit1 in the membranes of phagocytic cups. Expression and knockout studies have shown that TBC1D10B decreases phagosome formation in both Rab-GAP activity-dependent and -independent manners. Notably, the expression of the GDP-locked Rit1 mutant or Rit1 knockout inhibited the dissociation of TBC1D10B from phagocytic cups. In addition, the expression of the GTP-locked Rit1 mutant promoted the dissociation of TBC1D10B in phagocytic cups and restored the rate of phagosome formation in TBC1D10B-expressing cells. These data suggest that Rit1-TBC1D10B signaling regulates FcγR-mediated phagosome formation in macrophages.

Simple isomeric pyridine-based fluorescence-off probes for in vitro monitoring γ-glutamyltranspeptidase activity: Design, synthesis and application

γ-Glutamyltranspeptidase (GGT) is a cell surface-associated enzyme, which has been proven to be closely related to many diseases. Thus, development of simple and effective GGT-activatable fluorescent probes for early diagnosis of diseases is of great significance. Herein, a series of simple isomeric pyridine-based GGT-activatable fluorescent probes has been successfully designed and synthesized. The preliminary experimental results indicate that Py-GGT-1 has a significant fluorescence response to GGT via a rare fluorescence-off approach. Further fluorescence response experiments, such as the time-/dose-dependent fluorescence change and the selectivity/specificity estimation, reveal that Py-GGT-1 has an excellent ability to monitor endogenous GGT activity. Mechanism studies through HRMS analysis, DFT calculations and molecular docking have theoretically verified the catalytic process between Py-GGT-1 and GGT. To develop its practical application, live-cell imaging and serum-sample analysis were carried out, demonstrating that Py-GGT-1 coud effectively track GGT within cells and organisms. Based its high selectivity and reasonable sensitivity, we expect that the probe will be applied in clinical diagnosis of GGT-related diseases in the future.

Abstract A013: Functional and clinical consequences of hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer

Abstract Recent whole-genome sequencing studies have unveiled intriguing hotspot mutations within non-coding genomic regions, yet their implications in cancer remain unknown. Here, we characterized the role of twin hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer: chr8: 29,952,919 G>A and chr8: 29,952,919 C>T. Either mutation is found in ~20% of bladder tumors, and the mutations do not typically co-occur. Through a comprehensive analysis integrating ChiP-seq, ATAC-seq, and Hi-C data from bladder cancer cell lines, we observed that these mutations co-localize with enhancer elements that interact with multiple nearby genes. In vitro dual luciferase reporter assays showed that both mutations are functional and likely affect gene expression in a genomic context. CRISPR-Cas9-mediated knocking out of the putative enhancer region identified LEPROTL1, DCTN6, and SARAF as target genes that are likely co-regulated, which was further supported by strong positive correlations of their mRNA expression in TCGA data. Isogenic cell clones generated by CRISPR-mediated base-editing showed that the mutations result in simultaneous upregulation of the three target genes, which were also expressed at higher levels in TCGA tumors harboring the non-coding mutations. Little is known about the function of LEPROTL1, DCTN6, and SARAF. Utilizing siRNA knockdown models complemented by live-cell imaging, viability assays, and cell cycle analysis, we found that they promote cell proliferation and cell cycle progression. Furthermore, our base-edited isogenic cell lines showed more rapid proliferation and cell cycle progression, as well as resistance to cisplatin. The mutations were also associated with shorter disease-specific survival in a TCGA cohort, and DCTN6 showed a negative association with overall survival. These results underscore the clinical significance of the non-coding mutations. Currently, we are assessing the effect of these mutations on tumor growth and cisplatin response in vivo using xenograft mouse models and evaluating the mechanisms by which LEPROTL1, DCTN6, and SARAF modulate cell cycle progression. Taken together, our data suggest that non-coding enhancer mutations near LEPROTL1 contribute to bladder carcinogenesis by increasing the expression of three co-regulated novel oncogenes, all of which were previously uncharacterized in bladder cancer. Since we found the mutations to be clinically significant, our study also raises the possibility of using the non-coding mutations and their target genes as clinical biomarkers and potential therapeutic drug targets. Citation Format: Kelly Butler, Bilal Lone, Alexandra Dobbins, Arup Chakraborty, Salah Boudajadi, Andrea Apolo, Rouf Banday. Functional and clinical consequences of hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A013.

Abstract PO1-26-04: Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients

Abstract Patient-derived organotypic tissue cultures (PD-OTC) are unique models for probing how individual patient’s tumor microenvironment (TME) influences cancer development and treatment responses. They retain the patient tumor architecture and TME, that are difficult to recapitulate in other models, while affording flexible treatment options and comparison of cancer (CA) versus matched non-cancer (NC) tissue responses to treatments. We have developed a culturing method for establishing PD-OTC of breast cancer patients that exhibit CA tissue growth and extensive metabolic reprogramming between CA versus matched NC tissues while enabling patient-derived organoids (PDO) and fibroblasts to be isolated. Pairs of CA and NC tissues freshly resected from six patients bearing early-stage estrogen receptor/progesterone receptor positive ductal carcinoma were sliced at 750 µm, embedded in Matrigel, and cultured in a 6-well plate with a Biopore membrane insert and custom medium at 37°C/5% CO2 with gentle rocking (16-40 mg wet weight; n=3). Excess tissue slices were cryopreserved and kept at -196 °C. Culture media were sampled periodically for metabolite analysis by NMR. Microscopic examination revealed significant tissue, organoid-like, and fibroblast outgrowth after 1-1.5 month of culturing. Two to three days prior to the harvest of two patients’ (CZ16 and 17) OTC, the culture medium was replaced with [U-2H]-glucose + [U-13C,15N]-glutamine-containing medium to allow the two stable isotope tracers to be metabolized, followed by Stable Isotope-Resolved Metabolomic (SIRM) analysis by NMR and ion chromatography coupled with ultra high-resolution Fourier transform mass spectrometry (IC-UHRFTMS). Two separate pieces of the OTC were cut and each was assayed for glucose uptake and mitochondrial membrane potential by live fluorescence spectroscopy via a portable custom-built fluorescence microscope, and by histochemical analysis. Histological analysis showed that both CA and NC OTC of all patients maintained their structural integrity. SIRM analysis of two patients’ OTC revealed active catabolic and anabolic metabolism including glucose uptake/glycolysis, the Krebs cycle/mitochondrial membrane potential, the pentose phosphate pathway (PPP), gluconeogenesis, glycogen synthesis, and purine/pyrimidine/sugar nucleotides synthesis. The CA OTC of both patients showed enhanced glucose uptake, glycolysis, mitochondrial membrane potential/Krebs cycle, PPP, glycogen synthesis, and purine nucleotide synthesis, when compared with the matched NC OTC. Treatment-naïve CA OTC of CZ16 showed much more significant metabolic reprogramming (NAD+ metabolism, in particular) than CA OTC of CZ17 who has undergone neoadjuvant treatment, chemotherapy, and hormone therapy prior to surgery. Distinct metabolic features of breast CA tissues can be exploited as therapeutic targets. Organoids and fibroblasts were isolated from CA and/or NC OTC cultures and their characterization is in progress. When revived, cryopreserved OTC exhibited active metabolism and tissue/organoid/fibroblast outgrowth similarly to fresh OTC. In conclusion, our culturing method enables PD-OTC models to be established efficiently for early-stage breast cancer patients for downstream functional studies including live cell imaging and SIRM analysis. Organoids and fibroblasts can also be derived from < 20 mg of tissues for further studies. Future direction includes therapeutic studies on these patient-derived models to better understand and predict individual patient’s responses to therapy. Citation Format: Teresa Fan, Carlos Goncalves, Jing Yan, Jahid Islam, Penghui Lin, Mohamed Kaddah, Richard Higashi, Andrew Lane, Caigang Zhu. Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-04.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Exploring fluorescent and colorimetric probes for analyte detection: Utilizing it in live cell imaging and real-time sample analysis

Fluorescent and colorimetric probes are gaining interest in analyte detection, imaging, and real-time applications. However, it faces several challenges in achieving high sensitivity and selectivity, maintaining a strong signal-to-noise ratio, detecting analytes at extremely low concentrations, ensuring stability under different conditions and addressing issues related to cost, accessibility and instrumentation. Additionally, ensuring biological compatibility and enabling real-time monitoring are also challenges that need to overcome. We have investigated the sensing application of probe molecules towards various analytes like metal ions, anions, biomolecules, reactive oxygen species (ROS), reactive sulphur species (RSS), toxic agents and nanoparticles performed by our group together with critically comparing with the existing reports. This featured article provides the impact of fluorescent and colorimetric probes in advancing analytical techniques, imaging and real-time applications that transcend traditional boundaries.

Abstract 1692: MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal type of pancreatic neoplasm. Unfortunately, most patients are diagnosed with unresectable or metastatic disease, making current treatments insufficient. As a result, the 5-year survival rate for PDAC is below 10%.Pancreatic neoplasms are primarily initiated by the KRAS mutation, accounting for 95% of cases. However, the progression of PDAC to a highly aggressive and therapy-resistant cancer is accelerated by chronic inflammation. The role of mutant KRAS signaling and NF-κB inflammatory signaling in promoting PDAC progression has been extensively studied. Yet, the transcriptional mechanism of their cooperation remains unclear. Additionally, the interaction between PDAC tumor cells and signals in the tumor microenvironment (TME) is not yet fully understood. Our study demonstrates that the simultaneous activation of KRAS/MAPK and NF-κB by epidermal growth factor (EGF) and TNFα, respectively, increases cell migration as shown through live-cell imaging analysis. An integrated analysis of epigenetic and transcriptomic data (ChIP-seq and RNA-seq) was performed after dual stimulation of both pathways. This revealed a high occupancy of the active mark, H3K27ac at transcription start sites of a subset of genes involved in cell migration and subtype switch. Through gain and loss-of-function studies, FOSL1 and RELA were identified as the key transcription factors. The epigenome mapping of FOSL1 and RELA after the stimulation of both pathways revealed their occupancy at RELA-dominant and FOSL1/RELA co-occupied regions. Inhibitor treatments and loss-of-function studies revealed that RELA binding controls RNA polymerase recruitment for transcription initiation and enhances cell migration. Further analysis of RNA-seq data on PDAC cell lines and cells from patient-derived xenografts highlighted that active inflammatory signaling contributes to a subtype switch from the classical to the more migratory basal subtype. Our study, combined with single-cell RNA-seq analysis of published data, shows that macrophages in the tumor microenvironment supply TNFα that activates NF-κB signaling, leading to changes in cell migration. Our study has provided a comprehensive understanding of the mechanism by which mutant KRAS and inflammatory signaling contribute to the progression of PDAC. These findings could offer a new perspective in the development of mechanism-based therapeutic approaches that can improve the efficacy of chemotherapeutic agents and make tumors more amenable to surgical resection. Citation Format: Joana E. Aggrey-Fynn, Meghana Manjunath, Steven A. Johnsen. MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1692.

Abstract 4141: Quantitative measurement of 3D tumor spheroid invasion using live cell time-lapse imaging

Abstract 90% of the death of cancer individuals is related to cancer metastasis1. The invasion and migration of cancer cells into surrounding tissues and chambers is the first step of metastasis2. To have a better and more in-depth understanding of the pathophysiological activities involved in metastatic cancer, accurate and reliable methods for evaluating cell invasion are urgently needed. Compared to the 2D cell model, the 3D system mimics the natural physiological properties and conditions, such as structure, physiology, biological signals of living tissues, cell-matrix interactions4. In this study, we followed the commonly used protocol for 3D tumor invasion assay3 to generate spheroids in Ultra-Low-attachment (ULA) round bottom 96-well plate and subsequently embedded them in Matrigel® (5 mg/mL). After polymerization, the Matrigel that contains tumor spheroid was submerged in 100 μL cell culture media. The invasion of the 3D spheroid in the presence or absence of drug treatment was monitored and quantified using the brightfield and fluorescence live cell time-lapse image acquisition and image analysis functions of the Agilent xCELLigence RTCA eSight. The progress of spheroid invasion and the drug-inhibitory effects on tumor invasion were quantified by tracking and calculating the area of invasive protrusion from the spheroid. Our data show that 1) different types of cancer cells had different metastatic potentials demonstrated by the level of invadopodium extending into the Matrigel at desired time points; 2) the invasion of 3D HT1080 spheroid was inhibited by cytochalasin D (Cyto D), an inhibitor of actin polymerization, and GM6001, a potent broad-spectrum inhibitor of matrix metalloproteinases (MMP), in a dose-dependent manner; 3) Cyto D exhibited higher efficacy of inhibiting tumor invasion than GM6001. In summary, our approach by combining the 3D tumor spheroid invasion model with the RTCA eSight’s capability of live cell imaging and automatic quantification of the area of invasive protrusion can facilitate and enhance the development of new treatments at the preclinical stage in the future. Citation Format: Grace Yang, Tian Wang, Peifang Ye, Xiaoyu Zhang. Quantitative measurement of 3D tumor spheroid invasion using live cell time-lapse imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4141.

GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells.

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.

Auxin co-receptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root solely by modulation of nuclear auxin pathway.

The nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots.

The response of human osteoblasts on bovine xenografts with and without hyaluronate used in bone augmentation

The aim of the in vitro study was to asses the effect of hyaluronate in conjunction with bovine derived xenografts on the viability, proliferation on day 4, 7 and 10, expression of early osteogenic differentiation marker Alkaline phosphatase on day 14 and 21, collagen, calcium deposition on day 14, 21 and 28 and cellular characteristics, as assessed through live cell image analysis, confocal laser scanning microscopy and scanning electron microscopy, in primary human osteoblasts compared to three bovine xenografts without hyaluronate. All experiments were performed in triplicates. Data were compared between groups and timepoints using one-way analysis of variance (ANOVA). Bonferroni post hoc test were further used for multiple comparison between groups (p < .05) An increase in cell viability (p < .05) and enhanced ALP activity was observed in all xenografts. Specimens containing hyaluronate showed a highest significant difference (23755 ± 29953, p < .0001). The highest levels of calcium (1.60 ± 0.30) and collagen (1.92 ± 0.09, p < .0001) deposition were also observed with hyaluronate loaded groups. The osteoblasts were well attached and spread on all xenograft groups. However, a higher number of cells were observed with hyaluronate functionalized xenograft (76.27 ± 15.11, (p < .0001) in live cell image analysis and they migrated towards the graft boundaries. The biofunctionalization of xenografts with hyaluronate improves their in vitro performance on human osteoblasts. This suggests that hyaluronate might be able to improve the bone regeneration when using such xenografts.

Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research.

The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducingTau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of thedynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons,using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.

Single-organoid analysis reveals clinically relevant treatment-resistant and invasive subclones in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases, characterized by a treatment-resistant and invasive nature. In line with these inherent aggressive characteristics, only a subset of patients shows a clinical response to the standard of care therapies, thereby highlighting the need for a more personalized treatment approach. In this study, we comprehensively unraveled the intra-patient response heterogeneity and intrinsic aggressive nature of PDAC on bulk and single-organoid resolution. We leveraged a fully characterized PDAC organoid panel (N = 8) and matched our artificial intelligence-driven, live-cell organoid image analysis with retrospective clinical patient response. In line with the clinical outcomes, we identified patient-specific sensitivities to the standard of care therapies (gemcitabine-paclitaxel and FOLFIRINOX) using a growth rate-based and normalized drug response metric. Moreover, the single-organoid analysis was able to detect resistant as well as invasive PDAC organoid clones, which was orchestrates on a patient, therapy, drug, concentration and time-specific level. Furthermore, our in vitro organoid analysis indicated a correlation with the matched patient progression-free survival (PFS) compared to the current, conventional drug response readouts. This work not only provides valuable insights on the response complexity in PDAC, but it also highlights the potential applications (extendable to other tumor types) and clinical translatability of our approach in drug discovery and the emerging era of personalized medicine.

Abstract B108: NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects

Abstract Adult-type diffuse gliomas are aggressive brain cancers. The widespread standard treatment for these high-grade gliomas (HGG) is the Stupp Protocol, consisting of maximal safe surgical resection followed by concurrent chemoradiotherapy. However, since the Stupp Protocol, no significant therapeutic advances have been as widely and easily adopted, halting improvements in HGG patient survival. Therefore, identifying new drug targets or treatments that enhance the Stupp Protocol would result in therapeutic benefit. HGG are classified by distinct genetic and metabolic alterations; the most well-known being somatic gain of function mutations in isocitrate dehydrogenase (IDH) 1/2 genes. These mutations distinguish the two most severe HGGs, namely Grade 4 IDH-Mutant Astrocytoma and IDH-Wildtype Glioblastoma (GBM). IDH-mutant tumors accumulate the oncometabolite D-2-Hydroxyglutarate, which alters metabolic pathways and DNA damage responses. One metabolic alteration is decreased nicotinamide adenine dinucleotide (NAD+) levels, a metabolite essential for cellular homeostasis, and crucial in IDH-mutants as targeting NAD+ homeostasis is effective against IDH-mutant tumors. Here, we investigated if additional metabolic vulnerabilities within NAD+ and purine biosynthetic pathways could be identified in HGG cells. For this, a panel of cell lines, including an isogenic IDH1-mutant cell line, was screened for their sensitivity to a range of metabolic inhibitors in combination or not with chemo- and radiotherapy. This screen identified NUDT5, a Nudix hydrolase that metabolizes ADP-Ribose into Ribose-5-Phosphate (R-5-P) to replenish NAD+ pools, and synthesizes nuclear ATP for hormone driven chromatin remodeling, as an important determinant in glioma cell survival. Cell viability assays determined NUDT5 inhibition (NUDT5i) sensitized IDH1-wildtype HGG cells to different radiotherapy modalities, specifically X-rays and alpha particles, whilst IDH1-mutant HGG were sensitive to NUDT5i alone. Live-cell imaging analysis indicated NUDT5i induced cytostasis followed by cytotoxicity in the IDH1-mutant, suggesting IDH1-mutations confer exploitable metabolic dependencies on NUDT5 for proliferation and survival. NUDT5i did not significantly deplete NAD+ levels and NAD+ precursor supplementation failed to rescue cell survival and proliferation, indicating effects of NUDT5i are NAD+ independent. Our results indicate NUDT5 plays a crucial role in HGG proliferation and survival. Given that NUDT5 produces R-5-P for downstream nucleotide synthesis, we hypothesize that NUDT5i impairs nucleotide homeostasis and leads to replication stress. Current investigations include nucleoside supplementation and characterization of replication stress signaling by western blotting. We anticipate our results to prelude further investigations into NUDT5 as a potential therapeutic target in HGG. We suggest NUDT5i could enhance the effectiveness of the Stupp Protocol regardless of IDH-Status and advance HGG therapies. Citation Format: Thomas J.R Cox, Eren Ozcagli, Giuseppe Schettino, Lisiane B Meira. NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B108.