SKBR3 Cells Research Articles

Accurate assessment of DNA strand breaks is essential for evaluating the efficacy of radiation therapy, yet most existing methods rely on indirect detection of reactive oxygen species (ROS), which are unreliable in hypoxic tumor environments and do not consistently correlate with DNA damage. Here, we report a nuclease-resistant, L-DNA-based Förster resonance energy transfer (FRET) probe that directly detects radiation-induced DNA strand cleavage. The probes consist of short single-stranded L-DNA labeled with FAM and TAMRA fluorophores, designed to lose FRET upon strand scission. Both two- and six-thymidine variants (L-T2 and L-T6) were synthesized and shown to resist enzymatic degradation while maintaining fluorescence under irradiation up to 50 Gy. Radiation exposure induces a dose-dependent increase in donor fluorescence, with L-T2 exhibiting greater sensitivity in physiological buffer. The probes are quantitatively responsive, with detectable signal shifts from as little as 1% cleavage and a linear relationship between donor/acceptor emission ratios and DNA breakage. In cells, however, the behavior is more complex. While L-T6 exhibits dose-dependent FRET loss in certain contexts, particularly in radiation-sensitive SKBR3 cells, the extent of cleavage is significantly reduced compared to in vitro conditions. Glutathione depletion failed to enhance intracellular cleavage, suggesting that other mechanisms of protection or sequestration are at play. These findings highlight the challenges of sensing strand scission in the cytoplasm and point to unanticipated barriers to detecting radiation-induced damage in living cells.

BackgroundBreast cancer remains a leading cause of cancer-related mortality in women globally. The main purpose of the research to develop, optimise and characterise a trastuzumab (TZ)-functionalized nanolipid carrier (NCs) encapsulating capecitabine, as a targeted strategy to breast cancer cells, to enhance therapeutic efficacy and reduce the severe side effects associated with conventional chemotherapy.MethodsCapecitabine encapsulated NCs (CBNCs) were prepared by thin-film hydration technique, optimized by Box-Behnken design. The optimized formulation CBNCs were subsequently conjugated with TZ by using EDC-NHS chemistry. The prepared formulations of NCs were evaluated by FTIR, DSC, XRD, FESEM, TEM, AFM, drug loading, entrapment efficiency, average particle size, PDI, zeta potential, in vitro drug release. The successful surface conjugation of TZ was tested by BCA assay and SDS-PAGE analysis. In vitro targeting efficiency and cytotoxicity initially tested in MCF-7 cells (HER2-low expressing) and subsequently validated in SKBR3 cells (HER2-overexpressing) to confirm receptor-mediated uptake and specificity.ResultsOptimized CBNCs were found spherical, nanosized (194.6 nm), with a zeta potential −25.55 mV for CBNCs, which increased to –57.76 mV upon TZ conjugation. The formulation showed 8.5% drug loading capacity and 84.26% drug release over 72 h. FTIR and DSC showed compatibility of drug and lipid components with no major shifting in characteristic peaks. TEM and AFM confirmed formation of stable, spherical discrete nanostructures. TZ conjugation showed minor alternation in average size/surface charge/morphology/texture. Successful TZ conjugation onto CBNCs was confirmed by BCA assay and SDS-PAGE. Fluorescence microscopy confirmed successful cellular internalization. MTT assay on SKBR3 cells demonstrated significantly higher cytotoxicity for TZ-CBNCs compared to CBNCs and free drug, thereby validating the HER2-specific targeting effect beyond preliminary results obtained in MCF-7 cells.ConclusionIn view of the desired physicochemical properties, controlled drug release, and in vitro anticancer effectiveness, further in vivo investigations should be prioritized to validate its clinical application in HER2-positive breast cancer treatment. Nonetheless, the use of HER2-low MCF-7 cells in early assays highlights the importance of complementary validation in HER2-overexpressing models, as addressed by SKBR3 testing in this study.

Background/Objectives: This study developed a targeted drug delivery nanoplatform for treating HER2-positive breast cancer. The nanoplatform encapsulated two hydrophobic anticancer agents, neratinib (NTB) and docetaxel (DTX), within nanoparticles (DTX+NTB−NP) functionalized for conjugation to trastuzumab to form trastuzumab-tagged nanoparticles (TRZ−NP). Trastuzumab is a HER2-specific monoclo-nal antibody that binds to HER2 receptors, blocking signal transduction and inducing an-tibody-dependent cellular cytotoxicity (ADCC). Upon receptor-mediated endocytosis, neratinib inhibits cytosolic HER2 signaling, while docetaxel disrupts mitotic cell division, collectively leading to tumor cell death. Methods: Nanoparticles were fabricated by the nanoprecipitation technique, followed by surface modification with a crosslinker and a targeting moiety. DTX+NTB−NP, TRZ−NP, and singly loaded nanoparticles (NTB−NP and DTX−NP) were characterized and their effects evaluated in HER2-positive cancer cell line and xenograft model. Results: In vitro antiproliferation assay in SKBR-3 cell line re-veals a dose and time-dependent cytotoxicity. There was no significant difference in cyto-toxicity observed between DTX+NTB−NP and its free form (DTX+NTB) [p = 0.9172], and between TRZ−NP and its free form (TRZ+DTX+NTB) [p = 0.6750]. However, TRZ−NP, at half the concentration of the singly loaded nanoparticles, significantly reduced the viabil-ity of SKBR-3 cells compared to pure trastuzumab (TRZ) [p < 0.001], NTB−NP [p = 0.0019], and DTX−NP [p = 0.0002]. In vivo evaluation in female athymic nude mice showed sig-nificant log relative tumor volume (%) reduction in groups treated with TRZ−NP and DTX+NTB−NP compared to PBS (phosphate-buffered saline) controls (p ≤ 0.001 and p ≤ 0.001), respectively. Notably, TRZ−NP demonstrated a statistically significant regression in the log relative tumor volume (%) compared to DTX+NTB−NP (p = 0.001). Conclusions: These findings underscore the therapeutic potential and suitability of these nanoplatforms for the precise and controlled targeting of HER2-positive tumors. This study is the first to synchronize the delivery of multiple agents-docetaxel, neratinib, and trastuzumab-within a nanoparticle system for treating HER2-positive tumors, offering a promising strategy to enhance treatment outcomes for HER2 positive breast cancer patients.

BackgroundBreast cancer remains one of the most prevalent and lethal cancers among women, with a notable risk of recurrence and metastasis. Meteorin (METRN) is a newly discovered cytokine that plays a critical role in neurodevelopment and immune regulation. However, its involvement in breast cancer, particularly in modulating the immune microenvironment, is not well understood. This study aims to investigate the role of METRN in breast cancer progression and to elucidate its potential function in regulating interleukin-6 (IL-6)-mediated cluster of differentiation 8 positive (CD8+) T cell activity.MethodsWe used bioinformatics tools to analyze METRN’s diagnostic and prognostic value in breast cancer, validated the findings with clinical samples, and examined METRN’s effects on cell behavior using MCF-7, MDA-MB-231, and SKBR3 cell lines. We assessed cell proliferation, migration, invasion, and apoptosis, and studied METRN’s interaction with IL-6 in modulating CD8+ T cell function using enzyme-linked immunosorbent assay (ELISA) and quantitative PCR (qPCR). In vivo, tumor growth was evaluated in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, which lack adaptive immunity and thus reflect tumor cell-intrinsic effects.ResultsBioinformatics analysis showed that METRN was highly expressed in breast cancer and correlated with poor prognosis. METRN inhibition reduced tumor cell proliferation, migration and invasion, while increasing apoptosis. In vitro co-culture assays showed that METRN knockdown increased IL-6 production and enhanced CD8+ T-cell effector function, whereas IL-6 silencing abrogated these effects. CD8 staining was also performed and was shown for completeness. However, given that NOD/SCID mice lacked adaptive immunity, these signals cannot be interpreted as functional CD8+ T-cell infiltration, but were presented as technical staining results.ConclusionsMETRN promotes breast cancer progression through tumor-intrinsic mechanisms, while in vitro and bioinformatics analyses further indicate that METRN regulates IL-6-linked CD8+ T-cell activity. These findings highlight METRN as a potential biomarker and therapeutic target in breast cancer, while further validation in immunocompetent or humanized mouse models will be required to assess its role in immunotherapy.

Background: Recent evidence suggests that alterations in circadian rhythm genes may lead to circadian rhythm disruption (CRD), which is a key mechanism in the progression of breast cancer. Therefore, investigating the role of circadian rhythm genes in the prognosis of breast cancer holds significant clinical value.Materials and Methods: We utilized expression profile data from the Gene Expression Omnibus (GEO) database to identify cancer features closely associated with CRD in breast cancer. Then, we analyzed publicly available datasets (including GEO, TCGA, and METABRIC) to identify alterations in core circadian genes significantly associated with patient survival across breast cancer and constructed a circadian‐related gene signature (CGS) based on these prognostic cancer features.Results: Circadian rhythm–related genes (CRGs) were selected to construct a risk gene signature associated with individual prognosis, which was validated in six independent cohorts and demonstrated good predictive ability. We identified three circadian rhythm–associated subtypes with distinct prognoses, which exhibited significant differences in immune checkpoint molecules, drug sensitivity, and molecular features. Additionally, the gene signature and clinicopathologic features were integrated to develop a risk model with enhanced predictive accuracy. To validate the functional role of signature genes, BMAL1 knockdown in SKBR3 cells disrupted circadian rhythms, with qPCR confirming altered risk gene expression. We found that the nomogram exhibited superior discriminative ability compared to the traditional breast cancer staging system.Conclusion: We developed a nomogram that can accurately predict the prognosis of breast cancer, and conclude that the expression of CRGs is crucial in breast cancer treatment decisions.

G protein-coupled estrogen receptor reduces the breast cancer cell survival by regulating the IRE1α/miR-17-5p/ TXNIP pathway.

Cinnamaldehyde is a natural compound known for its antimicrobial and anticancer properties. Fourteen novel cinnamaldehyde-chalcone analogues (5a-5n) were synthesized and evaluated for anti-cancer, anti-bacterial, and anti-fungal activities. Among these, bromoethane chalcone 5n exhibited significant cytotoxicity against DU145 (IC50: 8.719 ± 1.8 μM), SKBR-3 (IC50: 7.689 μM), and HEPG2 (IC50: 9.380 ± 1.6 μM) cell lines, surpassing other derivatives. Compounds para methyl benzyl chalcone 5j and 2,3-dichloro benzyl chalcone 5b also demonstrated notable activity against SKBR-3 (IC50 7.871 μM) and HEPG2 (IC50 9.190 μM) cell lines. Erythrocyte osmotic fragility (EOF) analysis showed higher erythrocyte fragility for 5n (MEF50 = 0.457) and 5b (MEF50 = 0.538), indicating membrane-disruptive potential compared to quercetin (MEF50 = 0.431). Studies on antimicrobial activity revealed that compounds 5a-5e and 5n demonstrated moderate effectiveness against Staphylococcus aureus, while compound 5l showed activity against Candida albicans and Candida tropicalis. Docking studies revealed that compound 5n binds to succinate dehydrogenase, a key enzyme in the TCA cycle, ETC, with greater affinity (-12.9 kcal mol-1) than the standard inhibitor, malonate (-4.8 kcal mol-1). Acute oral toxicity assessment of 5n in Swiss albino mice demonstrated its safety at doses up to 1000 mg kg-1 body weight with no morbidity, mortality, or significant changes in haematological, biochemical, and pathological parameters. These findings highlight 5n's potential as a lead compound for further preclinical studies targeting cancer therapeutics.

To study the molecular mechanisms of LDH-loaded si-NEAT1 for regulating paclitaxel resistance and tumor-associated macrophage (TAM) polarization in breast cancer. qRT-PCR and Western blotting were used to detect the expression of lncRNA NEAT1, miR-133b, and PD-L1 in breast cancer SKBR3 cells and paclitaxel-resistant SKBR3 cells (SKBR3-PR). The effects of transfection with si-NEAT1 and miR-133b mimics on MRP, MCRP and PD-L1 expressions and cell proliferation, migration and apoptosis were investigated using qRT-PCR, Western blotting, scratch and Transwell assays, and flow cytometry. Rescue experiments were conducted using si-NEAT1 and miR-133b inhibitor. Human THP-1 macrophages were cultured in the presence of conditioned media (CM) derived from SKBR3 and SKBR3-PR cells with or with si-NEAT1 transfection for comparison of IL-4-induced macrophage polarization by detecting the surface markers. LDH@si-NEAT1 nanocarriers were constructed, and their effects on MRP, MCRP and PD-L1 expressions and cell behaviors of the tumor cells were examined. THP-1 cells were treated with the CM from LDH@si-NEAT1-treated tumor cells, and the changes in their polarization were assessed. SKBR3-PR cells showered significantly upregulated NEAT1 and PD-L1 expressions and lowered miR-133b expression as compared with their parental cells. Transfection with si-NEAT1 and miR-133b mimics inhibited viability, promoted apoptosis and enhanced MRP and BCRP expressions in SKBR3-PR cells. NEAT1 knockdown obvious upregulated miR-133b and downregulated PD-L1, MRP and BCRP expressions. The CM from SKBR3-PR cells obviously promoted M2 polarization of THP-1 macrophages, which was significantly inhibited by CM from si-NEAT1-transfected cells. Treatment with LDH@si-NEAT1 effectively inhibited migration and invasion, promoted apoptosis, and reduced MRP, BCRP and PD-L1 expressions in the tumor cells. The CM from LDH@si-NEAT1-treated SKBR3-PR cells significantly downregulated Arg-1, CD163, IL-10, and PD-L1 and upregulated miR-133b expression in THP-1 macrophages. LDH@si-NEAT1 reduces paclitaxel resistance of breast cancer cells and inhibits TAM polarization by targeting the miR-133b/PD-L1 axis.

Chimeric antigen receptor (CAR)-engineered NK cells are a promising approach for targeted immunotherapy in Her2-positive cancers. This study aimed to generate anti-Her2 CAR-NK92 cells, to evaluate their selective cytotoxicity against Her2-positive cancer cells, and to isolate and characterize their released exosomes. NK92 cells were electroporated with piggyBac transposon vectors encoding anti-Her2 CAR and the helper transposase. Puromycin selection was performed to enrich the transduced cells. CAR and GFP expression were assessed by flow cytometry, and exosomes were isolated and characterized in terms of protein cargo and surface protein expression. Cytotoxicity was evaluated using real-time cell analysis against Her2-positive SK-BR3 cells and Her2-negative MCF-7 cells. Electroporation did not significantly affect NK92 cell viability. Puromycin selection efficiently enriched for CAR-expressing cells, with GFP positivity reaching 99.8% and a 15-fold increase in CAR surface expression compared to wild-type cells. CAR-NK92 cells demonstrated robust, Her2-specific cytotoxicity in a E:T-dependent manner, with the greatest effect observed at a 10:1 effector-to-target ratio. Exosomes derived from CAR-NK92 cells contained CAR molecules and selectively targeted Her2-positive cells. Anti-Her2 CAR-NK92 cells and their exosomes exhibit potent and selective cytotoxicity against Her2-positive cancer cells, supporting their potential as innovative immunotherapeutic agents for solid tumors.

Glutaminase-driven ZnO nano-biohybrids as smart protein scaffolds for targeted and precision therapeutics in breast cancer.

Purpose: The incorporation of Nanoparticles (NPs) in Computed Tomography (CT) imaging significantly enhances the contrast, clarity, and sensitivity of CT scans, leading to a substantial improvement in the accuracy and reliability of diagnostic information obtained from the images. The objective of the current research was to investigate the application of gold (Au) NPs in enhancing the imaging capabilities of Breast Cancer (BC) cells. Materials and Methods: Au NPs were synthesized by loading Trastuzumab (TZ) on PEGylated Au NPs. Firstly, Au NPs were produced and coated with PEG-SH to form PEG-Au NPs. Next, TZ was coupled with OPSS-PEG-SVA to enable its attachment to the PEG-Au NPs. The resulting NPs were characterized for their structure, size, and morphology using standard analytical techniques. To assess the potential of the developed NPs for CT scan imaging of BC cells, SKBr-3 cells were treated with Au NPs and TZ-PEG-Au NPs. Additionally, the cytotoxicity of the NPs was evaluated with the MTT technique. Results: The SEM and TEM analyses revealed that the synthesized NPs exhibited a spherical shape and displayed a relatively uniform size distribution (approximately 45 nm). The results showed that the developed Au NPs have acceptable biocompatibility and superior X-ray attenuation properties compared to a commonly used contrast agent. Conclusion: Based on our results, it can be concluded that the proposed TZ- Polyethylene Glycol-Au NPs are suitable for CT imaging of BC cells.

The effects of a methanol extract of Nymphaea odorata (MeNO) rhizomes, its fractions and the active compound (methyl gallate, MeG) were investigated in estrogen receptor-positive (ER+) breast cancer cell lines MCF-7 and T47-D:A18, as well as ER-negative line SKBr3. Cell viability and cytotoxicity were determined using CellTiter-Glo® 2.0 assays at concentrations ranging from 1 to 100 μg/mL. Caspase activity and apoptosis were determined using Caspase-Glo® 3/7, Caspase-Glo® 8, and ApoTox-Glo™ triplex assays, as well as qPCR. Total RNA was isolated from MCF-7 cells treated with MeG. RNA-seq libraries were prepared using a Universal Plus mRNASeq kit, and sequencing was performed on a NovaSeq 6000. MeNO inhibited the growth of MCF-7 cells with an IC50 of 14.1 μg/mL, as well as T47-D:A18 (IC50 of 25.6 μg/mL) and SKBr3 cells (IC50 of 35.5 μg/mL). Bioassay-guided fractionation of MeNO in MCF-7 cells identified the active fraction containing one compound, namely methyl gallate (MeG). MeG had an IC50 of 8.6 μg/mL in MCF-7 cells. Transcriptomic analysis of MeG-treated MCF-7 cells showed differential expression of 10,634 genes, with 5643 upregulated and 4991 downregulated (FDR < 0.05). Ingenuity pathway analysis revealed the involvement of 43 canonical pathways, with the top upregulated pathways including apoptosis, autophagy, and the unfolded protein response pathways.

Background/Objectives: The dual targeting of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) represents an effective approach for cancer treatment. The current study involved the design, synthesis, and biological evaluation of a new series of purine-containing hydrazones, 6–24 (a,b), as anticancer agents targeting EGFR and HER2 kinases. Methods: The proposed compounds were initially screened in silico using molecular docking to investigate their binding affinity to the active sites of EGFR and HER2 kinase domains. Subsequently, the compounds were synthesized and evaluated in vitro for their antiproliferative activity, using the MTT assay, against the various cancer cell lines A549, SKOV-3, A2780, and SKBR-3, with lapatinib as the reference drug. The most active derivatives were then examined to determine their inhibitory activity against EGFR and HER2 kinases. Results: Among the assessed compounds, significant antiproliferative activity was demonstrated by 19a, 16b, and 22b. 19a exhibited substantial anticancer efficacy against A549 and SKBR-3, with IC50 values of 0.81 µM and 1.41 µM, respectively. This activity surpassed lapatinib, which has an IC50 of 11.57 µM on A549 and 8.54 µM on SKBR-3 cells. Furthermore, 19a, 16b, and 22b exhibited superior EGFR inhibitory efficacy compared with lapatinib (IC50 = 0.13 µM), with IC50 values of 0.08, 0.06, and 0.07 µM, respectively. Regarding HER2, 22b demonstrated the greatest potency with an IC50 of 0.03 µM, equipotent to lapatinib (IC50 = 0.03 µM). Flow cytometry analysis of A549 cells treated with 19a and 22b indicated their ability to arrest the cell cycle during the G1 phase and to trigger cellular apoptosis. Conclusions: Compounds 19a, 16b, and 22b represent intriguing candidates for the development of an anticancer agent targeting EGFR and HER2 kinases.

Through network pharmacological prediction and in vitro experimental verification, the mechanism of action of Lianqiao Jinbei Decoction (LJD) in inhibiting HER2-positive breast cancer cells was clarified, providing experimental evidence for its treatment of HER2-positive breast cancer. Network pharmacology method was used to construct the potential target network of LJD in the treatment of HER2+ breast cancer. After cell culture in vitro, the proliferation of HER2+ SK-BR3 breast cancer cells was investigated using CCK-8 technique. The apoptotic potential of SK-BR3 cells was detected by flow cytometry, and the migration of SK-BR3 cells was detected by cell scratch assay. The expression of HER2 protein in SK-BR3 breast cancer cells was detected by ELISA. HER2 was identified as the central gene and quercetin, β-sitosterol, and luteolin were the primary active ingredients using network pharmacology analysis. Serum-containing LJD medication can stop SK-BR3 cells from proliferating (P<0.05). Serum-containing LJD drugs at high, medium, and low concentrations may induce SK-BR3 cell death (P<0.05). LJD serum at high, medium, and low concentrations reduced the migration of SK-BR3 cells (P<0.05). The expression of HER2 protein was decreased by LJD high, medium, and low concentration drug-containing serum (P<0.05). Regarding treating HER2-positive breast cancer, LJD has a multi-component, multi-target, and multi-pathway mode of action. The primary target of LJD's activity is the HER2 protein. Serum-containing LJD medication can prevent SK-BR3 cells from proliferating and migrating while encouraging their apoptosis. This effect may be attained by preventing HER2 protein expression.

Multivalency can drive high-avidity binding of ligand-functionalized nanoparticles to cells with high target receptor expression, but it can also contribute to off-target binding to low-expression non-target cells. We explored how ligand affinity and liposome valency shape the resulting binding performance index (BPI), defined as the product of the proportion of liposome-bound target cells and that of non-bound non-target cells. Designed ankyrin repeat proteins (DARPins) spanning a wide range of HER2-binding affinities were tethered onto PEGylated liposomes at varying concentrations. BPI was initially evaluated in mixed-cell suspensions of HER2high SKBR3 (target) cells and HER2low T47D (non-target) cells, with the highest BPI (> 0.8) observed for high-valency liposomes displaying high-affinity DARPins. To further map the BPI landscape, we measured particle binding to HEK293T cells transiently transfected with HER2-EGFP, leveraging the inherent transfection heterogeneity to generate continuous binding response curves as a function of HER2 expression. HER2high (target) and HER2low (non-target) populations were defined by a HER2 threshold, which was varied across the range of HER2 expression to determine maximum BPI values (> 0.85) and corresponding HER2 threshold optima (HER2OPT). BPI generally tracks with traditional binding selectivity, but BPI is more sensitive to off-target effects or poor on-target binding and thus may better assess particle performance. We further demonstrate that HER2OPT can be rationally increased or decreased by adjusting DARPin valency and affinity (separately or synergistically) to lower or higher values, respectively. The approach outlined here enables rapid testing and optimization of ligand parameters for nanoparticle binding toward a given therapeutic target.

Abstract Background: Metastatic breast cancer (MBC) is considered an incurable disease and understanding the molecular mechanisms at the basis of tumor progression has become mandatory. Circulating tumor cells (CTCs) constitute a population of rare cells that are shed in the bloodstream by primary and metastatic tumors and migrate towards distant organs. The molecular characterization of CTCs is a powerful tool to better investigate the mechanisms underlying metastasis. In this study, we combine phenotypic and transcriptional profiling of CTCs isolated from MBC patients to infer the mechanisms involved in their biology, also in relation to the metastatic site and their phenotypic features. Methods: Blood samples for CTC analysis were collected from luminal MBC patients recruited in the KENDO study (Protocol code: IRST174.19); CTCs were enriched using the RosetteSep CTC Enrichment Cocktail and frozen until downstream analyses as viable cells. To validate the antibody staining for phenotypic analysis, tests were conducted using the cancer cell line SKBR-3. In brief, SKBR-3 cells were spiked in peripheral blood of a healthy volunteer and enriched using RosetteSep CTC Enrichment Cocktail. Enriched SKBR-3 and patients’ CTCs were incubated with antibodies targeting CD45 (leukocyte marker) and epithelial markers (E-tag; EpCAM, E-cadherin) for phenotypic analysis using the DEPArray NxT platform. For transcriptomic profiling, single CTCs were isolated as single, viable cells. Libraries were prepared using the QIAseq UPX 3’ Transcriptome Kit and sequenced using MiSeq on V3-150 cycles cartridges. Bioinformatic and statistical analyses were carried out using the CLC Genomic Workbench. Results: Through feasibility analysis conducted on SKBR-3 cells, we demonstrate that our workflow involving phenotypic analysis on DEPArray NxT is an efficient tool to identify cancer cells (CD45-/E-tag+) and white blood cells (WBCs; CD45+/E-tag-). In MBC samples, we detected different cell populations: WBCs (CD45+/E-tag-), CTCs (CD45-/E-tag+), and dual-positive CTCs (CD45+/E-tag+; dpCTCs). Transcriptomic profiling was successfully performed on 37 single CTCs isolated from 7 patients. The transcriptional profile of CTCs is consistent with breast malignancy for different databases including ClinVar2019, DisGeNet, and Human Phenotype Ontology. Moreover, Gene Set Enrichment analysis highlighted pathways associated with synapse organization and calcium channel activity, suggesting the implication of the interplay between cancer cells and neurons in metastasis. Compared to a commercial RNA, CTCs express at high levels transcripts associated with cancer dissemination and genome architecture (i.e., STAG2 and H2AFZ). In CTCs from patients with bone metastasis we detected the expression of genes associated with metastatic osteotropism (i.e., S100A4, VAPA, HMGB1), while CTCs from a single patient with gastric metastasis expressed genes associated with gastric cancer metastasis and cell redox homeostasis. Transcriptional profiling of dpCTCs revealed the expression of transcripts encoding for members of the CD47/SIRPα axis, supporting their macrophage-cancer cell fusion origin, and increased glycogen biosynthetic and metabolic activity. Conclusions: Our data suggest the potential of CTC based transcriptomics in providing new insights on the clinical-biological evaluation of MBC and its mechanisms underlying metastatic cascade and organotropism. In addition, our data provide hints for the characterization of dpCTCs, a poorly studied population whose role in metastasis is still far to be fully elucidated. Citation Format: Michela Palleschi, Tania Rossi, Sara Bandini, Michele Zanoni, Michela Cortesi, Erika Bandini, Andrea Rocca, Giulia Gallerani, Ivan Vannini, Meropi Plousiou, Lorenzo Gerratana, Giovanni Tallini, Giovanni Martinelli, Ugo De Giorgi, Paola Ulivi. Transcriptomic and phenotypic profiling of circulating tumor cells from metastatic breast cancer patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-01-17.

Abstract Background: Triple Negative Breast Cancer (TNBC) continues to be the deadliest subtype of breast cancer. There is an urgent need to identify novel molecular targets to be exploited. We previously identified the small G proteins Ras-Like Oncoproteins A and B, or RALA and RALB, as novel molecular targets of TNBC. The RALs, particularly RALA, were critical for tumor growth and metastasis in multiple models of TNBC. More recently, we found that TNBC cell lines are reliant on RALA and RALB expression. However, current generation RAL inhibitors are not suitable for clinical applications. There is an urgent need for better RAL inhibitors. Here, we show the discovery and validation of a novel RAL inhibitor and its efficacy in TNBC models. Methods: Molecular docking studies were completed with drug-like and lead-like molecules from the ZINC15 database and 3D models for inactive GDP-RALA (Protein Data Bank, - 2BOV). Virtual screening was performed with 500,000 molecules targeting the allosteric binding pocket of the RALA-GDP crystal structure and the homology models of RALB-GDP using Autodock. Surface Plasmon Resonance (SPR) was performed on the top 54 compounds (Reaction Biology). GTP-Pulldown assays were performed using a RAL-Activation Kits (BK040, Cytoskeleton, Inc. and #17-300 MilliPore-Sigma). Viability assays were perform using MTT (MilliPore-Sigma, 11465007001) and Growth in Low Adhesion (GILA) (G9682, Promega). Small G-Protein Assays were performed against RAS (BK008, Cytoskeleton), RAC/CDC42 (BK035, Cytoskeleton), RHOA (BK036, Cytoskeleton), and ARF (BK032-S, Cytoskeleton). For the measuring in vivo efficacy of OSURALi, MDA-MB-231 tumors in the mammary fat pad were allowed to reach ∼100mm3 before being randomized into DMSO and OSURALi groups. Mice were injected with 50mg/kg OSURALi 5x/week. When early removal criteria were met, all mice were sacrificed, perfused, and organs were harvested for histological analysis. Results: We identified a novel small molecule inhibitor we have named OSURALi. We assessed the efficacy of OSURALi against breast cancer cell lines and found OSURALi to be potent against RALA-dependent TNBC cell lines while RALA-independent SKBR3 cells and normal cell lines were more resistant to OSURALi. Current generation RAL inhibitors RBC8 and BQU57 were indiscriminately toxic to all breast cancer cell lines and normal cells regardless of their RAL-dependency. We further interrogated the drug-like properties of OSURALi. Excitingly, we found that OSURALi was well tolerated in mice and can significantly slow tumor growth and spontaneous lung metastasis in the MDA-MB-231 model. Conclusion: Together these data suggests that OSURALi could serve as a lead compound for further drug optimization and may lead to a bona fide targeted therapy for TNBC. Citation Format: Dillon Richardson, Jonathan M. Spehar, David T. Han, Prathik A. Chakravarthy, Sumudu Leelananda, Chad Bennett, Gina M. Sizemore, Steffen Lindert, Steven T. Sizemore. Discovery and Validation of Novel Small G Protein Inhibitor OSURALi for Targeting Triple Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-28.

Breast cancer is a prevalent and deadly disease affecting women worldwide. Recent studies have shown that γ-glutamyl cyclotransferase (GGCT), an enzyme involved in glutathione metabolism, is consistently upregulated in various cancers. However, its specific role in breast cancer remains poorly understood. This study aimed to investigate the functional role of GGCT in breast cancer. Bioinformatics, immunohistochemistry and immunoblotting analysis revealed that GGCT is significantly upregulated in breast cancer tissues, and its high expression is associated with poor survival outcomes. The knockdown of GGCT significantly suppressed MCF-7 and SKBR-3 cell activities. Cell proliferation decreased by 29.4–45.9%, and colony formation reduced by 51.5–56.6%. Migratory ability diminished by 36.8–49.1%, while invasion capability declined by 35.2–55.0%. Moreover, GGCT silencing reduced epithelial-mesenchymal transition (EMT) and inhibited the PI3K/AKT/mTOR signaling pathway. Notably, E-cadherin expression significantly increased in MCF-7 and SKBR-3-shGGCT cells, with changes ranging from 2.1-fold to 5.4-fold. Conversely, N-cadherin expression decreased by 54.2–84.2% in both cell lines. Vimentin expression also decreased significantly, with reductions of 58.8–83.0%. Further analyses indicated that p-AKT expression in MCF-7 and SKBR-3-shGGCT cells decreased by 51.4–84.8%. Additionally, p-mTOR expression was reduced by 71.2–87.2% in both cell lines, compared to shCtrl. Our data highlights the crucial role of GGCT in regulating EMT and the progression of breast cancer. The findings suggest that GGCT not only serves as a valuable prognostic marker but also represents a potential target for therapeutic interventions in breast cancer patients.

Abstract NANOG transcription factor, a molecule associated with cancer cell resistance, was targeted using decoy oligodeoxynucleotides (ODNs) loaded nanoparticles. We designed decoy ODN to mimic a human gene promoter, aiming to disrupt the function of NANOG. Silica (SiO2) nanoparticles were coated with polydopamine (PDOPA) and loaded with decoy ODNs. These nanoparticles were then functionalized with a targeting agent, the NL2 peptide, to achieve specific binding to HER2‐positive cells. Following this, their physicochemical properties were characterized using Fourier‐transform infrared spectroscopy (FT‐IR), dynamic light scattering (DLS), scanning electron microscopy (SEM), UV–visible spectroscopy (UV–vis), and drug release assays. The effectiveness of these nanoparticles was then tested on SKBR3 (HER2 positive) and MDA‐MB‐468 (HER2 negative) breast cancer cells. The results were encouraging the nanocomposites were successfully absorbed by the cancer cells. SiO2@PDOPA/DEC‐NL2 significantly inhibits cell growth and increased cell death (apoptosis). These findings suggest that the synthesized nanosystem targeting the HER2 receptor can potentially suppress the cancerous properties of SKBR3 cells in comparison with MDA‐MB‐468 cells. In conclusion, this study presents a promising approach for targeted drug delivery in breast cancer treatment. The developed nanoparticles loaded with NANOG decoy ODNs effectively targeted HER2‐positive cancer cells and demonstrated significant anticancer properties.

Antibody-drug conjugates (ADCs) represent a promising class of targeted cancer therapies, combining monoclonal antibody specificity with cytotoxic drug potency. Despite significant ADC technology advancements, challenges such as limited efficacy, often attributed to tumor heterogeneity and resistance development, remain present. In this study, we developed homogeneous dual-payload ADCs by combining site-selective conjugation methods: second-generation AJICAP technology for lysine modification and conventional interchain-break conjugation. Using trastuzumab as a model antibody, we successfully synthesized a drug-to-antibody ratio (DAR) of 10 (2 + 8) dual-payload ADC with monomethyl auristatin E and deruxtecan, displaying low aggregation and stable physicochemical properties. The dual-payload ADC exhibited superior in vitro cytotoxicity against HER2-positive SKBR-3 cells, compared to T-DXd, and demonstrated enhanced tumor suppression in vivo in a NCI-N87 xenograft model. These results highlight the potential of multipayload ADCs in enhancing therapeutic efficacy while maintaining stability, thereby providing a new strategy to overcome traditional ADC-related limitations.