xCELLigence Real-time Cell Analysis Research Articles

An in vitro assessment of teething gels’ effects on human gingival mesenchymal stem cells

BackgroundThe aim of this study is to examine the cytotoxic effects of dental gels with different contents, which are frequently used during teething, on gingival mesenchymal stem cells (G-MSCs).MethodThe teething gels used in this study were Dentinox, Gengigel, Osanite, and Jack and Jill. The human gingival mesenchimal stem cells (hG-MSCs) were incubated with these teething gel solutions (0.1%, 50% and 80% concentrations). Reproductive behavior of G-MSCs was monitored in real time for 72 h using the xCELLigence real-time cell analyzer (RTCA) system. Two-way repeated Anova test and post hoc Bonferroni test were used to evaluate the effect of concentration and dental gel on 0-hour and 72-hour viability. Significance was evaluated at p < 0.05 level.ResultsTeething gels prepared at 50% concentration are added to the G-MSC culture, the “cell index” value of G-MSCs to which Dentinox brand gel is added is significantly lower than all other groups (p = 0.05). There is a statistically significant difference between the concentrations in terms of cell index values at the 72nd hour compared to the 0th hour (p = 0.001).ConclusionsThe local anesthetic dental gels used in children have a more negative effect on cell viability as concentration increases.

Polyphyllin I enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth downregulating the Wnt/β-catenin pathway.

To investigate the synergistic effects of polyphyllin I (PPI) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway. Cell viability, apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays. The morphology of cancer cells was observed with inverted phase contrast microscope. The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays. The expressions of poly (adenosine diphosphate-ribose) polymerase, C-Myc, Cyclin B1, cyclin-dependent kinases 1, N-cadherin, Vimentin, Active-β-catenin, β-catenin, p-glycogen synthase kinase 3β (GSK-3β) and GSK-3β were determined by Western blotting assay. PPI sensitized TRAIL-induced decrease of viability, migration and invasion, as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells. The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/β-catenin signaling pathway. The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Liquid chromatography–mass spectrometry and xCELLigence real time cell analyzer revealed anticancer and antioxidant metabolites in Trianthema portulacastrum L. (Aizoaceae)

BackgroundThe rising incidence of cancer is a major concern globally being a leading cause of early mortality in most countries. This burden is projected to increase to 28.4 million cases worldwide by 2040 and yet, may be worsened by continuous exposure to risk factors due to a sheer shift from consumption of natural nutrients. To reverse this trend, it is imperative to source novel anticancer metabolites from plants that could minimize carcinogenic effects by suppressing the proliferation and survival of cancer cells. PurposeThis study aimed at characterizing and quantifying index anticancer and antioxidant metabolites, in the cultivars of T. portulacastrum. MethodsThe Ultra-High Performance Liquid Chromatography Mass Spectroscopy (UHP-LCMS) was used to quantify and characterize index anticancer and antioxidant metabolites in shoot, root and whole plant extracts of Trianthema portulacastrum (T01,T02,T03,T04,T05,T06). The biological activity of the tested samples was monitored on the xCELLigence Real Time Cell Analyzer to determine the cytotoxicity of the extracts as a function of cell index at a specific time post dose using the concentration range of 0.16–2.5 %. ResultsBased on the responses observed 48 h post exposure, all extracts showed high levels of cytotoxicity as determined by the xCELLigence apart from the root extracts. Rapid decreases in cell index (CI) values observed at higher extract percentages are highly indicative of cell death. These biological activities may be attributed to different anticancer metabolites such as epigallocatechin, glucaric acids, byakangelicin, xanthotoxin, apaensin, acetoxy-6-gingerol among other compounds of significant therapeutic benefits that were quantified with UHP-LCMS. ConclusionThese findings suggest that T. portulacastrum contains diverse anticancer metabolites which could be leveraged to develop plant-based anticancer drugs or novel chemicals with extended therapeutic utility.

Abstract 2681: Blockade of SIRPα in glioblastoma cells inhibits mitochondrial fatty acid β-oxidation and increase immune cell mediated cytotoxicity

Abstract Glioblastoma (GBM) is the most malignant brain tumor with limited immunotherapeutic options, likely due to significant GBM-associated systemic and intra-tumoral immunosuppression. The binding of SIRPα on myeloid cells to CD47 on cancer cells restricts immuno-phagocytosis against cancer cells. However, less attention has been given to SIRPα is also expressed in cancer cells, and the role of SIRPα on cancer cells is still unclear. Immunohistochemistry (IHC) staining was used to examine the expression of SIRPα in normal brain tissues and GBM. A single-cell database on the Broad Institute Portal was utilized to analyze the SIRPα expression in different cell types. SIRPα antisense morpholino was applied for gene knockdown. Established stable SIRPα overexpressed GBM cell lines were used to examine the mitochondrial function through the Seahorse Mito Stress test. In vitro, immune cell-mediated cytotoxicity was measured by Agilent xCELLigence Real-Time Cell Analysis. Patient biopsies show a 2-fold increase in SIRPα expression in GBM tumors compared to the normal adjacent (n=14-21; p &amp;lt; 0.01). Single-cell RNA-seq data on GBM tumors shows that SIRPα is expressed not only in the myeloid compartment but also in malignant cells. Studies have shown that mitochondrial function and fatty acid β-oxidation (FAO) play a critical role in GBM progression. Respirometry data revealed that blocking SIRPα in murine GBM cell line (CT2A) decreases mitochondrial ATP production. Seahorse Mito Stress test combined with fuel inhibitors showed that SIRPα expression mainly affects the usage of FAO (approximately 50%). In addition, stably, the GFP-tagged SIRPα overexpressed GBM cell line showed an increase of mitochondrial respiration with enhanced FAO ratio compared to the GFP control cell line. These suggested that SIRPα regulates mitochondrial FAO. Studies have shown that tumor-intrinsic FAO confers cell resistance to immune cell-mediated cytotoxicity. Interestingly, by coculturing with microglia, the innate immune population in the brain, our data showed that SIRPα blockade on GBM enhanced microglia-mediated cytotoxicity. Our data showed that targeting SIRPα on GBM may be a prospective therapeutic option by inhibiting FAO in mitochondria and increasing immune cell-mediated cytotoxicity. Citation Format: Yu-Ting Tsai, Glenn J. Lesser, David R. Soto-Pantoja. Blockade of SIRPα in glioblastoma cells inhibits mitochondrial fatty acid β-oxidation and increase immune cell mediated cytotoxicity [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 2681.

Abstract 1640: The role of CDC20 in the progression of colorectal cancer

Abstract Colorectal cancer (CRC) is the one of most common cancers and a major leading cause of cancer-related death worldwide. CDC20 is a key cell cycle regulator required during mitosis. Pathologically, CDC20 has been found to be overexpressed in cancer cells of various malignancies including breast, lung, brain and hepatic cancers and high level of CDC20 expression is associated with poor prognosis and disease progression in these cancers. Although it has been reported that CDC20 is associated with CRC progression and metastasis, it is still largely unknown how CDC20 regulates CRC cancer progression. Thus, the goal of this study is to identify the role of CDC20 in the progression of colorectal cancer. Our gene expression analysis revealed that CDC20 is highly expressed in colon cancer tissues compared to their adjacent normal colon in a cohort of CRC patients, suggesting the involvement of CDC20 in the CRC progression. To further understand the role of CDC20 in CRC progression, we experimentally targeted CDC20 genetically and pharmacologically. To perform CDC20 Loss of function studies, we screened various CRC cell lines for CDC20 protein levels and CDC20 highly expressing cell lines were utilized to establish CDC20-knockdown cells using shRNA targeting CDC20 and scrambled shRNA as controls. CDC20 Down-regulation levels were confirmed using both western blot and RT-PCR analysis. The effect of CDC20 knockdown/CRC cells on CRC cell proliferation was investigated using xCELLigence real-time cell analysis instrument and colony formation assay. Our results revealed that CDC20 knockdown CRC cells have lower proliferation capacity compared to the control, suggesting involvement of CDC20 in regulating tumorigenic phenotypes of CRC cells. Pharmacologically, we targeted CDC20 using a specific molecular inhibitor, Apcin and we assessed the Apcin treatment effect on cell viability and migration of CRC cells. Our results showed that Apcin significantly reduced CRC cell viability and migration in a dose-dependent manner. Taken together, our results suggest that CDC20 is a key regulator in the tumorigenic behavior of CRC and is considered a potential therapeutic target for treating patients with CRC. Citation Format: Nouran Abualsaud, Nada Gazzaz, Amal Alhamid, Deemah Alenizy, Bahauddeen M. Alrfaei, Mana Alshehri. The role of CDC20 in the progression of colorectal cancer [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 1640.

In vitro EVALUATION of THE EFFECTS of POTENTIAL GSK-3β INHIBITORS TERBUTALINE and ORCIPRENALINE

Lung cancer is a type of cancer that is mostly diagnosed at an advanced stage and has a short survival time despite standard chemotherapy and targeted therapies. Terbutaline and Orciprenaline are bronchodilator agent that are potent and selective β2 receptor agonists. The purpose of this study was to investigate by continuous monitoring to assay the effects of Terbutaline and Orciprenaline on human lung carcinoma cell line. Cells were treated 1, 10, 100, 200, 400 µM concentrations with Terbutaline/Orciprenaline. MTT assay and xCELLigence real-time cell analyzer were used to determine these compounds effect on cell viability. The cell index was monitored continuously by visualizing the impedance of the E-plate wells. Because of GSK3β roles in a diverse range of cellular processes like metabolism, cell proliferation, differentiation and survival and its key position at several signaling pathways, GSK3β inhibition was investigated of these molecules. It has been determined that Terbutaline and Orciprenaline inhibits GSK-3β.&#x0D; This is the first known study to demonstrate the effects of Terbutaline and Orciprenaline on A549 and the human bronchial epithelial cell line Beas-2b in real time. In this study, it was detected that Terbutaline and especially Orciprenaline may have potential therapeutic effects in lung carcinoma.

Synthesis, Anticancer Activity, and In Silico Modeling of Alkylsulfonyl Benzimidazole Derivatives: Unveiling Potent Bcl-2 Inhibitors for Breast Cancer.

A series of alkylsulfonyl 1H-benzo[d]imidazole derivatives were synthesized and evaluated for anticancer activity against human breast cancer cells, MCF-7 in vitro. The cytotoxic potential was determined using the xCELLigence real-time cell analysis, and expression levels of genes related to microtubule organization, tumor suppression, apoptosis, cell cycle, and proliferation were examined by quantitative real-time polymerase chain reaction. Molecular docking against Bcl-2 was carried out using AutoDock Vina, while ADME studies were performed to predict the physicochemical and drug-likeness properties of the synthesized compounds. The results revealed that compounds 23 and 27 were the most potent cytotoxic derivatives against MCF-7 cells. Gene expression analysis showed that BCL-2 was the most prominent gene studied. Treatment of MCF-7 cells with compounds 23 and 27 resulted in significant downregulation of the BCL-2 gene, with fold changes of 128 and 256, respectively. Docking analysis predicted a strong interaction between the compounds and the target protein. Interestingly, all of the compounds exhibit a higher binding affinity toward Bcl-2 than the standard drug (compound 27 vina score = -9.6 kcal/mol, vincristine = -6.7 kcal/mol). Molecular dynamics simulations of compounds 23 and 27 showed a permanent stabilization in the binding site of Bcl-2 for 200 ns. Based on Lipinski and Veber's filters, all synthesized compounds displayed drug-like characteristics. These findings suggest that compounds 23 and 27 were the most promising cytotoxic compounds and downregulated the expression of the BCL-2 gene. These derivatives could be further explored as potential candidates for the treatment of breast cancer.

Efficacy of MUC1-targeted CAR-NK cells against human tongue squamous cell carcinoma.

The clinical efficacy of CAR-NK cells against CD19-expressing blood cancers has been demonstrated, and they have shown potential for treating solid tumors as well. However, the efficacy of CAR-NK cells for treating human oral tongue squamous cell carcinoma (OTSCC) has not been examined. We assessed MUC1 expression in human OTSCC tissue and a cell line using immunohistochemistry and immunofluorescence. We constructed NK cells that express CAR targeted to MUC1 from pluripotent stem cells (iPSC-derived MUC1-targeted CAR-NK cells) and evaluated their effectiveness against OTSCC in vitro using the xCELLigence Real-Time Cell Analysis system and CCK8 assay, and in vivo by measuring xenograft growth daily in BNDG mice treated with MUC1-targeted CAR-NK cells. As controls, we used iPSC-derived NK cells and NK-free media, which were CAR-free and blank, respectively. MUC1 expression was detected in 79.5% (66/83) of all OTSCC patients and 72.7% (24/33) of stage III and IV. In stage III and IV MUC1 positive OTSCC, 63.6% (21/33) and 48.5% (16/33) patients had a MUC1-positive cancer cell rate of more than 50% and 80%, respectively. The iPSC-derived MUC1-targeted CAR-NK cells exhibited significant cytotoxicity against MUC1-expressing OTSCC cells in vitro, in a time- and dose-dependent manner, and showed a significant inhibitory effect on xenograft growth compared to both the iPSC-derived NK cells and the blank controls. We observed no weight loss, severe hematological toxicity or NK cell-mediated death in the BNDG mice. The MUC1-targeted CAR-NK cells had significant efficacy against human OTSCC, and their promising therapeutic response warrants further clinical trials.

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

BackgroundMesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities.MethodsA total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring.ResultsBy comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring.ConclusionsWe established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

Muscarinic Receptors as Targets for Metronomic Therapy in Ovarian Cancer

Aim: In this study, the effects of muscarinic acetylcholine receptor (mAChR) agonist carbachol on the proliferation of cisplatin-resistant (A2780cis) and cisplatin-free (SKOV-3) ovarian cancer cell line were for the first time investigated to further evaluate the potential therapeutic effect of metronomic chemotherapy.&#x0D; Material and Methods: The inhibitory effect of carbachol on cell proliferation was detected using the xCELLigence Real-Time Cell Analyzer (RTCA) dual plate (DP) system. A preliminary study was conducted to determine the dose of carbachol 100 µM, cisplatin 1 µM, and two combination studies were carried out with 100 µM carbachol + cisplatin 1 µM and 100 µM carbachol + 10 µM atropine, over cancer cells without drugs was used as the control group. The cell proliferation curve was monitored for 96 hours. The cell index value of inhibition in cell proliferation was automatically measured every hour for each well using RTCA 1.2.1 software.&#x0D; Results: Co-administration of carbachol with cisplatin caused a decrease in cell number in both A2780cis and SKOV-3 cell lines in a time-dependent manner (p

Evaluation of kinetic effects of Gliotoxin in different breast cancer cell lines.

In the present research, the antiproliferative properties of Gliotoxin, which is obtained from marine fungus and thought to be a promising metabolite, on MCF-7 and MDA-MB-231 breast cancer cells, which have different molecular subtypes, were evaluated. Different cell kinetic parameters were employed for this aim. In experiments, cell viability, cell index, mitotic index, BrdU labeling index, and apoptotic index were assessed. Gliotoxin concentrations of 1.5625 µM, 3.125 µM, and 6.25 µM were used in studies for both cell lines. As a result of the values obtained from cell viability and xCELLigence Real-Time Cell Analysis (RTCA) System, 1.5625 µM concentration was determined as IC50 dose. This concentration was applied to all other parameters and anticancer activities were observed.

Anti-Nasopharyngeal carcinoma mechanism of sanguinarine based on network pharmacology and molecular docking.

The purpose of this study was to investigate the mechanism of sanguinarine (SAN) against nasopharyngeal carcinoma (NPC) by means of network pharmacology, molecular docking technique, and experimental verification. The SAN action targets were predicted using the Swiss Target Prediction database, the related NPC targets were determined using the GEO database, and the intersection of drug and disease pathway targets were considered to be the potential targets of SAN against NPC. The target-protein interaction network map was constructed using the STRING database, and the core target genes of SAN against NPC were obtained via topological network analysis. "R" language gene ontology (GO) function and Kyoto encyclopedia of genes and genome (KEGG) pathway enrichment analyses were used to dock the core target genes with SAN with the help of AutodockVina. Cell proliferation was detected using MTT and xCELLigence real-time cell analysis. Apoptosis was identified via Hoechst 33342 staining, JC-1 mitochondrial membrane staining, and annexin V-FITC/PI double fluorescence staining, while protein expression was quantified using western blotting. A total of 95 SAN against NPC targets were obtained using target intersection, and 8 core targets were obtained by topological analysis and included EGFR, TP53, F2, FN1, PLAU, MMP9, SERPINE1, and CDK1. Gene ontology enrichment analysis identified 530 items, and 42 items were obtained by Kyoto encyclopedia of genes and genome pathway enrichment analysis and were mainly related to the PI3K/AKT, MAPK, and p53 signaling pathways. Molecular docking results showed that SAN had good binding activity to the core target. SAN inhibited the proliferation of NPC cells, induced apoptosis, reduced the expression levels of survivin and Bcl2, and increased the expression levels of Bax and cleaved caspase-8. It also decreased the expression levels of the key proteins p-c-Raf, p-MEK, and p-ERK1/2 in the MAPK/ERK signaling pathway in NPC cells. SAN inhibits the proliferation and induces the apoptosis of NPC cells through the MAPK/ERK signaling pathway.

Placental deficiency of the (pro)renin receptor ((P)RR) reduces placental development and functional capacity.

The (pro)renin receptor ((P)RR; also known as ATP6AP2) is a multifunctional receptor. The (P)RR activates the tissue renin-angiotensin system (RAS) and is also involved in regulating integral intracellular pathways such as V-ATPase and Wnt/β-catenin signalling. Given this, the (P)RR may be associated with essential pathways in placentation, however its role within the context of pregnancy remains poorly characterised. The first trimester/extravillous trophoblast cell line, HTR-8/SVneo, underwent an siRNA knockdown where they were incubated for 24h with a negative control siRNA or siRNA targeting ATP6AP2 mRNA. xCELLigence real-time cell analysis was performed to assess the effect of ATP6AP2 mRNA knockdown on HTR-8/SVneo cell proliferation, migration, and invasion. In subsequent experiments, GFP-encoding lentiviral packaged gene-constructs were used to knockdown (P)RR expression in the trophectoderm of C57/BL6/CBA-F1 mouse blastocysts. Blastocysts were incubated for 6h with vehicle (no-virus), control virus (non-targeting shRNA and GFP), or (P)RR-knockdown virus ((P)RR shRNA and GFP) before transfer into recipient pseudo-pregnant Swiss CD1 female mice. Fetal and placental tissues were collected and assessed at embryonic age (EA) 10 and 18. (P)RR levels were measured in the labyrinth zone of day 18 placentae and stereological Merz grid analysis was performed to determine the volumetric distribution of trophoblasts, fetal capillaries, and the maternal blood space. We showed that a reduction of ATP6AP2 expression in HTR-8/SVneo cells in vitro, impaired trophoblast proliferation, migration, and invasion. In vivo, decreasing placental labyrinth (P)RR expression adversely effected placental physiology, decreasing placental trophoblast number and total surface area available for exchange, while also increasing maternal blood space. Additionally, decreased (P)RR affected placental efficacy evident by the reduced fetal-placental weight ratio. Our study shows that the (P)RR is necessary for appropriate placental development and function.

Enhancing the functional maturity of hiPSC-derived cardiomyocytes to assess inotropic compounds

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) present an attractive in vitro platform to model safety and toxicity assessments—notably screening pro-arrhythmic compounds. The utility of the platform is stymied by a hiPSC-CM contractile apparatus and calcium handling mechanism akin to fetal phenotypes, evidenced by a negative force-frequency relationship. As such, hiPSC-CMs are limited in their ability to assess compounds that modulate contraction mediated by ionotropic compounds (Robertson, Tran, & George, 2013). To address this limitation, we utilize Agilent's xCELLigence Real-Time Cell Analyzer ePacer (RTCA ePacer) to enhance hiPSC-CM functional maturity. A continuous, progressive increase of electrical pacing is applied to hiPSC-CMs for up to 15 days. Contraction and viability are recorded by measurement of impedance using the RTCA ePacer. Our data confirms hiPSC-CMs inherently demonstrate a negative impedance amplitude frequency that is reversed after long-term electrical pacing. The data also indicate positive inotropic compounds increase the contractility of paced cardiomyocytes and calcium handling machinery is improved. Increased expression of genes critical to cardiomyocyte maturation further underscores the maturity of paced cells. In summary, our data suggest the application of continuous electrical pacing can functionally mature hiPSC-CMs, enhancing cellular response to positive inotropic compounds and improving calcium handling. SummaryLong-term electrical stimulation of hiPSC-CM leads to functional maturation enabling predictive assessment of inotropic compounds.

Cytotoxic and antiproliferative effects of hellebrin on breast and lung cancer cells

This study aimed to investigate the impact of hellebrin on human lung cancer cell (H1299) and breast cancer cell (MCF-7) lines over time. The viability of various concentrations of hellebrin (100 nM-400 nM) on two cancer cell lines was measured using the MTT method. The cellular proliferation over time was examined using xCELLigence real-time cell analysis (RTCA). The results showed a significant decrease in cell viability with increasing concentrations of hellebrin in both cancer cell lines compared to the control group (p˂0.0001). While the viability of both cancer cells decreased after a certain period of hellebrin application, the viability of the control groups increased over time. These findings indicate that hellebrin has high antiproliferative and cytotoxic effects on breast and lung cancer cells. Consequently, this study suggests that hellebrin may have potential as a treatment for other cancers, especially lung cancer, and further in vitro and in vivo experiments are needed to investigate this possibility.

Effects of SPARC and Possible Receptors on Colon Cancer Cell Line

Objective: The aim of this study was to observe the apoptotic/cytotoxic effects of exogenous SPARC on colon cancer cell line HT-29, then to investigate the function of stabilin-1 and integrin αvβ3, which are possible receptors for SPARC in colon cancer cells and to determine the quantitation of their receptor numbers.&#x0D; Methods: Appropriate doses of exogenous SPARC and it’s inhibitor, cilengitide added to HT-29 cell line were determined by xCELLigence Real-Time Cell Analysis system, SPARC-mediated caspase 3 expressions were measured. Using the RT-PCR system, gene expression levels of SPARC, stabilin-1 and integrin αvβ3 receptors (silenced/nonsilenced with cilengitide) were detected then the numbers of receptors per cell were quantitated by flow cytometry.&#x0D; Results: IC50 value of SPARC was determined as 4.57 μg/mL and IC50 value of cilengitide was determined as 50 nM. 5 μg/mL exogenous SPARC caused increased apoptosis in the HT-29 line. Significant increase in gene expression of integrin αvβ3 receptor was observed in the group incubated with 5 μg/mL SPARC, contrarily, the addition of cilengitide decreased gene expressions. The integrin αvβ3 receptor numbers&#x0D; increased approximately 2-fold with SPARC compared to the control. No significant changes were observed in the gene expression and receptor numbers of stabilin-1.&#x0D; Conclusion: Exogenous SPARC was shown to reduce proliferation and induce apoptosis in colon cancer cells. Integrin αvβ3 is thought to be the possible receptor mediating SPARC in colon cancer cells. Quantification of surface receptors per cell, which we think we have done first, can be considered as a marker in the follow-up of anticancer treatments.

DELTA-9 TETRAHYDROCANNABINOID AND GEMSITABIN COMBINATION ON NON-SMALL CELL (SQUAMOUS CELL, SK-MES-1) LUNG CANCER CELL LINE

Squamous cell carcinoma falls within the category of non-small cell lung cancers, presenting a considerable challenge in terms of treatment management. There has been growing interest in the potential anti-cancer properties of Delta-9 tetrahydrocannabinol. Gemcitabine, a nucleoside analog, has shown effectiveness against diverse cancer types. This study aimed to assess the combined efficacy of Delta-9 tetrahydrocannabinol and Gemcitabine in treating cancer. The squamous lung cancer cell line required for our study was provided by the stem cell unit. The doses were determined as 5µm/L and 10µm/L for Delta-9 tetrahydrocannabinol, 20 and 40 µm/Lfor Gemcitabine by performing a literature review. In our study, CellViabilityAnalysis by MTT, Xcelligence Real-Time Cell Analysis, Annexin V Apoptosis Flow Cytometry Analysis, Total Oxidant, Antioxidant Status, and Caspase-3 Detection Analysis was performed. Upon evaluating the rates of apoptotic cell death, it was observed that the THC 5 and THC 10 treatment groups exhibited a 30% and 60% increase, respectively, compared to the alcohol group. A significant difference in cytotoxic effect, as determined by MTT, was found between the control group and the Gemsitabin 20, Delta-9 Tetrahydrocannabinol 5µm/L, and Gemsitabin 20+Delta-9 Tetrahydrocannabinol 5µm/L groups (p&lt;0.001). There was a statistically significant difference between the groups regarding TOS (p&lt;0.001). All experimental groups exhibited a higher level of caspase 3 activations, in comparison to the control group. We observed a significant cytotoxic effect of Gemcitabine on squamous cell lung cancer cells. Delta-9 tetrahydrocannabinol, when used alone, exhibited a relatively low cytotoxic effect. However, no significant difference was observed in the groups where Delta-9 tetrahydrocannabinolwas combined with Gemcitabine.

A simplified workflow with end-point validation of real-time electrical cell-substrate impedance sensing of retinoic acid stimulated neurogenesis in human SH-SY5Y cells in vitro

ObjectiveRetinoic acid (RA) is known to transition proliferating SH-SY5Y neuroblastoma cells towards functional neurons. However, the activity of RA is restricted due to its photolability where any findings from prolonged time course observations using microscopy may alter outcomes. The aim of the study was to establish a real-time, long-term (9-day) protocol for the screening of differentiation events using Electrical cell-substrate impedance sensing (ECIS).Results and discussionA differentiation baseline for SH-SY5Y cells was established. Cells were seeded and exposed to repeated spikes of RA using the xCELLigence real-time cell analyser single plate (RTCA-SP) for real-time monitoring and identification of differentiation activity over a 9 day period in order to be more representative of differentiation over a prolonged timeline. Specific features associated with differentiation (growth inhibition, neurite outgrowths) were confirmed by end-point analysis.RA-induced growth inhibition and assumed phenotypic changes (i.e. neurite outgrowth) were identified by the xCELLigence analysis and further confirmed by end-point metabolic and phenotypic assays. Change in cellular morphology and neurite outgrowth length was identified by end-point fluorescence detection followed by computational analysis. Based on this it was possible to identify SH-SY5Y phenotypic differentiation with distinct phases observed over 9 days using Electric cell-substrate impedance sensing (ECIS) cell index traces providing a path to application in larger scale neurotrophic factor screening using this scalable technology.

House dust mite and Th2 cytokine-mediated epithelial barrier dysfunction attenuation by KL001 in 16-HBE cells

ABSTRACT House dust mite (HDM) is a common aeroallergen that can disrupt the airway epithelial barrier leading to dysregulated immune response, resulting in allergic lung diseases such as asthma. Cryptochrome (CRY), a circadian clock gene, plays an important role in the regulation of metabolism, and immune response. It remains unclear whether stabilizing CRY using KL001 can attenuate HDM/Th2 cytokine-induced epithelial barrier dysfunction in 16-HBE cells. We evaluate the effect of KL001 (20 µM) pre-treatment (4 hrs) in HDM/Th2 cytokine (IL-4 or IL-13)-mediated change in epithelial barrier function. HDM and Th2 cytokine-induced changes in transepithelial electrical resistance (TEER) were determined by an xCELLigence real-time cell analyzer and delocalization of adherens junction complex (AJC: E-cadherin and β-catenin) and tight junction proteins (TJP: Occludin and Zonula occludens-1) by immunostaining and confocal microscopy. Finally, quantitative real-time PCR (qRT-PCR) and Western blotting were used to measure altered gene expression and protein abundance of the epithelial barrier function and core clock genes, respectively. HDM and Th2 cytokine treatment significantly decreased TEER associated with altered gene expression and protein abundance of the selected epithelial barrier function and circadian clock genes. However, pre-treatment with KL001 attenuated HDM and Th2 cytokine-induced epithelial barrier dysfunction as early as 12–24 hrs. KL001 pre-treatment showed attenuation of HDM and Th2 cytokine-induced alteration in the localization and gene expression of AJP and TJP (Cdh1, Ocln, and Zo1) and core clock genes (Clock, Arntl/Bmal1, Cry1/2, Per1/2, Nr1d1/Rev-erbα, and Nfil3). We demonstrate, for the first time, the protective role of KL001 in HDM and Th2 cytokine-mediated epithelial barrier dysfunction.

Abstract 1771: Characterization of RHOA inactivation as a driver of CAR-T therapy resistance in diffuse large B-cell lymphoma

Abstract Background: Front-line chemoimmunotherapy for diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, is effective, however at least 40% of patients relapse or are refractory (r/r). Of this r/r DLBCL cohort, approximately 10% will be cured with conventional second-line treatments consisting of salvage chemotherapy and autologous stem cell transplant. CD19-directed chimeric antigen receptor T-cell (CAR-19) therapies are promising new second-line options in diffuse large B-cell lymphoma (DLBCL) and show exciting 30-40% durable complete responses in heavily pretreated patients. Recent whole-genome sequencing data published by our lab revealed that deletion of Ras homolog family member A (RHOA) is strongly associated with poor response to CAR-19 therapy in r/r DLBCL patients (p = 0.0013). In newly diagnosed DLBCL, 20% of patients harbor a RHOA deletion and another 5% have loss-of-function mutations. To date, no work has comprehensively identified the role of RHOA in DLBCL or how its inactivation may protect CAR-T resistant tumors. Methods: We created a novel in vivo model of RHOA deficient lymphoma by transplanting Eμ-Myc transgenic hematopoietic stem cells (HSCs) transduced with a shRhoa vector. We also created a syngeneic model of RHOA-deficient lymphoma with MYC-driven BL3750 cells. In vitro flow cytometry and Xcelligence real-time cell analysis experiments were utilized to assess cytotoxicity of CAR-19 against RHOA-deficient lymphoma cell lines. RNAseq analysis was conducted on Rhoa knockdown cell lines to elucidate cell-intrinsic mechanisms of lymphomagenesis. Cell proliferation and migration assays were performed to characterize the phenotype of RHOA-deficient DLBCL cell lines. Results: 18 mice were transplanted (11 shRhoa, 7 shRenilla control) and are being monitored for lymphoma development. In vitro CAR-T experiments demonstrated that while RHOA-deficient cells demonstrate a similar degree of killing initially, a population of RHOA-deficient cells persists and enrich at later time points. RNA-seq analysis of A20 shRhoa murine lymphoma cells uncovered enrichment of gene sets associated will cell-cycle progression and interferon (INF) signaling pathways. Cell proliferation assay and Western blot of RHOA inactivated cell lines revealed increased proliferation in shRhoa cells through the AKT/mTOR pathway compared to controls. Conclusions: RHOA-deficient lymphomas, while not entirely evading CAR-19 cells in vitro, demonstrate resistance mechanisms that allows a small population to persist. RNA-seq analysis reveals upregulated oncogenic and immunoinhibitory pathways, consistent with in vitro cell proliferation and Western blot assays. Further research will challenge in vivo RHOA-deficient tumors with CAR-19 cells and employ scRNA-seq to reveal transcriptomic changes within RHOA-deficient lymphoma responsible for CAR-19 resistance. Citation Format: Austin Newsam, Yitzhar Goretsky, Evan Roberts, Daniel Bilbao, Jonathan Schatz. Characterization of RHOA inactivation as a driver of CAR-T therapy resistance in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1771.