Cell Line 4T1 Research Articles

Platycodon grandiflorum-derived extracellular vesicles suppress triple-negative breast cancer growth by reversing the immunosuppressive tumor microenvironment and modulating the gut microbiota

Despite the approval of several artificial nanotherapeutics for the treatment of triple-negative breast cancer (TNBC), significant challenges, including unsatisfactory therapeutic outcomes, severe side effects, and the high cost of large-scale production, still restrict their long-term application. In contrast, plant-derived extracellular vesicles (PEVs) exhibit promising potential in cancer therapy due to their negligible systemic toxicity, high bioavailability and cost- effectiveness. In this study, we developed an alternative strategy to inhibit TNBC via Platycodon grandiflorum (PG)-derived extracellular vesicles (PGEVs). The PGEVs were isolated by ultracentrifugation and sucrose gradient centrifugation method and contained adequate functional components such as proteins, lipids, RNAs and active molecules. PGEVs exhibited remarkable stability, tolerating acidic digestion and undergoing minimal changes in simulated gastrointestinal fluid. They were efficiently taken up by tumor cells and induced increased production of reactive oxygen species (ROS), leading to tumor cell proliferation inhibition and apoptosis, particularly in the TNBC cell line 4T1. Additionally, PGEVs facilitated the polarization of tumor-associated macrophages (TAMs) toward M1 phenotype and increased the secretion of pro-inflammatory cytokines. Further in vivo investigations revealed that PGEVs efficiently accumulated in 4T1 tumors and exerted significant therapeutic effects through boosting systemic anti-tumor immune responses and modulating the gut microbiota whether administered orally or intravenously (i.v.). In conclusion, these findings highlight PGEVs as a promising natural, biocompatible and efficient nanotherapeutic candidate for treating TNBC.Graphical

Effect of Gadolinium Doping on the Optical and Magnetic Properties of Red-Emitting Dual-Mode Carbon Dot-Based Probes for Magnetic Resonance Imaging.

Bioimaging probes based on carbon dots (CDs) can become a useful replacement for existing commercial probes, benefiting clinical diagnostics. While the development of dual-mode CD-based probes for magnetic resonance imaging (MRI), which provides the ability for photoluminescence (PL) detection at the same time, is ongoing, several challenges have to be addressed. First, most of the CD-based probes still emit at shorter wavelengths (blue/green spectral range), which is harmful to biological objects or have very low PL intensity in the biological window of tissue transparency (red/near-infrared spectral range). Second, the relaxation characteristics of the proposed CD-based probes are still similar or only slightly larger than those of commercial contrast agents. Herein, we introduce a dual-mode probe allowing for simultaneous PL detection and MRI analysis, based on CDs doped with gadolinium ions (Gd3+) with a PL peak in the red (640 nm), a PL quantum yield up to 24%, and high longitudinal and transverse relaxivities reaching 25.76 and 40.57 L mmol-1 s-1, respectively. Moreover, Gd-doped CDs show good biocompatibility, which was studied on H9c2 and 4T1 cell lines with a cell viability above 70%. The developed Gd-doped red-emissive CDs can be utilized as efficient and nontoxic dual-mode probes for PL and MRI measurements carried out simultaneously.

A Three-agent Regimen for Triple Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) has a poor prognosis with current treatment options. Novel therapeutic strategies are urgently needed to enhance treatment outcomes for TNBC. This study evaluated the efficacy of a three-agent regimen compared to existing treatment regimens in a TNBC mouse model, and elucidated its potential mechanisms of action. The TNBC xenograft tumor mouse model was established using a 4T1 cell line in female BALB/c mice. Mice were treated with the three-agent regimen and other comparative treatments. Tumor volume was monitored to assess the anti-tumor effects. Biochemical and pathological evaluations were conducted to examine the impact of the regimen on anti-tumor immunity, anti- tumor angiogenesis, and tumor cell apoptosis. The three-agent regimen consisting of SIN+BEV+PAB demonstrated significant anti-tumor efficacy compared to controls, PAB alone, SIN+PAB, and BEV+PAB groups from day 9 of drug administration. The superior anti-tumor effect of SIN+BEV+PAB was primarily attributed to enhanced anti-tumor immunity, evidenced by increased percentages of CD4+ and CD8+ T cells, elevated IFN-γ levels, and decreased percentages of Tregs, reduced levels of TGF-β, IL-6, and IL-10. Additionally, the regimen showed potent anti-angiogenic effects by reducing VEGF expression and micro vessel density (MVD). Furthermore, it promoted tumor cell apoptosis through upregulation of BAX and cleaved caspase3, while downregulating Bcl2. These findings suggest that the novel three-agent combination of SIN+BEV+PAB may prove beneficial in improving treatment outcomes for patients with TNBC. The development of this regimen, which may be eligible for patent protection, could facilitate its introduction as a new therapeutic option for advanced TNBC in clinical practice.

White Turmeric Acetone Extract (Curcuma zedoaria) Effectivity on 4T1 Breast Cancer Cell Line Proliferation

The development of breast cancer therapy has now reached the utilization of complementary alternative medicine (CAM) as an adjuvant to breast cancer therapy. One of the natural ingredients that has the potential for further research is white turmeric (Curcuma zedoaria). The purpose of the research is to determine the effects of white turmeric extract on breast cancer cell line 4T1. The research involved extraction by maceration and evaporation methods. White turmeric extract with acetone solvent was tested at 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg/ml compared to the control to see the most potent concentration in providing therapeutic effects. The results of the test analysis of water content and ash content of acetone extract of white turmeric meet the standards as standardized herbal ingredients with a percentage of 15.935% and 5.523%. Higher concentrations specifically showed a better breast cancer proliferation inhibition effect (mean cell absorbance proportion of 0.096 ± 0.0008). White turmeric extract has the potential to be developed as an alternative therapy for 4T1 breast cancer. Higher concentrations showed a better breast cancer proliferation inhibition effect. White turmeric extract has the potential to be developed as an alternative therapy for 4T1 breast cancer. Keywords: acetone extract, breast cancer, cell proliferation, white turmeric

A general strategy towards activatable nanophotosensitizer for phototoxicity-free photodynamic therapy.

Background: Photodynamic therapy (PDT) has gained widespread attention in cancer treatment, but it still faces clinical problems such as skin phototoxicity. Activatable photosensitizers offer a promising approach to addressing this issue. However, several significant hurdles need to be overcome, including developing effective activation strategies and achieving the optimal balance between photodynamic effects and related side effects. Herein, we present a novel and general strategy for the construction of tumor-targeted activatable nanophotosensitizers (TNP1/PSs). Methods: TNP1/PSs were constructed through simple nanoprecipitation method, leveraging the strong cation-π interaction between cationic polymers and aromatic photosensitizers. We conducted a comprehensive characterization and investigation of the photoactivity, as well as the mechanisms underlying both OFF state and switched-on properties of TNP1/PSs. Additionally, we thoroughly evaluated the cytotoxicity, tumor-targeted ability, and anti-tumor efficacy of TNP1/PSs in the 4T1 cell line. Results: TNP1/PSs exhibit a markedly fully OFF state of photoactivity, subsequent to self-assembly through cation-π interactions in aqueous media. The mechanism study reveals a multi-pathway process induced by cation-π complexes, which includes reduced absorption and radiative decay, as well as enhanced thermal decay and intermolecular charge transfer. Upon targeting tumor cells, TNP1/PSs were effectively endocytosed and predominantly traversed the lysosomes, where degradation of the cationic polymer occurs, resulting in the spontaneous switch-on of PDT activity. In vivo studies employing small animal models demonstrated that the as-synthesized nanophotosensitizer possesses remarkable anti-tumor activity while completely avoiding skin phototoxicity. Conclusion: This work provides a powerful platform for efficiently constructing tumor-targeted activatable nanophotosensitizers, paving the way for safe and effective photodynamic therapy in cancer treatment.

Organelle-Level Trafficking and Metabolism Kinetics for Redox-Responsive Paclitaxel Prodrug Nanoparticles Characterized by Experimental and Modeling Analysis.

Redox-responsive self-assembled prodrug nanoparticles have received extensive attention for their high loading efficiency and environmentally responsive properties. However, the intracellular metabolism and transportation kinetics were poorly understood, which limited the rational design and development of this delivery system. Herein, tetraphenylporphyrin-paclitaxel (TxP) prodrugs with thioether, disulfide, and dicarbon linkers (TsP, TssP, and TccP) were synthesized and self-assembled as nanoparticles. The redox responsiveness was investigated both in the simulated medium and in tumor cells via mass spectrometry. TxP NP and PTX concentrations in 4T1 whole cells, endosomal systems, and cytoplasm over time were quantified by UPLC-MS/MS and modeled using the nonlinear mixed effect (NLME) approach. Cytotoxicity was studied in 4T1 and MCF-7 cell lines, and antitumor efficacy was analyzed in 4T1 tumor-bearing mice. Mass spectrometry identified both oxidative and reductive metabolites in redox simulants for TssP NPs and TsP NPs. In 4T1 cells, only reductive metabolites for TssP NPs were detected, while both oxidative and reductive metabolites for TsP NPs were detected. The developed subcellular pharmacokinetic model suggested that the estimated metabolism rates of TxP NPs in endosomal systems were 10 to 27 times of the rates in cytoplasm, indicating that endosomal systems were the dominant place for intracellular metabolism. These rates were numerically higher for TsP NPs than TssP NPs in endosomal systems (1.7-fold) and the cytoplasm (2.5-fold). The internalization of nanoparticles was identified to be slow (kmax,int, 0.015 h-1) and saturable. The transportation rate constant across the endosomal membranes was fast for PTX (27.1 h-1) and slow for TxP NPs (0.098 h-1). TsP and TssP NPs had comparable in vivo antitumor efficacy, which was higher than that of TccP NPs. This study quantified the organelle-level transportation and metabolism kinetics for three prodrug nanoparticles with redox-responsive or inert linkers using a combined experimental and modeling approach. These findings and the modeling framework might inform future studies for redox-responsive prodrug design and drug delivery systems.

Nanomedicine based on chemotherapy-induced immunogenic death combined with immunotherapy to enhance antitumor immunity.

Chemo-immunotherapy based on inducing tumor immunogenic cell death (ICD)with chemotherapy drugs has filled the gaps between traditional chemotherapy and immunotherapy. It is verified that paclitaxel (PTX) can induce breast tumor ICD. From this basis, a kind of nanoparticle that can efficiently deliver different drug components simultaneously is constructed. The purpose of this study is for the sake of exploring the scheme of chemotherapy-induced ICD combined with other immunotherapy to enhance tumor immunogenicity and inhibit the growth, metastasis, and recurrence of breast tumors, so as to provide a research basis for solving the tough problem of breast cancer treatment. Nanomedicine loaded with PTX, small interference RNA that suppresses CD47 expression (CD47siRNA, siCD47), and immunomodulator R848 were prepared by the double emulsification method. The hydrodynamic diameter and zeta potential of NP/PTX/siCD47/R848 were characterized. Established the tumor-bearing mice model of mouse breast cancer cell line (4T1) in situ and observed the effect of intravenous injection of NP/PTX/siCD47/R848 on the growth of 4T1 tumor in situ. Flow cytometry was used to detect the effect of drugs on tumor immune cells. NP/PTX/siCD47/R848 nano-drug with tumor therapeutic potential were successfully prepared by double emulsification method, with particle size of 121.5 ± 4.5nm and surface potential of 36.1 ± 2.5mV. The calreticulin on the surface of cell membrane and extracellular ATP or HMGB1 of 4T1 cells increased through treatment with NPs. NP/PTX-treated tumor cells could cause activation of BMDCs and BMDMs. After intravenous injection, NP/PTX could quickly reach the tumor site and accumulate for 24h. The weight and volume of tumor in situ in the breast cancer model mice injected with nanomedicine through the tail vein were significantly lower than those in the PBS group. The ratio of CD8+/CD4+ T cells in the tumor microenvironment and the percentage of dendritic cells in peripheral blood increased significantly in breast cancer model mice injected with nano-drugs through the tail vein. Briefly, the chemotherapeutic drug paclitaxel can induce breast cancer to induce ICD. The nanomedicine which can deliver PTX, CD47siRNA, and R848 at the same time was prepared by double emulsification. NP/PTX/siCD47/R848 nano-drug can be enriched in the tumor site. The experiment of 4T1 cell tumor-bearing mice shows that the nano-drug can enhance tumor immunogenicity and inhibit breast tumor growth, which provides a new scheme for breast cancer treatment. (Graphical abstract).

Exposure to Hypoxic Conditions Up-regulates HER2 in Breast Cancer Cell Lines.

Tissue specimen quality is becoming increasingly important for basic research and routine clinical results. Warm ischemia time (WIT) affects human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) scores. However, the role of WIT on HER2 modulation remains unclear. We hypothesized that the WIT-mediated increase in HER2 IHC scores was caused by hypoxia. Therefore, this study aimed to determine the mechanism by which WIT mediates the increase in HER2. HER2 mRNA expression was measured in 4T1, SKBR3, and HCC1954 breast cancer cell lines using real-time PCR following hypoxia exposure. The membrane proteins were isolated and extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) or evaluated through non-permeabilized immunofluorescent analysis. Hypoxic conditions up-regulated GLUT1 mRNA expression but not HER2 expression. The HER2 membrane protein fraction increased in response to hypoxic conditions. Nonpermeabilized immunofluorescence analysis showed that membrane-bound HER2 was also promoted under hypoxic conditions. HER2 is not regulated at the mRNA level; however, the level of membrane-bound HER2 increases in response to hypoxia.

Photocatalytic activity and anticancer properties of green synthesized ZnO-MgO-Mn2O3 nanocomposite via Ocimum basilicum L. seed extract

Green synthesis using plant extracts such as Ocimum basilicum L. (OBL) seed has attracted attention due to its sustainable and environmentally friendly nature. In this study, ZnO-MgO-Mn2O3 nanocomposites were synthesized using OBL seed extract at two different calcination temperatures of 500 °C and 600 °C, and evaluated in terms of photocatalytic application and cytotoxicity. Phytochemicals in OBL seeds act as reducing and masking agents in the production route, which leads to the formation of nanomaterials with unique properties. Characterization techniques, including XRD, FE-SEM, and DRS were used to analyze the structural, morphological, and optical features of the nanocomposite. XRD results confirmed increasing crystal size from ~ 32 nm (500 °C) to ~ 84 nm (600 °C). Also, FE-SEM images showed formation of irregularly shaped nanocomposites and the EDX spectra of the samples confirmed the presence of zinc, magnesium, manganese, and oxygen elements. The photocatalytic behavior of the nanocomposite cacined at 500 °C was investigated for different organic pollutants. Removal percentages of 97% for Eriochrome Black T dye (pH = 10 for 90 min), 99% for methylene blue dye (pH = 10 for 60 min), 89% for methyl orange dye (pH = 10 for 105 min), and 86.9% for Rhodamine B dye (pH = 3 for 90 min) were obtained. Moreover, the cytotoxicity of the synthesized nanocomposite at 500 °C was evaluated on the 4T1 cell line to investgate its effect on biological systems, and the IC50 value was obtained around 323 µg/mL.

Organic Moiety on Sn(IV) Does Matter for In Vitro Mode of Action: nBu3Sn(IV) Compounds with Carboxylato N-Functionalized 2-Quinolones Induce Anoikis-like Cell Death in A375 Cells.

Objectives: New tributyltin(IV) complexes containing the carboxylate ligands 3-(4-methyl-2-oxoquinolin-1(2H)-yl)propanoic acid (HL1) and 2-(4-methyl-2-oxoquinolin-1(2H)-yl)acetic acid (HL2) have been synthesized. Methods: Their structures have been determined by elemental microanalysis, FT-IR and multinuclear NMR (1H, 13C and 119Sn) spectroscopy and X-ray diffraction study. A solution state NMR analysis reveals a four-coordinated tributyltin(IV) complex in non-polar solvents, while an X-Ray crystallographic analysis confirms a five-coordinated trigonal-bipyramidal geometry around the tin atom due to the formation of 1D chains. A theoretical structural analysis was performed by optimization employing B3LYP-D3BJ functional and 6-311++G(d,p)/def2-TZVP(Sn) basis sets for H, C, N, O/Sn, respectively. The interactions between tin(IV) and surrounding atoms were examined by QTAIM approach. The in vitro antiproliferative activity of the synthesized compounds was evaluated by MTT and CV assays versus MCF-7 (human breast adenocarcinoma), HCT116 (human colorectal carcinoma), A375 (human melanoma), 4T1 (mouse breast carcinoma), CT26 (mouse colon carcinoma) and B16 (mouse melanoma) tumor cell lines. Results: Both synthesized compounds (nBu3SnL1 and nBu3SnL2) exerted powerful micromolar IC50 cytotoxicity values and demonstrated high selectivity toward malignant cells. Both experimental drugs affected cell adhesion and induced anchorage independent apoptosis, a favorable type of cell death with an essential role in cancer dissemination prevention. The BSA-binding affinity of the obtained organotin compounds was followed by spectrofluorometric titration and molecular docking simulations. Conclusions: The tributyltin(IV) compounds selectively induce anoikis-like cell death in A375 cells, also highlighting the importance of the organic moiety on the tin(IV) ion in the mechanism of action.

The Free Radical Scavenging and Cytotoxic Properties of Ardisia crenata and Peliosanthes teta in the 4T1 Breast Cancer Cell Line

Background: Ardisia crenata (A. crenata) and Peliosanthes teta Andrew (P. teta) are popular medicinal plants in Malaysia that are claimed to have several biological effects. Objective: To assess the anticancer and radical-scavenging properties of A. crenata and P. teta methanolic extracts. Methods: TPC was quantified using the Folin-Ciocalteu test; free radical scavenging was evaluated using the 2,2'-azino-bis3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays; and cytotoxic activity was assessed using the MTT assay. Results: In both ABTS and DPPH systems, A. crenata exhibits much higher radical scavenging activity than ascorbic acid. P. teta showed less significant antioxidant activity. Furthermore, A. crenata methanolic extracts demonstrated free radical scavenging properties in a concentration-dependent manner, quenching ABTS and DPPH radicals with no lag phase observed in the experiments. Furthermore, A. crenata had a strong cytotoxic effect on the breast cancer 4T1 cell line but had no suppression of cell viability in the 3T3 normal fibroblast cell line. Interestingly, the TPC of A. crenata was significantly higher than that of P. teta methanolic extracts. Conclusions: A. crenata showed antioxidant and anticancer action against breast cancer 4T1 cells, indicating that it could be a valuable source for future pharmacological and medical research.

Construction of a stable 4T1 cell line expressing UL19 by the PiggyBac transposon system

To investigate the mechanism of the major capsid protein VP5 (encoded by the UL19 gene) of oncolytic herpes simplex virus type Ⅱ (oHSV2) in regulating the antitumor function of immune cells, we constructed a mouse breast cancer cell line 4T1-iRFP-VP5-GFP stably expressing VP5 protein, near-infrared fluorescent protein (iRFP), and green fluorescent protein (GFP) by using the PiggyBac transposon system. Flow cytometry and Western blotting were employed to screen the monoclonal cell lines expressing both GFP and VP5 and examine the expression stability of UL19 in the constructed cell line. The results of SYBR Green I real-time PCR and Western blotting showed that the copies of UL19 and the expression level of VP5 protein in the 15th passage of 4T1-iRFP-VP5-GFP cells were significantly higher than those in the 4T1 cells transiently transfected with UL19, demonstrating the stable insertion of UL19 into the 4T1 cell genome. The real-time cell analysis (RTCA) was employed to monitor the proliferation of 4T1-iRFP-VP5-GFP cells, which showed similar proliferation activity to their parental 4T1 cells. Further studies confirmed that NK92 cells exhibited stronger cytotoxicity against 4T1-iRFP-VP5-GFP cells than against 4T1 cells. This study layed a foundation for elucidating the role of VP5 protein in regulating immune cells, including T cells and NK cells, via HLA-E in 4T1 cells to exert the anti-tumor function.

Cytotoxic activity of water and ethanol extract of Curcuma caesia (black turmeric) on breast cancer cell line

Breast cancer is one of the cancers with the highest incidence and mortality rate in the world. Curcuma caesia (Black turmeric) is known to have neuroprotective, anthelmintic, sunscreen activity, anti-diabetic and also anti-cancer activity. This study aimed to determine the phytochemical content and possible cytotoxic activity of C. caesia water extract (EWCC) and C. caesia ethanol extract (EECC) on breast cancer cell lines. Samples were extracted using two extraction methods, maceration technique using 96% ethanol as solvent and double boiling using water as a solvent. The phytochemical content, total phenolic content (TPC), and total flavonoid content (TFC) of each extract were determined using spectrophotometer method. Cytotoxic activity was measured using MTT assay on MCF-7 and 4T1 breast cancer cell lines (24 hrs and 48 hrs). Results showed that EWCC and EECC contained flavonoids, alkaloids, and polyphenols. The values of TPC and TFC were 106.08 mg GAE/100 g and 4.32 mg QUE/g for EECC, respectively, and 73.29 mg GAE/100 g and 3.40 mg QUE/g for EECC, respectively. Meanwhile, the cytotoxic activity test found that EWCC did not provide cytotoxic activity on MCF-7 and 4T1 cells. In the MCF-7 cell line, EECC exhibited IC50 values of 212.90 µg/mL at 24 hrs and 311.91 µg/mL at 48 hrs. Similarly, in the 4T1 cell line, EECC exhibited IC50 values of 161.06 µg/mL at 24 hrs and 150.89 µg/mL at 48 hrs. From these results, it can be concluded that EECC has cytotoxic activity in MCF-7 and 4T1 breast cancer cell models, so this extract can be studied more deeply to determine its pathway of action.

Doubly self-assembled dermatan sulfate/chitosan nanoparticles for targeted siRNA delivery in cancer therapy

RNA interference, a naturally occurring regulatory mechanism in which small interfering RNA (siRNA) molecules are responsible for the sequence-specific suppression of gene expression, emerged as one of the most promising gene therapies in cancer. In this work, we investigate a microfluidics double self-assembly method based on micellization and polyelectrolyte complex formation for the encapsulation of siRNA targeting the BIRC5 gene, a member of the inhibitor of apoptosis gene family, that codes for survivin a protein of theinhibitorof apoptosis protein family that is involved in triple-negative breast cancer (TNBC) proliferation and metastasis within nanoparticles of an amphiphilic chitosan-graft-poly(methyl methacrylate) copolymer and low-molecular weight dermatan sulfate, a polysaccharide targeting the CD44 receptor overexpressed in this tumor. Nanoparticles are spherical and display a hydrodynamic diameter of ∼ 200 nm, as measured by dynamic light scattering and scanning electron microscopy. In addition, these colloidal systems exhibit a strongly negative zeta-potential that confers them excellent physical stability for at least four months owing to electrostatic repulsion and evidences the exposure of the polyanionic dermatan sulfate on the surface. The key role of dermatan sulfate in the active targeting and intracellular delivery of the cargo in the murine breast cancer cell line 4T1, a model of TNBC, is confirmed by confocal laser scanning microscopy and imaging flow cytometry. Finally, the silencing efficiency is demonstrated in 4T1 cell viability, migration, proliferation and spheroid formation assays in vitro. Overall results highlight the promise of this simple, reproducible and scalable method for the nanoencapsulation of siRNA and other therapeutic nucleic acids.

Abstract B040: Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer

Abstract Breast cancer is one of the most frequently diagnosed cancers and the second leading cause of women’s cancer death. Although the five-year survival rate for women in the United States with nonmetastatic invasive breast cancer is 90%, this rate drops to less than 30% for those with tumors that have metastasized to distant organs. Epithelial-to-Mesenchymal Transition (EMT) plays a critical role in tumor metastasis by enabling the loss of epithelial characteristics and the gain of mesenchymal traits, leading to increased cell motility, invasion, and resistance to therapy. Consequently, targeting the EMT process may prevent tumor cell migration and improve patient outcomes. Rather than being a binary switch from an epithelial to a mesenchymal state, cells undergoing EMT can assume a spectrum of intermediate states, some of which may reshape the tumor microenvironment by recruiting pro-tumor immunosuppressive cells, such as regulatory T cells, M2-like macrophages, and myeloid-derived suppressor cells. However, the impact of EMT on the immune composition of the tumor microenvironment and the causative mechanisms remain poorly understood. Using multi-omics approaches in a mouse model of triple-negative breast cancer, we assessed the interaction between tumor and immune cells in the tumor microenvironment driven by the EMT. A heterogeneous mouse breast cancer cell line (4T1) was used to isolate five distinct single-cell-derived clonal populations residing in distinct states within the EMT spectrum. These EMT clones, along with the parental 4T1 cell line, were orthotopically transplanted into the mammary fat pads of BALB/c mice. Primary tumors were subsequently harvested and dissociated for single-cell RNA-sequencing (scRNA-seq) and DNA methylation analysis. Lungs were histologically evaluated for metastases. Tumors derived from an intermediate EMT clone exhibited increased tumor growth kinetics and a higher incidence of metastasis compared to epithelial or mesenchymal clones. In scRNA-seq data, tumors derived from different EMT clones largely retained their original EMT states in vivo and recruited distinct immune cell profiles. DNA methylation analysis further revealed that these tumors displayed unique methylation patterns in EMT-related CpGs. This study introduced a model that captures the complexity of the tumor microenvironment across the EMT spectrum. This was accomplished through immunophenotyping using scRNA-seq and identifying unique epigenetic patterns through DNA methylation. Our findings highlight the potential for distinct immune cell populations to be recruited to tumors in different EMT states, thereby influencing survival and metastasis outcomes. Furthermore, our results open new opportunities for developing cancer combination treatment strategies that target specific tumor-immune cell interactions. Citation Format: Hanxu Lu, Meisam Bagheri, Todd Miller, Diwakar Pattabiraman, Brock Christensen. Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B040.

Synthesis, characterisation, single crystal structure and evaluation of a redox innocent carbazate functionalized phenanthroline for antimycobacterial and anticancer activity

Development of bioactive candidates for cancer and bacterial infections are ever demanding challenges due to the resistance shown by these cancer and bacterial cells for already exposed drug molecules. Methyl carbazate derivatized phenanthroline compound 6-[2-(methoxycarbonyl)diazen-1-yl]-1,10-phenanthroline-5-one (MCDPO) is synthesized in a three step reaction from 1,10-phenanthroline. The spectroscopic features of MCDPO are studied by HRMS, IR, 1H NMR and 13C NMR, UV-visible spectroscopy. The three-dimensional molecular structure of the MCDPO is confirmed by single crystal XRD study. The MCDPO crystallized in a triclinic system P-1 space group with the following unit cell parameter: a = 5.0347(2) Å, b = 10.3509(4) Å, c = 11.8816(4) Å, α = 84.431(3)°, β = 84.172(3)°, γ = 81.380(4)°, and V = 606.92(4) Å3 at T = 133 K. The MCDPO is containing aromatic rings, hydrogen bonding donors as well as hydrogen bonding acceptor groups forming supramolecular molecular arrangements through C-H⋯O, CH⋯N, C-O⋯C, π-π and π⋯C non-covalent interactions. Electrochemical redox and electrochromic features of MCDPO are studied through cyclic voltammetry and UV-vis based spectroelectrochemistry. This compound shows good anticancer activity with IC50 values 1.438, 6.576 and 2.901 µM against 4T1, MCF-7 and PC-3 cancer cell lines respectively. The flow cytometry study on 4T1 cells suggests that the MCDPO promoted cancer cell death by inducing apoptosis, and cell cycle arrest in the G0/G1 phase. It also induces nuclear fragmentation and reactive oxygen species (ROS) generation, which was studied by DAPI, AO, and DCFH-DA based cellular staining studies by fluorescence confocal imaging. The MCDPO also studied for antibacterial activity against Escherichia Coil bacteria and Mycobacterium tuberculosis (Mtb). The MCDPO shows minimum inhibitory concentration (MIC) of 0.39 µg/mL against Mtb which is slightly better than the one of the clinically used drug candidates Ethambutol.

“Development, optimization, and characterization of Eudragit-based nanoparticles for Dasatinib delivery”

This study focused on developing and evaluating dasatinib-loaded nanoparticles (DST-NPs) using Eudragit L100 as a polymer matrix for enhanced breast cancer treatment. The optimized formulation exhibited a particle size of 202.1 ± 5.7 nm, a zeta potential of −18 ± 1.01 mV, and an entrapment efficiency of 93.11 ± 0.2%. In-vitro release studies demonstrated sustained drug release from DST-NPs, following Fickian diffusion. Pharmacokinetic studies in rats revealed higher Cmax and AUC0-t for DST-NPs compared to pure DST, indicating improved bioavailability. Tissue distribution studies showed enhanced targeting of DST-NPs, with higher concentrations in the liver and spleen. In vivo efficacy in a DMBA-induced mammary carcinoma model demonstrated that DST-NPs significantly reduced tumor volume, maintained stable body weight, and improved survival rates compared to pure DST. Hematologic analysis indicated a favorable blood profile with DST-NPs, and histopathological examinations confirmed the restoration of normal mammary gland and liver architecture. MTT assays showed higher cytotoxicity of DST-NPs against MCF-7, MDA-MB231, and 4T1 cell lines, with lower IC50 values than pure DST. Stability studies indicated that DST-NPs maintained their properties over six months at various storage conditions. These findings highlight the potential of DST-NPs as an effective nanocarrier system for cancer therapy.

Effective Synthesis of C20-Epi-Isothiocyanato-Salinomycin and its Thiourea Derivatives as Potential Anticancer Agents.

Salinomycin, a naturally occurring polyether ionophore antibiotic isolated from Streptomyces albus, has been demonstrated potent cytotoxic activity against a variety of cancer cell lines. In particular, it exhibits selective targeting of cancer stem cells. However, systemic toxicity, drug resistance and low bioavailability of the drug significantly limit its potential applications. In this study, the C20-epi-isothiocyanate of salinomycin was designed and synthesized, and then reacted with amines as a versatile synthon to assemble a series of salinomycin thiourea derivatives, which improved the druggability of salinomycin. The antiproliferative activities of the compounds were evaluated in vitro against A549, HepG2, HeLa, 4T1, and MCF-7 cancer cell lines using the CCK-8 assay. The pharmacological results showed that some salinomycin thiourea derivatives exhibited excellent inhibitory activity against at least one of the tested tumor cells and high selectivity. Further mechanistic studies showed that compound 9 f, containing a 3,5-difluorobenzyl moiety, could directly induce apoptosis, probably by increasing caspase-9 protein expression and cell cycle arrest in G1 phase in a concentration dependent manner.

Improved Therapeutic Efficacy of Doxorubicin Chemotherapy With Cannabidiol in 4T1 Mice Breast Cancer Model.

High dose chemotherapy is one of the therapeutic strategies for breast cancer and doxorubicin (DOX) as a chemotherapy agent is widely used. DOX indication is limited due to its dose-depended cardiotoxicity. Recently, cannabidiol (CBD) shows antitumoral and cardioprotective effects, so we hypothesized that CBD administration with high-dose DOX chemotherapy can improve anticancer activity and reduce cardiotoxic side effects. Mice breast cancer model established by injecting 4T1 cell lines. One group was not injected by 4T1 cells as a not cancerous group and received normal saline (NS, 0.1 mL). In cancerous groups, first group was considered as cancerous control and received NS (0.1 mL); the second group received CBD (5 mg/kg, IP) on Days 1,7, and 14; in the third group DOX (5 mg/kg, IV) as CBD schedule was administrated; the fourth group treated with CBD 1 day before DOX injection as pretreatment, and the last group was treated with CBD and DOX at same time with previous doses and schedules. On Day 21, all mice were sacrificed, heart and lungs tissues were obtained and histological sections were isolated. SOD2, iNOS, MMP2, MMP9 were evaluated through western blot and TUNEL test preformed for breast tumor. Tumor size and weight significantly decreased in DOX, pretreatment CBD + DOX and CBD + DOX groups. Administration of CBD with DOX could not prevent weight loss. TUNEL test demonstrated the highest tumor cell apoptosis in pretreatment CBD + DOX and CBD + DOX. In lungs belonged to CBD + DOX, there was not any sign of metastasis. Cardiac histopathological examination of pretreatment CBD + DOX and CBD + DOX did not show any sign of congestion or inflammation. In CBD + DOX SOD2 increased, also iNOS, MMP2, and MMP9 decreased compared to DOX. This study demonstrated that simultaneous administration of CBD and DOX can increase antitumoral effect and reduce DOX cardiotoxicity. Nevertheless, CBD can induce cardiotoxicity as administrated alone.

Strong Affinity between Astatine and Silver: An Available Approach to Anchoring 211At in Nanocarrier for Locoregional Oncotherapy.

Recently, 211At-related endoradiotherapy has emerged as an important oncotherapy strategy. Conjugating 211At with a nanocarrier provides a vital candidate for radionuclide therapy of various malignant tumors. In this study, we proposed utilizing the intrinsically high affinity of heavy halogens and sulfhydryl compounds for metallic silver to achieve highly efficient conjugation between 211At and Ag-based nanoparticles in a simple way. 211At@Ag-PEG-FA was obtained via a one-pot assembly of 211At, Ag, and SH-PEG-FA in extremely high radiolabeling yield (>95%) within 15 min and maintained excellent stability in simulated physiochemical media. Additionally, the prepared 211At@Ag-PEG-FA demonstrated specific binding to the breast cancer cell line (4T1), with a high endocytosis rate and low reflux, leading to significant cell growth inhibition. 211At@Ag-PEG-FA exhibits an excellent antitumor effect that completely suppressed tumor growth during the first week, effectively prolonging the median survival of mice to 44 days, relative to 18 days in the control group. All of the mice exhibited minimal side effects from 211At@Ag-PEG-FA in the experiment, indicating its acceptable biosafety. Our work shows that the strong affinity of Ag can be utilized to produce radioactivated nanomedicines with excellent stability and high efficiency, which also provides some valuable insights for the 211At radiolabeling of general compounds.