Hoechst Nuclear Staining Research Articles

Abstract 2060: Automation of 3D cancer spheroid assay for compound screening

Abstract Finding efficient drug combinations to treat cancer patients is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient testing drug efficacy to discover new therapeutic targets. 3D cancer models are highly valuable tools for cancer research and drug development. However, the complexity of performing 3D assays remains a hurdle for adopting these methods for compound screening. To alleviate the bottlenecks that come with labor-intensive manual protocols we developed cell culture automation methods using CellXpress.ai instrument. The instrument enables automation of 2D or 3D culture for prolonged complex workflows. The CellXpress.ai provides automated plating, passaging, media exchanges, monitoring organoids, alone with compound treatment and the end point assays with high content image analysis. In the present study we describe full automation of imaging, analysis, and cell culture process which enables scaling up complex 3D cell-based assays and compound screening. As an example of 3D protocol, we automated culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell line in U-shape low attachment plates. HCT116 cells were expanded in 2D, then spheroids were formed after automated dispensing of cell suspension into U-shape 96 or 384 plates. After 48h spheroids were treated with 12 anti-cancer compounds, at multiple concentrations for 3-5 days, then stained and imaged. Cell plating, compound additions, media exchange, and staining were performed automatically by CellXpressAI instrument. During the culture spheroids were monitored using transmitted light, with immediate analysis of phenotypic changes, including inhibition of growth or spheroid disintegration. For endpoint assay spheroids were stained with combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD, then imaged, and analyzed for multiple read-outs including spheroid size, marker intensities, and live-dead cell scoring. We observed concentration-dependent inhibition of spheroid growth and cell death in response to anti-cancer compounds and evaluated effective compound concentrations for cytotoxic or cytostatic effects. Cell culture process automation for 3D assays, powered by imaging and image analysis has a potential to bring 3D biology into another level, allowing to increase throughput and reproducibility, and enabling variety of high throughput drug discovery and precision medicine applications. Citation Format: Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Zhisong Tong, Emilie Keidel, Felix Spira. Automation of 3D cancer spheroid assay for compound screening [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 2060.

Characterization of the C17.2 cell line as testing system for endocrine disruption-induced developmental neurotoxicity.

Hormone signaling plays an essential role during fetal life and is vital for brain development. Endocrine-disrupting chemicals can interfere with the hormonal milieu during this critical time-period, disrupting key neurodevelopmental processes. Hence, there is a need for the development of assays that evaluate developmental neurotoxicity (DNT) induced by an endocrine mode of action. Herein, we evaluated the applicability of the neural progenitor C17. 2 cell-line, as an in vitro test system to aid in the detection of endocrine disruption (ED) induced DNT. For this, C17.2 cells were exposed during 10 days of differentiation to agonists and antagonists of the thyroid hormone (Thr), glucocorticoid (Gr), retinoic acid (Rar), retinoic x (Rxr), oxysterols (Lxr), estrogen (Er), androgen (Ar), and peroxisome proliferator activated delta (Pparβ/δ) receptors, as well as to the agonist of the vitamin D (Vdr) receptor. Upon exposure and differentiation, neuronal morphology (neurite outgrowth and branching), and the percentage of neurons in culture were assessed by immunofluorescence. For this, the cells were incubated with Hoechst (nuclear staining) and stained for βIII-tubulin (neuronal marker). The C17.2 cells were responsive to the Rar, Rxr and Pparβ/δ agonists which decreased neurite outgrowth and branching. Additionally, exposure to the Gr agonist increased the number of cells differentiating into neurons, while exposure to the Rxr agonist had the opposite effect. With this approach, we have identified that the C17.2 cells are responsive to Gr, Rar, Rxr, and Pparβ/δ agonists, hence contributing to the development of test systems for hazard assessment of ED-induced DNT.

Preparation, characterization, and anticancer effects of an inclusion complex of coixol with β-cyclodextrin polymers

Context Coix [Coix lacryma-jobi L. var. mayuen (Roman.) Stapf (Poaceae)], a crop of medicinal and edible significance, contains coixol, which has demonstrated anticancer properties. However, the limited solubility of coixol restricts its potential therapeutic applications. Objective This study prepared a water-soluble coixol-β-cyclodextrin polymer (CDP) inclusion compound and evaluated its anticancer effect. Materials and methods The coixol-CDP compound was synthesized through a solvent-stirring and freeze-drying technique. Its coixol content was quantified using HPLC, and its stability was tested under various conditions. The anticancer effects of the coixol-CDP compound (4.129, 8.259, 16.518, and 33.035 mg/L for 24, 48, and 72 h) on the proliferation of non-small cell lung cancer (NSCLC) A549 cells were evaluated using an MTT assay; cell morphology was examined by Hoechst nuclear staining; apoptosis and cell cycle was detected by flow cytometry; and the expression of apoptosis-related proteins was assessed by Western blots. Results The water-soluble coixol-CDP inclusion compound was successfully prepared with an inclusion ratio of 86.6% and an inclusion yield rate of 84.1%. The coixol content of the compound was 5.63% and the compound remained stable under various conditions. Compared to coixol alone, all 24, 48, and 72 h administrations with the coixol-CDP compound exhibited lower IC50 values (33.93 ± 2.28, 16.80 ± 1.46, and 6.93 ± 0.83 mg/L) in A549 cells; the compound also showed stronger regulatory effects on apoptosis-related proteins. Discussion and conclusions These findings offer a new perspective for the potential clinical application of Coix in NSCLC therapy and its future research.

Murine Infusion Studies of Infused Human Megakaryocytes Support a “Catch-and-Release” Model of Thrombopoiesis

Introduction: Thrombopoiesis, the process by which megakaryocytes (Mks) release platelets, is still not well understood, including where it occurs and how a Mk releases its platelet content. The present study, based on infused human Mks (hMks) into immunocompromised mice, and measurements of the subsequent presence of nucleated Mks and anucleated cytoplasmic fragments in the lungs and in the peripheral blood were carried out to provide new insights into this process. Method: To gain a deeper understanding of the role of the lung in thrombopoiesis, we performed quantitative, temporal measurements of the size of human CD41 +CD42 + nucleated Mks and anucleated cytoplasmic fragments after tail-vein infusion of either mature (Day 12 of culture from CD34 + hematopoietic stem/progenitor cells (HSPCs)) hMks or immature (Day 7 of culture from CD34 + HSPCs) in NOD-scid gamma (NSG) mice. Data was collected from both the pulmonary bed by in situ microscopy ( Figure 1) and in the peripheral blood by flow cytometry ( Figure 2). To distinguish in the peripheral blood large cytoplasmic fragments from recirculating hMks, Hoechst-nuclear staining was done prior to flow cytometric analysis For size references, isolated donor platelets and input Hoechst-stained hMks, containing platelet-like particles (PLPs), were concurrently analyzed ( Figure 2, bottom left). Results: By pulmonary in situ microscopy, we found that infused mature hMks were entrapped on their first passage through the lungs. Over the subsequent hour, anucleate cytoplasmic fragments ( Figure 1, left) recycle to the lungs becoming increasingly smaller in size over the hour-long study. Infused immature hMks also shed cytoplasmic fragments, but these began much smaller in size though these, too, reduced in size over time ( Figure 1, right). In the peripheral blood, mature CD41 +CD42 + hMks made up <2% of all the circulating fragments ( Figure 2, top left). These hMks initially were comparable in size to the input hMks ( Figure 2, bottom), but decreased in size over time, presumably due to shedding of their cytoplasm. Already by 10 mins post-infusion, a dominant group of large-sized (L), anucleated CD41 +CD42 + cytoplasmic fragments were observed, slightly smaller than the input Hoechst-stained hMks. Their presence decreased, reaching near-zero in <20 hrs. In contrast, mid-sized (M), cytoplasmic fragments, comparable in size to donor platelets, increased in abundance over time. Small-sized (S), cytoplasmic fragments were only present at 10 mins and were largely the input PLPs. In contrast, up to 34% of the circulating CD41 +CD42 + fragments after infusing immature hMks were nucleated ( Figure 2, right), suggesting that unlike mature hMks that are efficiently entrapped by the murine lungs, many immature hMks were small enough to pass through the murine vascular beds and recycle multiple times without completely releasing their cytoplasm. Conclusions: In the murine lungs, mature hMks are efficiently entrapped and within minutes shed large cytoplasmic fragments. These cytoplasmic fragments then recycle back to the lung for further processing, resulting in a near complete release of platelet-size fragments by 90 mins. These findings are consistent with the recent observations of Zhao X, et al., using a distinct in vivo murine lung/heart system (PMC10329040) except that infused murine Mks recycled nucleated. Whether this difference is a true species-difference or related to hMks traversing the murine vasculature needs to be explored, but in aggregate, these two studies demonstrate that thrombopoiesis is a complex process that begins when whole Mks and/or large cytoplasmic fragments are released intravascular from the marrow, spleen and/or lung. Subsequently, the Mks and large cytoplasmic fragments are then converted into smaller fragments and finally platelets by repetitive cycles of “catch-and-release” in the pulmonary bed. The mechanistic basis controlling this process needs better elucidation, but at the same time, offers insights of potential clinical application.

In vitro anticancer efficacy of Cyperus rotundus (L.) on breast adenocarcinoma cells via the induction of DNA fragmentation and apoptosis

Introduction and Aim: Cyperus rotundus (L.), commonly known as nutgrass, has been in use in Indian medicine traditional system against several ailments. This investigation was performed to assess the in vitro antineoplastic activity of extracts prepared from water and ethanol with C. rotundus rhizomes. Materials and Methods: Toxic effects induced by the C. rotundus extracts on MCF-7 breast adenocarcinoma cells was evaluated using Sulphorhodamine- B (SRB) Assay, Trypan blue assay, Hoechst nuclear staining assay, Acridine Orange-Ethidium bromide (AO-EB) differential fluorescence staining assay and DNA fragmentation assays. Results: The results of SRB assay indicated the IC50 value of aqueous extract as 510.887 µg/mL and that of ethanol extract as 122.98 µg/mL. Trypan blue assay also indicated the similar results, wherein ethanol extract exerted higher cytotoxicity compared to the aqueous extract. MCF-7 cells treated with aqueous and ethanol extracts of C. rotundus rhizomes were exposed to Hoechst staining and AO-EB nuclear staining assays, wherein and ethanol extract induced clear apoptotic bodies in the tested cells. Further, the cells were treated with the ethanol extract and DNA fragmentation was confirmed by DNA ladder assay. MCF-7 cells administered with ethanol extract were subjected to DNA fragmentation assay for assessing the apoptosis induction nature of the extract. Conclusion: Results from our study indicate a promising approach into the possibility of developing C. rotundus ethanol extract into an effective anticancer moiety after appropriate pre-clinical and clinical validations.

Xanthatin suppresses pancreatic cancer cell growth via the ROS/RBL1 signaling pathway: In vitro and in vivo insights

BackgroundAs a malignant digestive system tumor, pancreatic cancer has a high mortality rate. Xanthatin is a sesquiterpene lactone monomer compound purified from the traditional Chinese herb Xanthium strumarium L. It has been reported that Xanthatin exhibits inhibitory effects on various cancer cells in retinoblastoma, glioma, hepatoma, colon cancer, lung cancer, as well as breast cancer. However, in pancreatic cancer cells, only one report exists on the suppression of Prostaglandin E2 synthesis and the induction of caspase 3/7 activation in Xanthatin-treated MIA PaCa-2 cells, while systematic in vitro and in vivo investigations and related mechanisms have yet to be explored. PurposeThis research aims to explore the in vitro and in vivo effects of Xanthatin on pancreatic cancer and its molecular mechanisms. MethodsThe anticancer effects and mechanisms of Xanthatin on pancreatic cancer cells were assessed through employing cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, carboxyfluorescein diacetate succinimidyl ester (CFDA SE) cell proliferation assay, colony formation assay, wound healing assay, transwell assay, Annexin V-FITC/propidium iodide (PI) dual staining, Hoechst nuclear staining, Western blot analysis, phosphoproteomics, and reactive oxygen species (ROS) measurement. The in vivo anticancer effects of Xanthatin on pancreatic cancer cells were studied using a nude mouse model. ResultsThe present study showed that Xanthatin can prevent the proliferation and metastasis of pancreatic cancer cells and trigger the exposure of phosphatidylserine (PS), chromatin condensation, and caspase activation, thereby inducing apoptosis. Phosphoproteomic analysis indicated that Xanthatin inhibits the phosphorylation of the proliferation-associated protein RBL1, and oxidative stress can lead to RBL1 dephosphorylation. Further investigation revealed that Xanthatin significantly upregulates ROS levels in pancreatic cancer cells, and the antioxidant N-acetylcysteine (NAC) can reverse Xanthatin-induced cell proliferation inhibition and apoptosis. In addition, Xanthatin can suppress pancreatic cancer cell growth in a xenograft nude mouse model with low toxicity to the mice. ConclusionXanthatin may inhibit the proliferation of pancreatic cancer cells and trigger apoptosis through the ROS/RBL1 signaling pathway.

Combined Epimedii Folium and Ligustri Lucidi Fructus with dexamethasone alleviate the proliferation of airway smooth muscle cells by regulating apoptosis/autophagy

Ethnopharmacological relevanceTraditional Chinese medicine (TCM) theory believes kidney deficiency is the root cause of chronic refractory asthma with pathological changes of airway remodeling. Our previous experiments confirmed that the combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) with the effect of nourishing Yin and Yang of the kidney could improve the pathological changes of airway remodeling in asthmatic rats, but the specific mechanism remains unclear. Aim of the studyThis research was designed to reveal the synergy of ELL and dexamethasone (Dex) in the proliferation, apoptosis, and autophagy of airway smooth muscle cells (ASMCs). Materials and methodsPrimary cultures of ASMCs from rats were prepared and induced with histamine (Hist), Z-DEVD-FMK (ZDF), rapamycin (Rap), or 3-Methyladenine (3-MA) at generation 3–7 for 24 or 48 h. Subsequently, the cells were treated with Dex, ELL, and ELL&Dex for 24 or 48 h. The effect of various concentrations of inducers and drugs on cell viability was detected by Methyl Thiazolyl Tetrazolium (MTT) assay, cell proliferation was tested using immunocytochemistry (ICC) by detecting Ki67 protein, cell apoptosis was measured by Annexin V-FITC/PI assay and Hoechst nuclear staining, cell ultrastructure was observed by transmission electron microscopy (TEM), and immunofluorescence (IF), Western blot (WB) combined with quantitative real-time PCR (qPCR) were used for measuring autophagy and apoptosis-related genes including protein 53 (P53), cysteinyl aspartate-specific proteinase (Caspase)-3, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, mammalian target of rapamycin (mTOR) and p-mTOR. ResultsIn ASMCs, Hist and ZDF promoted cell proliferation, significantly decreased Caspase-3 protein expression, and up-regulated Beclin-1 levels; Dex alone and in combination with ELL promoted Beclin-1, Caspase-3, and P53 expression, enhancing autophagy activity and apoptosis in Hist and ZDF-induced AMSCs. In contrast, Rap inhibited cell viability, increased Caspase-3, P53, Beclin-1, and LC3-II/I and decreased the levels of mTOR and p-mTOR with promoting apoptosis and autophagy; ELL or ELL&Dex reduced P53, Beclin-1, and LC3-II/I to down-regulate apoptosis and the excessive autophagic state of ASMCs induced by Rap. In the 3-MA model, cell viability and autophagy were reduced; ELL&Dex significantly upgraded the expression of Beclin-1, P53, and Caspase-3 and promoted apoptosis and autophagy of ASMCs. ConclusionsThese results suggest that ELL combined with Dex may regulate the proliferation of ASMCs by promoting apoptosis and autophagy and be a potential medicine for the treatment of asthma.

Temporal relationship of the orphan receptor TR3 translocation and expression with zinc-induced apoptosis in prostate cancer cells.

This study aimed to investigate the precise mechanism of zinc-induced apoptosis in human prostate cell lines PC-3 and LNCaP and explore its relationships with the translocation and expression of the orphan receptor TR3 and cytochrome c. The effects of zinc exposure on apoptosis levels were examined in both PC-3 and LNCaP cells. These cells were treated with exogenous ZnCl2 (100 µM) for 0, 4, 8, 12, and 24 h. Dansylaminoethyl-cyclen (DAEC) fluorescent probe was applied to visualize cellular zinc localization. Zinc-induced apoptosis was identified by Hoechst nuclear staining and flow cytometry analysis. TR3 messenger RNA (mRNA) expression levels were detected by real time-quantitative polymerase chain reaction (PCR). TR3 protein localization and mitochondrial release of cytochrome c were identified by immunofluorescence microscopy. Mitochondrial membrane potential collapse under fluorescence microscopy was examined with a MitoLight probe. Zinc exposure led to gradually increasing apoptosis levels in both LNCaP and PC-3 cells over the 24-h treatment period. The apoptotic effects could be observed as early as after 4 h of zinc treatment in PC-3 cells, with this being seen at 8 h in LNCaP cells. The apoptosis levels of both PC-3 and LNCaP cells began to increase significantly from 8 to 24 h when necrotic cells were also found. TR3 protein translocation from the nucleus to the mitochondria was noted and was accompanied by cytochrome c release into the cytosol from the mitochondria. Interestingly, no significant changes in TR3 mRNA expression levels were observed after zinc treatment. However, the mitochondrial membrane potential of both PC-3 and LNCaP cells gradually disappeared following extended zinc exposure. Zinc-induced apoptosis could be regulated through TR3 protein translocation from the nucleus to the mitochondria in prostate cancer cells without notable changes in TR3 mRNA levels. The direct effect of zinc on the mitochondria was associated with the release of cytochrome c into the cytosol through the mitochondrial targeting of TR3 protein.

The Complement System Mediates COVID-19 Endothelial Cell Injury, Modifying Cell Permeability

Background Coronavirus Disease 2019 (COVID-19) is a (SARS-CoV-2)-mediated respiratory disease. The severity of COVID-19 has been classified as mild, moderate, severe and critical per the WHO-China Joint Mission. Among a total of 72 314 case records from the Chinese Center for Disease Control and Prevention, approximately 81% of COVID-19 cases were defined as mild, 14% of as severe, and 5% as critical. The overall hospital mortality of COVID-19 cases is approximately 15-20%, but it is up to 40-49% among critical cases requiring intensive care unit admission. The complement system, is a key component of the innate immune response, consists of more than 50 soluble and membrane-bound proteins that provide defense against microbes and mediate inflammatory responses. The activation of the complement system involves a series of enzymatic transformations, where C3b acts as a mediator. The final step of the complement cascade is the formation of a membrane attack complex (MAC, C5b-9) on the plasma membrane (PM) of the cells. Several pathologies including thrombotic, inflammatory, autoimmune and age-related diseases are associated with abnormal complement activation. There is evidence linking the activation of the complement system and endothelial injury to organ damage and complications that increase the risk of mortality in COVID-19. We hypothesize that endothelial cell injury resulting from complement activation contributes to COVID-19-associated vascular injury and organ damage. Aims To identify differences in complement activation associated with mild and severe forms of COVID-19 To determine the role of differential complement activation by COVID-19 patient sera in mediating endothelial cell damage. Methodology Clinical information, and sera from SARS-CoV-2 positive patients with mild and severe COVID-19 were obtained from the Canadian COVID-19 Prospective Cohort Study (CANCOV) to identify complement activation and endothelial cell (EC) injury. Complement activation was determined using a previously established protocol (Noris et al., 2017). Briefly, primary human umbilical vein endothelial cells (HUVECs) were treated with adenosine diphosphate (ADP), followed by treatment with healthy control or COVID-19 patient serum. To confirm the biological activity of complement, we measured the deposition of C3b and C5b-9 on the plasma membrane of HUVECs via immunofluorescence (IF) on cells exposed to healthy or patient sera. In addition, a permeability assay using a transwell model was used to measure the changes in permeability induced by healthy or patient sera via complement activation. Briefly, HUVECs were seeded on the top part of the insert, allowing them to form a monolayer. Monolayer integrity was assessed by imaging cells stained with Hoechst (nuclear stain) and phalloidin (cytoskeleton stain) on the day of the experiment, as well as before and after incubation with healthy or patient sera. Results We were able to detect complement activation/deposition when exposing the cells to COVID-19 sera. As for C3b deposition, cells exposed to patient sera showed no difference compared to those treated with sera from healthy donors. However, we observed increased C5b-9 deposition on the PM of cells treated with patient sera compared to those treated with sera from healthy donors, with further increased deposition when cells are treated with deceased patient sera. We observed a trend demonstrating increased permeability when cells are treated with deceased COVID-19 patient sera compared to healthy donors. Cellular permeability is significantly lower in those cells treated sera from patients with mild or severe COVID-19. Conclusion There is a differential biological activity of complement on endothelial cells depending on the severity of COVID-19, this complement activation leads to an increase in cell permeability. While more research needs to be done, these results contribute to a better understanding of the consequences of complement activation on endothelium in COVID-19.

High-Efficiency Multi-Drug Functional Profiling in a Microfluidic Device Towards Personalized Predictions in Pediatric Leukemia

Treatment of pediatric acute lymphoblastic leukemia (ALL) has seen dramatic improvements over the last several decades, leading to overall survival rates of over 85%. However, for patients with relapsed/refractory disease, achieving complete remission remains extremely challenging. Improving treatment outcomes for these patients will require increasingly personalized treatment strategies that make use of functional screens in addition to molecular profiling technologies for treatment selection. Given the past success of combination chemotherapy in cancer treatment and recent promise for newly developed targeted agents, functional assays that are able to evaluate patient response to combinations of therapies will likely be most effective in identifying clinically actionable treatment protocols. Moreover, assays that can achieve this with minimal patient sample and reduced experimental workload will have the greatest potential for clinical translation and integration into clinical workflows. To that end, this work aims to develop a microfluidic assay to directly test candidate combinations of therapeutics in patient derived ALL cells. We aim for this to serve as a more effective way to screen for drug combination response and higher order interactions relative to standard wellplate formats. Once validated, this assay can be used to identify biomarkers of response to standard and experimental combination regimens in pediatric ALL, and ultimately, could be used to prospectively guide therapy in newly diagnosed patients. The developed device achieves this by enabling simultaneous exposure to 3 superimposed small molecule drug gradients across a 3D culture of leukemia cells such that each location in the device comprises a unique combination of all 3 drugs within a simplex (Fig 1A). Single cell drug concentration exposure is determined from experimentally validated steady state computational fluid dynamics (CFD) model predictions, and single cell viability is analyzed via Calcein (live), PI (dead) and Hoechst (nuclear) staining following 48 hours of treatment. Using this system, we then develop a novel experimental and computational framework for evaluating drug combination efficacy and synergy across the full range of combination ratios captured in our device. Specifically, we evaluate response across multiple devices at optimally spaced dose levels, increasing coverage of the 3-drug combination space and allowing for custom implementation of established synergy models. This enables synergy assessment in a continuous gradient of drug combination ratios, as opposed to the discrete combination ratios commonly evaluated in standard assays, and may further improve detection of drug synergy and identification of the most potent and efficacious combination ratios. To demonstrate this, we analyze response to a combination of daunorubicin, vincristine, and nelarabine in Jurkats, a T-ALL cell line, across 7 different devices at 7 different dose levels (Fig 1B). We quantify viability as a function of concentration across each device and each dose level, and generate dose response curves at distinct zones within the device. Specifically, we generate dose response curves in zones dominated by daunorubicin, vincristine, and nelarabine alone and find half maximal effective concentration values (EC50 values) similar to those reported in literature. We further evaluate response at varying ratios of each 2-drug combination, and generate dose response curves as shown in Fig 1B. Analyzing the 2-drug combination dose responses relative to single-drug dose responses allows for quantification of 2-drug combination synergy based on Loewe and DiaMOND synergy models, and future work will extend our developed method to analyze emergent 3-drug combination synergy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anticancer and Antimicrobial Evaluations on Alternative Reading Frame (ARF) Peptides and their Derivatives.

Alternative reading frame (ARF) protein up-regulates the intracellular level of a tumour suppressor protein, p53, by blocking MDM2 mediated p53 ubiquitination. The two homologous forms of ARF proteins are p19ARF in mice and p14ARF in humans. In our study, p19ARF-derived peptide ARF (26-44) and its cell-penetrating peptide conjugate Tat-ARF (26-44), p14ARF-derived peptide ARF (1-22), and its NrLS conjugate ARF (1-22)-NrLS were designed, and their anticancer properties were investigated. Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates. Peptides synthesized using solid-phase peptide synthesis (SPPS) were purified using RPHPLC and characterized using Bruker MALDI-TOF mass spectrometry. Cytotoxicity was evaluated on HeLa and BE(2)-C cells by cell viability IC50 determination. Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Morphological studies were carried out using SEM and TEM techniques, live/dead staining, ROS and Hoest staining. Peptides Tat-ARF (1-22) and ARF (1-22)-NrLS exhibited potent cytotoxic effects, comparable to the known standard cisplatin. Cellular morphological studies showed signs of apoptosis which were confirmed by reactive oxygen species (ROS) generation and Hoechst nuclear staining. ARF peptides showed potent antimicrobial activities at low micromolar concentrations without haemolysis. Tat modification improved the activity of ARF (26-44) by 9 folds against HeLa and 5 folds against BE(2)-C cells. NrLS modification of ARF (1-22) imparted 12 fold potency against HeLa and 2-fold potency against BE(2)-C cells. This study helps to further understand the effect of these peptides on MDM2 proteins and their role in the apoptosis signalling pathway.

Abstract 1406: FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer

Abstract Introduction: We previously reported that an aggressive subpopulation of highly tumorigenic, drug-resistant bladder cancer cells can arise from the bulk tumor cells without mutational events, and that this phenotypic plasticity is driven at least in part by epigenetic mechanisms. In the current work, we analyzed the chromatin accessibility of enhancers to identify transcription factors that contribute to this transition to a drug-resistant state. Materials and methods: We used Hoechst nuclear staining and fluorescence activated cell sorting to isolate the Hoechst-low drug resistant side population (SP) cells from the less drug resistant Hoechst-high non-side population (NSP) cells in bladder cancer cell lines. We performed RNA sequencing and AcceSssIble assay to profile RNA expression, global DNA methylation and chromatin accessibility in SP and NSP cells. Putative enhancer regions were identified using H3K27ac ChIPseq data in bladder cancer cell lines, which are publicly available. We next performed transcription factor motif analysis on accessible enhancers near genes overexpressed in SP cells relative to NSP cells. The differentially expressed candidate target transcription factor by RNA-seq was validated using qRT-PCR. Functional validation was done using CRISPR knock out and drug resistance assays. Results: A higher percentage of enhancers were differentially accessible between SP and NSP cells compared with the promoters. We identified 746 enhancers that are accessible only in SP cells and 465 enhancers only in NSP cells. Transcription factor motif analysis showed that FOX family motif was enriched in accessible enhancers near SP-overexpressed genes. Among FOX family transcription factors, FOXC1 was the only overexpressed transcription factor, and it was also the most significantly overexpressed gene in SP cells. FOXC1 was consistently overexpressed in bladder tumor tissues relative to normal bladder tissues in three independent studies in BaseSpace Correlation Engine. CRISPR knock-out of FOXC1 in multiple bladder cancer cell lines resulted in decreased cisplatin resistance. Conclusion: We found that FOXC1 is overexpressed and its motif is enriched on the accessible enhancers when bladder cancer cells shift to a drug-resistant tumorigenic phenotype. Conversely, FOXC1 knock-out attenuates cisplatin resistance in bladder cancer cells. These observations suggest that differential expression and enhancer binding of FOXC1 promotes the previously observed, mutation-independent shift towards cisplatin resistance in bladder cancer. Citation Format: Yi-Tsung Lu, Tong Xu, Tien-Chan Hsieh, Maheen Iqbal, Zhifei Luo, Peggy J. Farnham, Suhn K. Rhie, Amir Goldkorn. FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1406.

Optimization of guar gum-gelatin bioink for 3D printing of mammalian cells

Bioprinting, as a fabrication technique, has witnessed significant progress over the past decade. Therefore, the development of bioink is crucial for the success of bioprinting as the process is governed by the properties of biomaterials used in bioink. Due to the lack of standards, only a limited number of bioinks are available, because of which direct comparison between two bioinks is not possible. This is the biggest hurdle in the advancement of extrusion-based bioprinting. This study focuses on providing a standard method for bioinks by analyzing the effect of storage modulus (G′), loss modulus (G″), tan ∂ (G”/G′), and bloom strength on their printability. A new economical bioink comprising gelatin and different concentrations of guar gum (a plant-based biopolymer) was studied. Guar gum is water soluble and is known for its ability to form highly viscous solutions even at lower concentrations. This new combination of biopolymer was formulated to obtain a broad range of bloom strength, G′ and G”. Tan ∂ was calculated using G’ and G”, followed by bloom test. These physical parameters were correlated with the printability of bioink, after evaluation of filament formation. Results suggested that the guar gum-gelatin bioinks having bloom strength in the range of 480–750 and tan ∂ in the range of 0.15–0.2, were able to form filament upon extrusion and were therefore considered as printable. Compared to oscillatory shear tests, the bloom test is a rapid and easy analytical technique for predicting the printability of bioinks. Cell examination is difficult when the scaffold possesses autofluorescence; hence two different assays were employed, viz. The live/dead assay and staining with Hoechst nuclear stain for analysis of the bioprinted cells. Apart from the bioink characterization, the methodology employed in this investigation can be used as a standard to analyze the printability of other extrusion-based bioinks.

Abstract A27: An apoptosis-based screen for targeted agents in rhabdomyosarcoma reveals potential combination therapies

Abstract Intensification of therapy for rhabdomyosarcoma (RMS) by combining conventional chemotherapies has not proven effective at improving long-term survival. More selective small molecules may improve outcomes when added to chemotherapeutic backbones, but the optimal actionable targets in RMS are not well defined. An alternative strategy to develop combination therapies is to identify agents with complementary methods of apoptotic induction that can be paired with synergistic results. We first set out to identify classes of drugs that induce apoptosis in representative fusion-positive (FP) and fusion-negative (FN) cell lines. We built a screening assay using high-throughput microscopy to quantify both cell number (Hoechst nuclear staining) and apoptosis (NucView 488 Caspase-3 Enzyme Substrate [Biotium]). Technical controls included DMSO and staurosporine. The assay had a SSMD β score of 6.4, demonstrating excellent quality for screening. We represented FP-RMS with RMS13 cells and represented FN-RMS with RD cells. We screened ~2,000 bioactive compounds spanning a wide variety of biochemical mechanisms and targets. We tested compounds at 5 μM to identify even weakly active agents, since they might synergize with other drugs. We deemed “active” compounds to be those that reduced cell number 4 SD below DMSO-treated wells. Specific drug targets were enriched among the set of active compounds. In RD cells, for example, 16 of 25 (64%) HDAC inhibitors reduced cell number and activated caspase 3, which is greater than the library average of 264/1,921 compounds (14%) (p<10-4 by Fisher exact test). Similarly, 11 of 15 (73%) mTOR inhibitors were active (p<10-4). By contrast, only a few neurotransmitter antagonists were active compounds, suggesting that these are random or noncanonical effects. Enriched targets included Aurora kinase, bromodomains, CDK, CHK, HDAC, HSP, kinesin/KSP, mTOR, PLK1, the proteasome, ROCK, tyrosine kinases, Wee1, and XPO1. We separately considered whether any classes of compounds showed differential activity against the two cell lines, which represent two distinct biologic types of RMS. We grouped compounds by their nominal target and compared the mean effects against RD vs. RMS13 cells for each group. MEK inhibitors were more effective against RD cells than RMS13 cells, consistent with the importance of an NRAS mutation in RD cells. Conversely, inhibitors of ROCK or CHK were more active against the FP RMS13 cells. This analysis supports investigation of MEK inhibition in FN-RMS and suggests new areas for biologic discovery in FP-RMS. In summary, the screen identified multiple druggable pathways that suppress apoptosis in RMS. The added depth of a functional readout (caspase activity) suggests that this screen has identified agents with greater than cytostatic effects. Ongoing work will identify the proximate biochemical mediators of cell death in relevant conditions, and test whether synergistic drug combinations can be explained by complementary modes of BH3 family member regulation. Citation Format: Amit J. Sabnis, Steven Chen, Carolina Morales, Benjamin S. Braun. An apoptosis-based screen for targeted agents in rhabdomyosarcoma reveals potential combination therapies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A27.

The antiproliferative effect of gambogic acid on HepG2 cells and its mechanism research

Objective To observe the inhibitory effect of gambogic acid on the proliferation of liver cancer cells HepG2 and explore its mechanism. Methods The vitro cultured liver cancer cells HepG2 were divided into the control group, low dose gambogic acid group, medium dose gambogic acid group and high dose gambogic acid group according to random number table method, 5 multiple holes in each group. The control group was cultured in normal medium and 0.5% DMSO was added as the solvent control. The 0.1, 1 and 10 μmol/L of gambogic acid were added to low, medium and high dose group of gambogic acid for intervention. MTT method was used to detect the inhibition rate of cell proliferation, flow cytometry was used to detect the apoptosis rate, Hoechst nuclear staining was used to observe the nucleus apoptosis, and Western blot was used to detect the expression of Bcl-2 and Bax protein. Results Compared with the control group, the HepG2 cells proliferation rate (68.00% ± 3.55%, 51.93% ± 4.36%, 47.16% ± 4.73% vs. 99.87% ± 4.53%) in low, medium and high dose gambogic acid group significantly decreased (P<0.01); apoptosis rate (23.00% ± 1.22%, 40.09% ± 4.65%, 70.32% ± 4.99% vs. 4.33% ± 0.57%) significantly elevated (P<0.01); Bcl-2 experssion (0.73 ± 0.10, 0.25 ± 0.10, 0.19 ± 0.08 vs. 0.97 ± 0.11) significantly decreased (P<0.01); and Bax expression (0.39 ± 0.14, 0.88 ± 0.15, 0.85 ± 0.13 vs. 0.22 ± 0.08) significantly elevated (P<0.01); Bax/Bcl-2 (0.34 ± 0.10, 1.87 ± 0.29, 1.63 ± 0.23 vs. 0.13 ± 0.06) significantly increased (P<0.01). Hoechst staining showed that the apoptosis rate of HepG2 cells increased in a dose-dependent manner with the increasing concentration of gambogic acid. Conclusions Gambogic acid could inhhibit HepG2 cells proliferation by regulating the expression of Bax and Bcl-2. Key words: Gambogic acid; Hep G2 cells; Cell proliferation; Apoptosis

Cytotoxic potentials of S. cumini methanolic seed kernel extract in human hepatoma HepG2 cells.

Syzygium cumini (Myrtaceae) is commonly called as Jamun or Jambolan. It has antidiabetic, anti-inflammatory, antipyretic, and antioxidant activities. Hepatocellular carcinoma is the most frequent and deadliest cancers worldwide. We investigated the cytotoxic potentials of S. cumini methanolic seed kernel extract against human hepatoma HepG2 cells. HepG2 cells were treated with 10, 20, and 40 μg/mL of seed kernel extract for 24 hours and cytotoxic analysis was performed by MTT assay. S. cumini induced apoptosis related morphological changes in HepG2 cells were analyzed by annexin V and propidium iodide double staining. Nuclear fragmentation and chromatin condensation were analyzed by Hoechst nuclear staining. Mitochondrial membrane potential (MMP) was investigated by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining. Protein expressions of hepatocyte nuclear factor-1α (HFN-1α) was performed using western blotting. S. cumini treatments caused a significant and a concentration-dependent increase in the cytotoxicity of HepG2 cells. S. cumini treatments increased the percentage of cells in an early and late apoptosis stage. This treatment also caused chromatin condensation and nuclear fragmentation. Further, S. cumini treatments decreased MMP and also caused a significant downregulation of HFN-1α protein expression. The present study demonstrated that S. cumini seed extract induced apoptosis in HepG2 cells through decrease in MMP and downregulation of HFN-1α.

Toll-like receptor 9 signaling promotes autophagy and apoptosis via divergent functions of the p38/JNK pathway in human salivary gland cells

Abnormal signaling transduction in salivary gland cells is associated with the pathogenesis of Sjögren's syndrome (SS). Previously, we identified aberrant expression of toll-like receptor 9 (TLR9) in gland cells of SS patients and mouse models. In this study, we investigated the role of TLR9 and its downstream p38/mitogen-activated protein kinase (MAPK) and c‐Jun N‐terminal kinase (JNK) signaling in mediating apoptosis and autophagy in human salivary gland (HSG) cells. We selected either CpG-Odn, a classical TLR9 activator, or lentivirus-packaged TLR9 full-length cDNA to activate TLR9 signaling transduction. Activation of TLR9 signaling induced phosphorylation of its downstream protein kinases, p38/MAPK and JNK, in a time-dependent manner, and decreased HSG cell viability. Western blotting of LC3B-II and p62 in both normal and autophagic flux-administered conditions revealed elevated autophagy upon TLR9 activation. Observing the cell cytoplasm through transmission electron microscopy and mRFP-GFP-LC3B-tagged fluorescence confirmed an increased number of autophagosomes and autolysosomes in TLR9-activated cells. Bax/Bcl-2 ratio calculations, caspase-3 activity assays and Hoechst nuclear staining were utilized to confirm the involvement of apoptosis in TLR9 signaling activation. Furthermore, we selected SB239063, a p38/MAPK signaling inhibitor, and SP600125, a JNK inhibitor, to identify the functions of p38/MAPK and JNK in TLR9-mediated signaling transduction. Multiple approaches, including Western blotting assays, fluorescence assessments and caspase-3 activity measurements, confirmed that inhibition of p38/MAPK signaling ameliorated both autophagy and apoptosis in TLR9-activated HSG cells, whereas inhibition of JNK signaling attenuated apoptosis but failed to modulate autophagy in the models mentioned above. Our results indicate a divergent function of p38/MAPK and JNK in TLR9-mediated autophagy and apoptosis in salivary gland cells.

Gracilaria foliifera (Forssk.) Børgesen ethanolic extract triggers apoptosis via activation of p53 expression in HepG2 cells

Background: Hepatocellular carcinoma is one of the most common types of malignancy and causes significant morbidity and mortality worldwide. Gracilaria foliifera (Forssk.) Borgesen, a brown marine alga, is shown to have growth inhibitory potential against various cancer cell lines other than human hepatoma HepG2 cells. Objective: To investigate the cytotoxic potentials of G. foliifera in HepG2 cells. Materials and Methods: HepG2 cells were fed with culture medium supplemented with different concentrations of ethanolic extract of G. foliifera (20, 40, and 80 μg/mL). After 24 h of treatment, the cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of early apoptosis was investigated by annexin V-fluorescein isothiocyanate immunofluorescence. Induction of late apoptosis and necroptosis was investigated by annexin V and propidium iodide (PI) staining. Nuclear chromatin condensation was evaluated by Hoechst staining. p53 protein expression was analyzed using Western blotting. Results: G. foliifera treatment in HepG2 cells caused a significant cytotoxic effect. Phosphatidylserine translocation confirms the induction of early apoptosis. Analysis of late apoptosis using annexin V/PI staining showed that the percentage of apoptotic cells was increased in a concentration-dependent manner. Hoechst nuclear staining further confirms the nuclear chromatin condensation. G. foliifera treatment also induced the tumor suppressor p53 protein expressions. Conclusion: The present study demonstrated that G. foliifera induced apoptosis in HepG2 cells through activation of p53. Abbreviations used: HCC: Hepatocellular carcinoma; DMEM: Dulbecco's minimum essential medium; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCCS: The National Center for Cell Science; FBS: Fetal bovine serum; PI: Propidium iodide; PS: Phosphatidylserine.

Dynamics of the Bone Marrow Microenvironment during Leukemic Progression Revealed By Codex Hyper-Parameter Tissue Imaging

IntroductionThe bone marrow (BM) microenvironment consists of various cell types such as mesenchymal stromal cells, endothelial cells, osteoblastic cells and multiple immune cell types including mature myeloid cells and lymphocytes. Recent studies have shown that leukemias can create and maintain a leukemia-supporting BM microenvironment, and vice versa, a dysfunctional BM microenvironment can contribute to leukemia development and progression. Moreover, in tumors the microenvironment is often immunosuppressive and restrains effective anti-tumoral immune responses by adaptive and innate immunity. A better understanding of the precise localization of microenvironmental and immune cell types in intact tissue, and how they physically interact with each other and with tumor cells, will improve our understanding of the mechanisms by which cancer reprograms its microenvironment and may form the basis for novel immunotherapies.MethodsCO-Detection by antibody indEXing (CODEX) is a multiplex fluorescence microscopy platform based on DNA-conjugated antibodies that allows the analysis of 50+ markers in a single tissue section. After staining with an antibody cocktail, tissues are imaged in a multi-cycle reaction using a microfluidics system and a fluorescence microscope with a computer automated X/Y/Z stage. DNA-conjugated antibodies are rendered visible using complementary fluorescent DNA probes, followed by imaging, probe stripping, washing and re-rendering. This process is repeated until all the antibodies present in the initial cocktail have been rendered and imaged. Here, we used CODEX to analyze intact BM at the single-cell level (~200nm resolution) during leukemic progression. Chronic myeloid leukemia (CML)-like disease was induced in non-irradiated mice using BCR-ABL1-GFP retrovirus. Tissue sections of femoral bones harvested at different time points after leukemia onset were stained using a 50+ marker CODEX antibody panel to simultaneously identify hematopoietic and leukemic stem and progenitor cells, multiple BM microenvironmental cell types, myeloid and lymphoid cells as well as functional markers.ResultsWe have built an integrated computational pipeline for the analysis of high-dimensional CODEX data that enables the identification and characterization of BM cell types as well as their spatial organization in situ. Raw images were concatenated and aligned using Hoechst nuclear stain as a reference, followed by deconvolution, segmentation, marker expression quantification and spatial compensation. Exported data were clustered in an unsupervised manner using VorteX algorithm, which identified 28 distinct cellular clusters based on marker expression values. All major BM compartments including stromal (vascular, pericytes, osteoblastic), lymphoid (T and B cell subsets), myeloid (megakaryocytes, macrophages, dendritic cells, granulocytes) and progenitor cell types, as well as leukemic cells, were represented. During leukemic progression, the BM microenvironment was dramatically rearranged. Besides the expected growth of the leukemic clone, we observed a massive increase in vascular and osteoblastic cell types, whereas immune cell clusters were significantly reduced. Interestingly, CD71, the transferrin receptor, was strongly up-regulated on tumor cells in advanced leukemia, indicating towards a role for iron metabolism in malignant progression. Furthermore, hierarchical clustering of tissue regions based on cellular composition using X/Y/Z positional information pointed towards the emergence of specific cell-cell interaction modules that developed during leukemic progression, including mutual attraction between B cells and central arterioles.ConclusionsHigh-dimensional imaging of the BM microenvironment by CODEX allows studying the abundance and distribution of cellular elements that are often underestimated or missed by traditional flow cytometry, such as stromal cells, vasculature and megakaryocytes. Importantly, CODEX identifies single cells in their tissue context during leukemic progression and facilitates the discovery of novel cell-cell interactions and cell types as well as unexpected marker constellations. DisclosuresSamusik:Akoya Biosciences: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Nolan:Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Goltsev:Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Distribution of FMRFamide-related peptides and co-localization with glutamate in Cupiennius salei, an invertebrate model system.

FMRFamide-related proteins have been described in both vertebrate and invertebrate nervous systems and have been suggested to play important roles in a variety of physiological processes. One proposed function is the modulation of signal transduction in mechanosensory neurons and their associated behavioral pathways in the Central American wandering spider Cupiennius salei; however, little is known about the distribution and abundance of FMRFamide-related proteins (FaRPs) within this invertebrate system. We employ immunohistochemistry, Hoechst nuclear stain and confocal microscopy of serial sections to detect, characterize and quantify FMRFamide-like immunoreactive neurons throughout all ganglia of the spider brain and along leg muscle. Within the different ganglia, between 3.4 and 12.6% of neurons showed immunolabeling. Among the immunoreactive cells, weakly and strongly labeled neurons could be distinguished. Between 71.4 and 81.7% of labeled neurons showed weak labeling, with 18.3 to 28.6% displaying strong labeling intensity. Among the weakly labeled neurons were characteristic motor neurons that have previously been shown to express ɣ-aminobutyric acid or glutamate. Ultrastructural investigations of neuromuscular junctions revealed mixed presynaptic vesicle populations including large electron-dense vesicles characteristic of neuropeptides. Double labeling for glutamate and FaRPs indicated that a subpopulation of neurons may co-express both neuroactive compounds. Our findings suggest that FaRPs are expressed throughout all ganglia and that different neurons have different expression levels. We conclude that FaRPs are likely utilized as neuromodulators in roughly 8% of neurons in the spider nervous system and that the main transmitter in a subpopulation of these neurons is likely glutamate.