Next, we performed immunohistochemistry analyses of key enzymes in nucleotide de novo synthesis reactions, i.e., carbamoyl-phosphate synthetase 2 (CAD), phosphoribosylpyrophosphate synthetase 2 (PRPS2), guanine monophosphate synthase (GMPS), and adenylosuccinate synthetase (ADSS), in primary and metastatic tumor foci spontaneously formed in mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) mice or formed in 4T1-inoculated syngeneic mouse model, confirming that these enzymes were up-regulated in metastatic cancer cells (Fig 2I, S1C Fig). Here, CAD is linked to metastatic malignant neoplasm.