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.