3D Multicellular Stem-Like Human Breast Tumor Spheroids Enhance Tumorigenicity of Orthotopic Xenografts in Athymic Nude Rat Model

Therapeutic targeting of stem cells needs to be strategically developed to control tumor growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes present, including cancer stem cells (CSCs). The development of 3D stem-like properties of human breast tumor spheroids in stem cell factor conditioned media was investigated in orthotopic xenografts for enhanced tumorgenicity in the athymic nude rat model. MCF-7, ZR-75-1, and MDA-MB-231 breast cancer cell lines were cultured in serum-free, stem cell factor-supplemented medium under non-adherent conditions and passaged to generate 3rd generation spheroids. The spheroids were co-cultured with fetal lung fibroblast (FLF) cells before orthotopic heterotransplantation into the mammary fat pads of athymic nude rats. Excised xenografts were assessed histologically by H&E staining and immunohistochemistry for breast cancer marker (ERB1), proliferation marker (Ki67), mitotic marker (pHH3), hypoxia marker (HIF-2α), CSC markers (CD47, CD44, CD24, and CD133), and vascularization markers (CD31, CD34). Breast cancer cells cultured in stem cell factor supplemented medium generated 3D spheroids exhibited increased stem-like characteristics. The 3D stem-like spheroids co-cultured with FLF as supporting stroma reproducibly and efficiently established orthotopic breast cancer xenografts in the athymic nude rat.

A novel heterocyclic compound inhibits human breast tumor cells via ROS mediated apoptosis

The new heterocyclic compound 2-((6-chloro-2-methylpyrimidin-4-yl)amino)-N-(2-chloro-6-methylphenyl) thiazole-5-carboxamide (1), designed using 2-chloro-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (2) as start material, was successfully obtained via multistep synthesis route and finally characterized by IR (infrared radiation), 1H NMR (nuclear magnetic resonance), and single crystal X-ray crystallography. The inhibitory effect of compound 1 on human breast tumor cell line BS524 was further explored. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and IC50 (half maximal inhibitory concentration) values suggested that compound 1 has significant anti-proliferation activity on BS524 cells and with low side effect. Then, serial experiments, such as the Annexin V-FITC/PI assay, TUNEL staining and autophagy detection revealed that compound 1 could inhibit cell proliferation via induce cells apoptosis, and the apoptosis is induced by (reactive oxygen species) ROS generation in BS524 cells.

Loss of the Metastasis Suppressor NME1, But Not of Its Highly Related Isoform NME2, Induces a Hybrid Epithelial–Mesenchymal State in Cancer Cells

Epithelial–mesenchymal transition (EMT) is important for the initial steps of metastasis. Although it is well accepted that the nucleoside diphosphate kinase NME1 is a metastasis suppressor, its effect on EMT remains poorly documented, as does that of its closely related isoform, NME2. Here, by using gene silencing, inactivation and overexpression strategies in a variety of cellular models of cancer, we show that NME1 is a powerful inhibitor of EMT. Genetic manipulation of NME2, by contrast, had no effect on the EMT phenotype of cancer cells, indicating a specific function of NME1 in EMT regulation. Loss of NME1 in epithelial cancer cells resulted in a hybrid phenotype intermediate between epithelial and mesenchymal cells, which is known to be associated with cells with a highly metastatic character. Conversely, overexpression of NME1 in mesenchymal cancer cells resulted in a more epithelial phenotype. We found that NME1 expression was negatively associated with EMT markers in many human cancers and was reduced in human breast tumor cell lines with the aggressive ‘triple-negative’ phenotype when compared to human breast tumor cell lines positive for estrogen receptor. We show that NME1, but not NME2, is an inhibitor of essential concerted intracellular signaling pathways involved in inducing EMT, including the AKT and MAPK (ERK, p38, and JNK) pathways. Additionally, NME1 depletion considerably altered the distribution of E-cadherin, a gatekeeper of the epithelial phenotype, shifting it from the plasma membrane to the cytosol and resulting in less E-cadherin on the cell surface than in control cells. Functional aggregation and dispersion assays demonstrated that inactivation of NME1 decreases E-cadherin-mediated cell–cell adhesion. We conclude that NME1, but not NME2, acts specifically to inhibit EMT and prevent the earliest stages of metastasis.