4.9. Subcutaneous Tumor Growth In Nude Mice

Subcutaneous tumor growth assay was conducted, as described previously [51]. Five to six-week-old male nude mice (BALB/cAnN.Cg-Foxn1^nu/CrlNarl) were purchased from National Laboratory Animal Center (NLAC, Taiwan). 10⁶ cells that were suspended in sterile PBS were subcutaneously injected into either left or right flank of the same mice. The tumors were measured weekly and tumor volume (mm³) was calculated using the formula 1/2 × (length) × (width)² to obtain the tumor growth curve. The mice were sacrificed at 98 days post-injection of M1-2 or M2-3 cells (n = 3/group) and 42 days after injection of NHRI-HN1 or NHRI-HN2 cells (n = 4/group). The subcutaneous tumors were weighed, photographed, processed by the Pathology Core Lab (National Health Research Institutes, Miaoli, Taiwan), and their histopathology examined by H&E staining. In accordance with the definition of 3R (replacement, reduction, and refinement), as well as the guidelines for design and statistical analysis of experiments using laboratory animals, the in vivo analysis has been presented by averaging all obtained data [52]. The mice were randomized to the group assignment.