BT474 Breast Cancer Cells Research Articles

Aptamer-Functionalized Nanoparticles Mediate PD-L1 siRNA Delivery for Effective Gene Silencing in Triple-Negative Breast Cancer Cells.

Small interfering RNA (siRNA) therapies require effective delivery vehicles capable of carrying the siRNA cargo into target cells. To achieve tumor-targeting, a drug delivery system would have to incorporate ligands that specifically bind to receptors expressed on cancer cells to function as portals via receptor-mediated endocytosis. Cell-targeting and internalizing aptamers are the most suitable ligands for functionalization of drug-loaded nanocarriers. Here, we designed a novel aptamer-based platform for the active delivery of siRNA targeting programmed cell death-ligand 1 (PD-L1) to triple-negative breast cancer (TNBC) cells. The generated nanovectors consist of PLGA-based polymeric nanoparticles, which were loaded with PD-L1 siRNA and conjugated on their surface with a new RNA aptamer, specific for TNBC and resistant to nucleases. In vitro results demonstrated that these aptamer-conjugated nanoparticles promote siRNA uptake specifically into TNBC MDA-MB-231 and BT-549 target cells, along with its endosomal release, without recognizing non-TNBC BT-474 breast cancer cells. Their efficiency resulted in an almost complete suppression of PD-L1 expression as early as 90 min of cell treatment. This research provides a rational strategy for optimizing siRNA delivery systems for TNBC treatments.

Analysis of RANK-c interaction partners identifies TRAF3 as a critical regulator of breast cancer aggressiveness

Breast cancer is a highly heterogeneous disease both at the histological and molecular levels. We have previously shown that RANK-c is a regulator of NF-κB signaling and exerts a suppressive effect on aggressive properties of ER negative breast cancer cells, while there is an opposite effect on ER positive cell lines. In order to identify molecular determinants that govern the opposing function of RANK-c in breast cancer cells we employed the two cell lines with the highest degree of phenotypic divergence upon RANK-c-expression (SKBR3 and BT474) and identified proteins that interact with RANK-c by affinity-enrichment mass spectrometry (AE-MS) analysis. Annotating enriched proteins with NF-κB signaling pathway revealed TRAF3 as an interacting partner of RANK-c in SKBR3 cell protein lysates, but not in BT474 breast cancer cells in which RANK-c induces cell aggressiveness. To determine the role of TRAF3 in the phenotype of BT474-RANK-c cells, we reconstructed the TRAF3/RANK-c interaction both in parental BT474 and RANK-c expressing cells and tested for aggressive properties through colony formation, migration and invasion assays. TRAF3 forced expression was able to reverse BT474 phenotypic changes imposed by RANK-c, rendering cells less aggressive. Finally, TRAF3 gene expression data and TRAF3 immunohistochemical (IHC) analysis on breast cancer samples indicated that TRAF3 expression correlates with Overall Survival (OS), Recurrence Free Survival (RFS) and several clinicopathological parameters (histological grade, proliferation index) of breast cancer disease.

APPLICATION OF SILICONE COATING TO OPTIMIZE THE PROCESS OF OBTAINING CELLULAR SPHEROIDS BY THE HANGING DROP METHOD

Purpose of the study . To study the effect of SIEL 159–330 coating on the cell clusters formation rate in a hanging drop method in combination with the use of methylcellulose (MC) and collagen as cell aggregation improving agents. Materials and methods . BT20 breast cancer cells were cultured in drops of 20 μL (104 cells per drop) on the lid of a polystyrene Petri dish coated with SIEL 159–330 silicone elastomer (GNIIKHTEOS, Moscow, Russia) or without coating. The study tested three concentrations of MC (0.1 %, 0.25 % and 0.4 %) and collagen (150 µg/ml, 300 µg/ml and 600 µg/ml). The rate of formation of cell conglomerates was assessed by evaluating their area after 4, 24, 48, and 72 hours of cultivation. Results . The use of SIEL 159–330 coating made it possible to obtain spheroids of the same size as the addition of 0.4 % MC over a time interval of 72 hours. The silicone coating additionally reduced the size of cell spheroids in the medium with 0.1 % MC at all time points; however, this effect disappeared with increasing concentration of MC. In addition, the use of SIEL 159–330 reduced the relationship between the size of cellular spheroids and the concentration of MC, which allows us to consider the use of this coating as an alternative to MC or a way to reduce its concentration. In the experiment with the addition of collagen to the culture medium, the sizes of cell conglomerates formed on the silicone coating were significantly smaller than on uncoated plastic in all variants of the experiment and time points. The effect was more pronounced for a collagen concentration of 600 μg/ml. The use of SIEL 159–330 coating, in addition, reduced the variability in the size and shape of the resulting cell conglomerates. Conclusion . Accelerated aggregation of cells and fibers of the extracellular matrix in hanging drops, as well as a reduction in the variability in the size and shape of the resulting cell clusters on SIEL 159–330, allows us to reduce the time of experiments and material costs, as in experiments with the addition of substances that accelerate the formation of spheroids (MC and collagen), as well as in their absence.

Synthesis, Spectroscopic Analysis, and In Vitro Anticancer Evaluation of 2-(Phenylsulfonyl)-2H-1,2,3-triazole

The 1,2,3-Triazole derivatives containing the sulfonyl group have proved their biological importance in medicinal chemistry and drug design. In this sense, we describe the regioselective synthesis of 2-(phenylsulfonyl)-2H-1,2,3-triazole 3 in good yield through a classical sulfonamidation reaction of 1H-1,2,3-triazole 1 with benzenesulfonyl chloride 2 in dichloromethane using a slight excess of triethylamine at 20 °C for 3 h. This procedure is distinguished by its short reaction time, high yield, excellent regioselectivity, clean reaction profile, and operational simplicity. The sulfonamide 3 was characterized by high-resolution mass spectrometry, FT–IR, UV–Vis, 1D and 2D NMR spectroscopy, and elemental analysis. The sulfonamide 3 exhibited moderate activity against UO-31 renal, SNB-75 central nervous system, HCT-116 colon, and BT-549 breast cancer cell lines, with growth inhibition percentages (GI%) ranging from 10.83% to 17.64%.

Abstract 367: Nano-immunotherapy in TNBC: Aptamer-based nanoparticles for PD-L1 siRNA delivery to cancer cells

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive group of breast cancers. The lack of specific actionable targets makes chemotherapy the main treatment for TNBC patients. However, chemotherapy has limited success due to scarce bioavailability, severe systemic side effects and drug resistance. Polymeric nanoparticles (PNPs) may efficiently deliver in vivo therapeutics to tumors when conjugated to specific targeting agents. Potential agents for targeting tumor cells are aptamers: short, single-stranded oligonucleotides that interact at high affinity with their targets. Here, we report the characterization of new multifunctional nanovectors consisting of safe and biodegradable PNPs, highly specific TNBC aptamers as delivery agents and artificial small interfering RNA (siRNA) as drug payload, designed to suppress programmed cell death-ligand 1 (PD-L1) expression, a major feature of immune evasion in cancer cells. We efficiently entrapped siRNA-PD-L1 into PNPs. To enable active targeting, siPD-L1-PNPs were functionalized with TN145-aptamer, which we previously generated by cell-SELEX and shown to bind with nanomolar affinity to TNBC cells distinguishing them from both normal breast cells and non-TNBC breast cancer cells. TN145 aptamer actively internalizes into target cells, thus representing a good candidate to deliver a therapeutic payload. We show that the aptamer-decorated nanovectors efficiently deliver fluorescein-labeled siRNA into TNBC MDA-MB-231 and BT-549 cells, as assessed by confocal microscopy. Unconjugated nanovectors or conjugated with scrambled aptamers were used as controls. Non-TNBC BT-474 and MCF7 breast cancer cells, were used to exclude unspecific binding. Importantly, a 30-min incubation of TN145-conjugated nanovectors on target cells, at a siPD-L1 concentration of 1 nM, results in stronger PD-L1 silencing than that achieved by siPD-L1 delivered via a commercial transfection reagent. Furthermore, TN145-PNPs loaded with both siPD-L1 and cisplatin were generated and the efficacy of combined treatment was tested on tumor cells and tumor and immune cell co-cultures. Chemotherapy, including cisplatin, has been reported to induce PD-L1 enrichment in TNBC cells, hence an aptamer-targeted nanosystem enabling the synergistic effect of siRNA that directly knocks down PD-L1 expression on tumor cells with a powerful chemotherapeutic drug could be the future way to eradicate TNBC cells. Citation Format: Lisa Agnello, Simona Camorani, Silvia Tortorella, Annachiara d’Argenio, Roberto Nilo, Monica Fedele, Antonella Zannetti, Mauro Comes Franchini, Laura Cerchia. Nano-immunotherapy in TNBC: Aptamer-based nanoparticles for PD-L1 siRNA delivery to cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 367.

Abstract 2404: Role of TPD52L2 in cell migration and triple negative breast cancer progression

Abstract The Golgi apparatus plays an important role in cell migration and the establishment of epithelial polarity. A dispersed (or fragmented) Golgi ribbon is often seen in stressed cells and in various pathologies, including neurodegeneration or cancer, but whether and how a fragmented Golgi apparatus affect cellular function is poorly defined. Previously our lab has performed and analyzed published RNAi screens to identify genes required to maintain the Golgi ribbon. These candidate genes were filtered for altered expression patterns in breast cancer patients and for their association with survival data. The selected genes reevaluated by RNAi in BT549 breast cancer cells to assess the effect on Golgi fragmentation. One of the most effective hits was TPD52L2, a poorly characterised gene that encodes a membrane-associated protein that is overexpressed in various types of cancer. The aim of this project is to investigate the role of TPD52L2 in cell migration and cell invasion in breast cancer in order to understand the role of Golgi fragmentation in tumorigenesis. In random cell migration assays in dense and sparse seeding conditions, TPD52L2 depleted cells showed an altered actin cytoskeleton and were found to migrate faster in dense seeding without any change in directional persistence. These findings suggest that TPD52L2 not only is required to maintain an intact Golgi structure but also plays an important role in cellular motility and potentially metastasis. Citation Format: Seyedamirabbas Parizadeh, Stephan Geley, Veronika Reiterer-Farhan, Hesso Farhan. Role of TPD52L2 in cell migration and triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2404.

Combining metabolic phenotype determination with metabolomics and transcriptional analyses to reveal pathways regulated by hydroxycarboxylic acid receptor 2

BackgroundThe adaptation of cellular metabolism is considered a hallmark of cancer. Oncogenic signaling pathways support tumorigenesis and cancer progression through the induction of certain metabolic phenotypes associated with altered regulation of key metabolic enzymes. Hydroxycarboxylic acid receptor 2 (HCA2) is a G protein-coupled receptor previously shown to act as a tumor suppressor. Here, we aimed to unveil the connection between cellular metabolism and HCA2 in BT-474 cells. Moreover, we intend to clarify how well this metabolic phenotype is reflected in transcriptional changes and metabolite levels as determined by global metabolomics analyses.MethodsWe performed both, siRNA mediated knockdown of HCA2 and stimulation with the HCA2-specific agonist monomethyl fumarate. Seahorse technology was used to determine the role of HCA2 in BT-474 breast cancer cell metabolism and its potential to induce a switch in the metabolic phenotype in the presence of different energy substrates. Changes in the mRNA expression of metabolic enzymes were detected with real-time quantitative PCR (RT-qPCR). Untargeted liquid chromatography-mass spectrometry (LC-MS) metabolic profiling was used to determine changes in metabolite levels.ResultsKnockdown or stimulation of HCA2 induced changes in the metabolic phenotype of BT474 cells dependent on the availability of energy substrates. The presence of HCA2 was associated with increased glycolytic flux with no fatty acids available. This was reflected in the increased mRNA expression of the glycolytic enzymes PFKFB4 and PKM2, which are known to promote the Warburg effect and have been described as prognostic markers in different types of cancer. With exogenous palmitate present, HCA2 caused elevated fatty acid oxidation and likely lipolysis. The increase in lipolysis was also detectable at the transcriptional level of ATGL and the metabolite levels of palmitic and stearic acid.ConclusionsWe combined metabolic phenotype determination with metabolomics and transcriptional analyses and identified HCA2 as a regulator of glycolytic flux and fatty acid metabolism in BT-474 breast cancer cells. Thus, HCA2, for which agonists are already widely used to treat diseases such as psoriasis or hyperlipidemia, may prove useful as a target in combination cancer therapy.

Effect of CDCA5 on Proliferation and Metastasis of Triple Negative Breast Cancer Cells under shRNA Interference Technology.

Objective It was to explore the effect of cell division cycle associated 5 (CDCA5) under shRNA interference on proliferation and metastasis of triple negative breast cancer (TNBC) cells. Methods MDA-ME-231 and BT549 cells were selected as the research objects. According to the different interference methods and CDCA5 interference sequences, they were divided into the interference group 1MDA-ME-231, the interference group 2MDA-ME-231, the interference group 1BT549, the interference group 2BT549 (using shRNA technology), the control group MDA-ME-231, and the control group BT549 (breast cancer cells under normal culture conditions). MCF10A cells were routinely cultured as the negative control group to analyze the effect of CDCA5 expression on the proliferation and migration of cancer cells. Results The expression of CDCA5 protein in MDA-ME-231 and BT549 cells in control group was significantly higher than that in negative control group (P < 0.05). Compared with the control group, the inhibition rates of CDCA5 expression in 1MDA-ME-231, 2MDA-ME-231, 1BT549, and 2BT549 cells in the interference group were 39.01%, 42.98%, 49.57%, and 60.98%, respectively (P < 0.05). From 12 h, the proliferation level of TNBC cells at different culture time was lower than that of the control group (P < 0.05). Compared with the number of staining cells in the control group, the positive staining cells in 1MDA-ME-231 (61.42%), 2MDA-ME-231 (72.06%), 1BT549 (52.53%), and 2BT549 (59.65%) in the interference group were significantly decreased (P < 0.05). Conclusion The results show that the expression of CDCA5 in TNBC is increased, which plays an important role in the proliferation and migration of cancer cells. shRNA interference technology can knock down the expression of CDCA5 and inhibit its “promoting cancer” effect.

Novel Graphene Oxide/Quercetin and Graphene Oxide/Juglone Nanostructured Platforms as Effective Drug Delivery Systems with Biomedical Applications.

In this paper, novel drug delivery systems (DDS) were designed based on graphene oxide (GO) as nanocarrier, loaded with two natural substances (quercetin (Qu) and juglone (Ju)) at different concentrations. The chemical structure and morphology of the synthesized GO-based materials were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and Raman spectroscopy. The antibacterial activity was evaluated against standard strains, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Candida albicans ATCC 10231. Results demonstrated excellent antimicrobial activity, with a 5 log reduction of E. coli and a 1 log to 3.04 log reduction of S. aureus populations. Reduction rates were above 90%. Biocompatibility tests were also performed on GO-based materials, and the results showed biocompatible behavior for both L929 fibroblast cell line and BT474 breast cancer cells at lower concentrations. The identity of Qu and Ju was demonstrated by matrix-assisted laser desorption/ionization (MALDI) analysis, showing the compounds’ mass with high accuracy. In addition, specific properties of GO made it a versatile matrix for the MALDI analysis. The results of this study indicated that GO-based platforms may be suitable for applications in many areas for the effective and beneficial use of hydrophobic compounds such as Ju and Qu.

Photo-curing of GelMA A bioink is more preferable than chemical curing for creating 3D models of breast cancer tumor growth.

e15067 Background: Biogels based on natural components of the extracellular matrix are traditionally used to obtain 3D models of breast cancer (BC) tumor growth. However, such biogels are difficult to use in 3D bioprinting due to their poor shape retention and suboptimal curing conditions. Artificial biogels based on gelatin methacrylate and/or alginate have a good printability and can include natural components of the extracellular matrix to improve interaction with breast cancer cells. Nevertheless, for successful reconstruction of the tumor microenvironment, not only the composition but also the microstructure of the resulting models is important. The aim of the study was to investigate the effect of gelatin methacrylate and alginate composed bioink curing method on the microstructure of the resulting 3D construct and the morphology of breast cancer cells enclosed in it. Methods: BT20 breast cancer cells were mixed with GelMA A bioink (Cellink, USA) at a concentration of 106 cells/ml. The construct was printed on a BIO X bioprinter (Cellink, USA) at an ink temperature of 26°C, a printing table temperature of 10°C, a pressure of 8 kPa, a print speed of 10 mm/s, and a needle diameter of 22G. Next, the printed constructs were cured chemically by immersion in a 100 mM CaCl2 solution for 1 min or photo-cured by irradiating with light at a wavelength of 405 nm for 20 seconds from a distance of 5 cm. After washing, the constructs with encapsulated cells were incubated in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA) for two weeks, after which they were fixed in 10% formalin and embedded in paraffin blocks. The sections were H&amp;E stained and photographed at a magnification of 400X, then the area and roundness of the pores were determined using the ImageJ software. Results: During photo-curing of the printed construct, the pore area on the section averaged 1.5±1.07 µm2 (M±SD, n = 500), which is significantly less than with chemical curing (4.5±0.9 µm2, n = 500) (α = 0.05, df = 998). At the same time, the pores had an irregular shape (roundness 2.5±0.6, n = 500), which indicates their communication, in contrast to the chemically cured construct, the pores of which were almost perfectly round (roundness 1.2±0.2 M±SD, n = 500). BT20 culture cells encapsulated in bioink had an elongated process shape, as if squeezing through the fine-mesh structure of the light-cured construct, while in the chemically cured construct they had a rounded shape, not going beyond the boundaries of the pores. Conclusions: When creating 3D tumor growth models of breast cancer using bioinks based on gelatin methacrylate and alginate, photocuring is preferable, as it allows creating a spongy microstructure of communicating pores. Such a structure supports cell migration and helps maintain cell morphology close to that observed in vivo.

MiR-150 expression in breast cancer attracts and activates immune cells, and is associated with better patient outcome.

570 Background: MicroRNA (miRNA) epigenetically regulate a large number of cancer-related genes and is known as a key player in cancer biology. MiR-150 promote cancer cell proliferation, migration, and invasion. However, there has been no study that investigated the role of miR-150 in the tumor microenvironment (TME) of breast cancer patients. Methods: Total of 1,961 breast cancer patients from multiple independent large cohorts were analyzed. These observational results were validated by in vitro experiments. Lymphocyte attraction was assessed using Transwell system. Results: miR-150 expression was the highest in triple negative breast cancer (TNBC) among subtypes and correlated with Nottingham histological grade (all p&lt;0.001). A high miR-150 was significantly associated with better overall survival (OS) in both METABRIC and TCGA cohorts regardless of subtypes (all p&lt;0.05 except for TNBC in TCGA). MiR-150 expression was strongly correlated with Hallmark immune-related gene sets (Allograft rejection, IL6 signaling, IFN-γ response, Inflammatory response, IL2 signaling, and apoptosis), with cytolytic activity, with infiltration of CD8+ T cells, CD4+ memory T cells, and dendritic cells, as well as with gene expressions of major immune checkpoint molecules (PD-1, CTLA4, IDO1, TIGIT, BTLA, and LAG3), consistently in both METABRIC and TCGA cohorts (all r&gt;0.65, p&lt;0.01). Unexpectedly, overexpression of miR-150 by mimic did not increase growth, migration, nor invasion of neither MDA-MB231 or BT-549 breast cancer cell lines. On the other hand, overexpression of miR-150 in either MDA-MB231 or BT-549 significantly increased attraction of lymphocyte cell line (Jurkart cells), which was abolished by addition of miR-150 inhibitor. By comparing the gene expression profile between miR-150 overexpressed and control cells, we found that many inflammation related gene sets including NF-kB signaling that enhance cell migration was enriched to miR-150 overexpressed cells, implicating that is how miR-150 high cells attract lymphocytes to TME. Conclusions: miR-150 expression is associated with immune cell infiltration and immune response, as well as with better survival in breast cancer patients. Overexpression of miR-150 in breast cancer cells did not promote cancer cell proliferation, migration, nor invasion; however, increased attraction of lymphocytes. Multiple inflammation-related genes were expressed higher in miR150 overexpressed MDA-MB231.

Transcriptome profiling and proteomic validation reveals targets of the androgen receptor signaling in the BT-474 breast cancer cell line

BackgroundAccumulating evidence suggests that the androgen receptor (AR) and its endogenous ligands influence disease progression in breast cancer (BCa). However, AR-mediated changes in BCa differ among the various BCa subtypes according to their hormone receptor profile [i.e., presence/absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2, (HER2)]. Thus, we explored the androgen-regulated transcriptomic changes in the ER+PR+HER2+ BCa cell line, BT-474, and compared them with PR-mediated changes.MethodsWe performed RNA sequencing analysis in treated BT-474 cells with dihydrotestosterone (DHT) and progesterone. Validation of the top ten differentially androgen-regulated genes and a number of other genes found in enriched signaling pathways was performed by qRT-PCR in BT-474 and other BCa cell lines. In addition, a parallel reaction monitoring targeted proteomic approach was developed to verify selected transcripts at the protein level.ResultsIn total 19,450 transcripts were detected, of which 224 were differentially regulated after DHT treatment. The increased expression of two well-known androgen-regulated genes, KLK2 (p < 0.05) and KLK3 (p < 0.001), confirmed the successful androgen stimulation in BT-474 cells. The transcription factor, ZBTB16, was the most highly upregulated gene, with ~ 1000-fold change (p < 0.001). Pathway enrichment analysis revealed downregulation of the DNA replication processes (p < 0.05) and upregulation of the androgen signaling and fatty acid metabolism pathways (p < 0.05). Changes related to progesterone treatment showed opposite effects in gene expression than DHT treatment. Similar expression profiles were observed among other BCa cell lines expressing high levels of AR (ZR75.1 and MBA-MB-453). The parallel reaction monitoring targeted proteomic analysis further confirmed that altered protein expression (KLK3, ALOX15B) in the supernatant and cell lysate of DHT-treated BT-474 cells, compared to control cells.DiscussionOur findings suggest that AR modulates the metabolism of BT-474 cells by affecting the expression of a large number of genes and proteins. Based on further pathway analysis, we suggest that androgen receptor acts as a tumor suppressor in the BT-474 cells.

Gefitinib-Tamoxifen Hybrid Ligands as Potent Agents against Triple-Negative Breast Cancer.

Anticancer drug conjugates may benefit from simultaneous action at two targets potentially overcoming the drawbacks of current cancer treatment, such as insufficient efficacy, high toxicity, and development of resistance. Compared to a combination of two single-target drugs, they may offer an advantage of pharmacokinetic simplicity and fewer drug-drug interactions. Here, we report a series of compounds connecting tamoxifen or endoxifen with the EGFR-inhibitor gefitinib via a covalent linkage. These hybrid ligands retain both ER antagonist activity and EGFR inhibition. The most potent analogues exhibited single-digit nanomolar activities at both targets. The amide-linked endoxifen-gefitinib drug conjugates 17b and 17c demonstrated the most favorable anti-cancer profile in cellular viability assays on MCF7, MDA-MB-231, MDA-MB-468, and BT-549 breast cancer cells. Most importantly, in TNBC cells 17b and 17c displayed nanomolar IC50-values (380 nM - 970 nM) and were superior in their anti-cancer activity compared to their control compounds and combinations thereof.

Применение покрытия из силикона для получения клеточных сфероидов методом висячей капли

In modern experimental oncology, 3D cell cultures have become particularly important, as they provide more relevant results compared to traditional 2D cultures. The problem of obtaining relevant cell models remains urgent for the study of breast cancer. One of the most common techniques for obtaining a 3D culture is the hanging drop method. Researchers are constantly developing its modifications to reduce variations in the shape and size of the resulting cell spheroids. One of the ways to solve this problem is to apply a hydrophobic coating to the surface of cell culture plastics. Silicones or Parafilm® laboratory films are often used for this purpose. As a result, the curvature of the drop surface increases, which leads to accelerated aggregation of cells in the centre of the drop. The purpose of this research was to evaluate the possibility of applying a coating made of the silicone elastomer SIEL 159-330 (Russia) to modify the hanging drop method. Materials and methods. We used the SIEL 159-330 coating cured at a temperature lower than that recommended by the manufacturer and investigated its cytotoxic properties as well as its effect on the formation of cell spheroids in a hanging drop. The material for the study was the BT-474 breast cancer cell line. Results. The research found that the tested elastomer has no effect on cell viability. At the same time, SIEL 159-330, compared to polystyrene, significantly reduces the time of cell aggregate formation in the lower part of the drop. In addition, cell spheroids of the breast cancer culture obtained on the SIEL 159-330 coating vary less in shape and size than spheroids obtained on the polystyrene or Parafilm coating.

Antibody-Functionalized Carnauba Wax Nanoparticles to Target Breast Cancer Cells.

Development of safer nanomedicines for drug delivery applications requires immense efforts to improve clinical outcomes. Targeting a specific cell, biocompatibility and biodegradability are vital properties of a nanoparticle to fulfill the safety criteria in medical applications. Herein, we fabricate antibody-functionalized carnauba wax nanoparticles encapsulated a hydrophobic drug mimetic, which is potentially interesting for clinical use due to the inert and nontoxic properties of natural waxes. The nanoparticles are synthesized applying miniemulsion methods by solidifying molten wax droplets and further evaporating the solvent from the dispersion. The pH-selective adsorption of antibodies (IgG1, immunoglobulin G1, and CD340, an antihuman HER2 antibody) onto the nanoparticle surface is performed for practical and effective functionalization, which assists to overcome the complexity in chemical modification of carnauba wax. The adsorption behavior of the antibodies is studied using isothermal titration calorimetry (ITC), which gives thermodynamic parameters including the enthalpy, association constant, and stoichiometry of the functionalization process. Both antibodies exhibit strong binding at pH 2.7. The CD340-decorated wax nanoparticles show specific cell interaction toward BT474 breast cancer cells and retain the targeting function even after 6 months of storage period.

Complement-Mediated Selective Tumor Cell Lysis Enabled by Bi-Functional RNA Aptamers.

Unlike microbes that infect the human body, cancer cells are descended from normal cells and are not easily recognizable as “foreign” by the immune system of the host. However, if the malignant cells can be specifically earmarked for attack by a synthetic “designator”, the powerful effector mechanisms of the immune response can be conscripted to treat cancer. To implement this strategy, we have been developing aptamer-derived molecular adaptors to invoke synthetic immune responses against cancer cells. Here we describe multi-valent aptamers that simultaneously bind target molecules on the surface of cancer cells and an activated complement protein, which would tag the target molecules and their associated cells as “foreign” and trigger multiple effector mechanisms. Increased deposition of the complement proteins on the surface of cancer cells via aptamer binding to membrane targets could induce the formation of the membrane attack complex or cytotoxic degranulation by phagocytes and natural killer cells, thereby causing irreversible destruction of the targeted cells. Specifically, we designed and constructed a bi-functional aptamer linking EGFR and C3b/iC3b, and used it in a cell-based assay to cause lysis of MDA-MB-231 and BT-20 breast cancer cells, with either human or mouse serum as the source of complement factors.

Abstract P139: An estrogen receptor beta agonist liquiritigenin potentiates inhibition of hormone-dependent breast-cancer growth by cholesterol biosynthesis inhibitor RO 48-8071

Abstract Hormone-dependent breast cancer represents approximately 70% of all diagnosed breast cancers. Estrogens and progestins stimulate breast cancer cell proliferation, angiogenesis and metastasis. Estrogen receptor (ER)-alpha positive breast cancers are treated with anti-estrogens and aromatase inhibitors, but resistance to these agents invariably develops during the course of therapy. Our goal was to identify novel treatment strategies to suppress breast cancer growth and avoid drug resistance. Cholesterol biosynthesis plays a vital role in tumor cell growth. Cholesterol is an integral part of membrane structure and is also a precursor of steroid hormones that are synthesized by tumor cells and cause cellular proliferation by interacting with specific receptors. Cancer cells have elevated levels of cholesterol, which could be responsible for anti-hormone resistance. During studies to determine the effects of the cholesterol biosynthesis inhibitor RO 48-8071 ([4’-[6-(Allylmethylamino)hexyloxy]-4-bromo-2’-fluorobenzophenone fumarate] (RO), which inhibits oxidosqualene cyclase (OSC), an enzyme that acts downstream of HMG-CoA reductase to convert 2, 3-monoepoxysqualene to lanosterol (a key step in the biosynthesis of cholesterol), it was serendipitously discovered that RO degraded ER-alpha (ERa), and elevated levels of ER-beta (ERb), a known tumor suppressor. We tested RO alone and in combination with liquiritigenin (LQ), a selective agonist for ERb, to ascertain their inhibitory effects against tumor cell progression. RO or LQ alone reduced significantly the in vitro viability of MCF-7 and BT-474 breast-cancer cells. BT-474 cells were more sensitive than MCF-7 cells to LQ (IC50 0.15 uM vs 0.25 mM, respectively). RO (5-10 uM) + LQ (0.1-0.3 mM) treatment reduced cell viability in an additive manner in MCF-7 cells and in a synergistic manner in BT-474 cells. Combination treatment was significantly more effective than treatment with a single agent. Administration of RO (10 mg/kg/iv) or LQ (20 mg/kg/ip) significantly inhibited growth of tamoxifen-resistant BT-474 tumor xenografts in vivo; combination treatment was even more effective. Both agents appeared to be non-toxic in vivo. Mechanistically, RO, LQ, or RO + LQ reduced ERa but induced ERb expression in tumor xenografts. Both compounds significantly reduced expression of markers of angiogenesis and increased apoptosis in tumor xenografts; use of RO + LQ significantly enhanced the effects observed with a single agent. The anti-tumor properties of RO may in part be due to an off-target effect that reduces ERa and increases ERb, the latter then interacting with LQ to more effectively promote its anti-proliferative effects. The potent anti-tumor properties of RO and LQ, particularly when used together, suggest that this combination has substantial potential as a means by which to clinically manage breast-cancer growth, including in disease types that are tamoxifen resistant. Citation Format: Yayun Liang, Salman Hyder. An estrogen receptor beta agonist liquiritigenin potentiates inhibition of hormone-dependent breast-cancer growth by cholesterol biosynthesis inhibitor RO 48-8071 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P139.

Design and Characterization of Liposomal Methotrexate and Its Effect on BT-474 Breast Cancer Cell Line.

Background: Breast cancer is the most common type of cancer among women worldwide. Traditional treatments, including chemotherapy, surgery, mastectomy, and radiotherapy, are commonly used. Because of the limitation of the aforementioned methods, novel treatment strategies are needed. Methotrexate is a chemotherapeutic drug, which is commonly used to treat breast cancer. Because of the side effects of the free drug, the liposomal form of the drug is suggested. Methods: Liposomal methotrexate was prepared and the encapsulation efficiency was measured. Moreover, the particle size and the zeta potential were measured. The liposome morphology was confirmed using transmission electron microscopy. The MTT assay was done to examine the cytotoxicity of free and encapsulated methotrexate on BT-474 cell line. The Annexin-V/PI dual staining assay was performed to assess the apoptosis in BT-474 breast cancer cells via the flow cytometry method. Results: The transmission electron microscopy results confirmed the integrated and spherical structure of the nanoparticles. The results of drug release showed that in acidic pH (5.4), more than 90% of the drug was released after 24 hours, which was higher than 2 other pHs. Furthermore, the IC50 value of liposomal methotrexate was determined as 2.15 and 0.82 mg/mL for 24 and 48 hours. The flow cytometry results confirmed that liposomal methotrexate had a greater cytotoxic effect on cancer cells compared with free methotrexate. Conclusion: Because of the advantages of liposomal based nanocarriers, in this study, liposomal methotrexate could be suggested as an appropriate candidate to treat breast cancer.

Combined Evaluation of MAP1LC3B and SQSTM1 for Biological and Clinical Significance in Ductal Carcinoma of Breast Cancer.

Breast cancer is the leading cause of cancer death in women worldwide. The microtubule-associated protein light chain 3B (MAP1LC3B) and adaptor sequestosome 1 (SQSTM1) are two major markers for autophagy. Increased protein levels of MAP1LC3B and SQSTM1 are considered to be causes of autophagy inhibition or activation in various types of cancers. However, the roles of MAP1LC3B and SQSTM1 in breast cancer are still not clear. Using a tissue microarray from 274 breast invasive ductal carcinoma (IDC) patients, we found that tumor tissues showed higher protein levels of MAP1LC3B and cytoplasmic SQSTM1 in comparison to those in adjacent normal tissues. Moreover, high levels of MAP1LC3B were associated with better survival, including disease-specific survival and disease-free survival (DFS) in IDC patients. Furthermore, high co-expression of MAP1LC3B and SQSTM1 was significantly associated with better DFS in IDC patients. Astonishingly, the autophagy inhibitor accumulated the protein levels of MAP1LC3B/SQSTM1 and enhanced the cytotoxic effects of cisplatin and paclitaxel in MCF7 and BT474 breast cancer cell lines, implying that autophagy inhibition might result in poor prognosis and chemosensitivity in IDC. Taken together, high co-expression of MAP1LC3B and SQSTM1 might serve as a potential diagnostic and prognostic biomarker for IDC patients.

Evaluation of protein functionalized gold nanoparticles to improve tamoxifen delivery: synthesis, characterization, and biocompatibility on breast cancer cells

In the study, human serum albumin functionalized gold nanoparticles (HSA-AuNPs) were fabricated benefiting from the unique properties of HSA and AuNP to achieve tamoxifen delivery to breast cancer cells for renewing tumor targeting with unconventional nanoparticles. The release pattern of tamoxifen (TMX), the cell viability, oxidative DNA damage, and total antioxidant capacity of HSA-AuNP with and without TMX were assessed on BT-474 and MDA MB-231 breast cancer cell lines. The outcomes indicated that TMX entrapped HSA-AuNP can induce cancer cell death in a dose-dependent aspect and could be proved via further studies as a potential drug delivery agent.