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

Abstract

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.

The Chorioallantoic Membrane Xenograft Assay as a Reliable Model for Investigating the Biology of Breast Cancer

“…However, in the CAM model, MCF-7 ovografts presented lower Ki-67 than the MDA-MB-231 ovograft, indicating a less aggressive biological behavior of the luminal breast cancer cell line compared with the triple-negative one, which was in line with our expectations. In contrast, a study investigating the features of breast cancer cell line-derived spheroid xenografts in athymic nude rats reported both higher Ki-67 levels and PHH3 counts in MCF-7 xenografts than in MDA-MB-231 xenografts [ 76 ]. Nevertheless, the mitosis marker PHH3 might be a good alternative for Ki-67 [ 77 ], but this marker is not used in routine breast cancer diagnostics.…”

EGFR Transgene Stimulates Spontaneous Formation of MCF7 Breast Cancer Cells Spheroids with Partly Loss of HER3 Receptor

“…Immunocytochemistry of spheroids was performed essentially as described in [ 42 ], with some modifications. Briefly, spheroids were collected by centrifugation, washed in cold PBS, transferred onto the slide, and gently flattened under a coverslip to expose the interior.…”

Green synthesis of fluorescent carbon nanodots from sage leaves for selective anticancer activity on 2D liver cancer cells and 3D multicellular tumor spheroids

“…The ability of cancer cells to group into spheroids in an in vitro setting indicates an enhanced tumorigenicity, resistance to chemotherapeutic drugs, and tumor-forming capacity in vivo. 73,74 We studied the effect of the synthesized CNDs on Hep3B cells' capacity to form spheroids. Cells were allowed to group into spheroids under non-treated conditions (Control).…”