Fig. 7: Genetic targeting of mitochondrial 1C metabolism reduces metastasis formation in vivo.

a 4T1 breast cancer cells transfected with non-targeting control (SCR, n = 9) and 4T1 cells with KD of Mthfd1l (n = 9) were injected into the mammary fat pads of immunocompetent female Balb/c mice. Primary tumor growth was monitored and lung metastasis formation was evaluated at end-point. b Primary tumor size was measured and mean tumor volume for each group was calculated; mean ± SEM (n = 8 (Scr) and 9 (Mthfd1l KD)). c Primary tumor weight was measured at the end-point. Dots indicate individual animals; mean ± SEM (n = 9); unpaired, two-tailed t-test with Welch’s correction. d Primary tumor tissue was analyzed for Mthfd1l protein expression by Western Blot and quantified relative to β-actin as loading control. Dots indicate individual animals; mean ± SEM (n = 7 (Scr) and 8 (Mthfd1l KD)); unpaired, two-tailed t-test with Welch’s correction. Protein lysates from tumors with significantly low Mthfd1l and β-actin expression relative to total protein were judged to be not pure tumor lysate and discarded from analysis (see Figure S7D–F for details). e Macroscopic lung metastases were counted and are depicted as number of metastases per lung. Dots indicate individual animals; mean ± SEM (n = 9); unpaired, two-tailed t-test with Welch’s correction. f Lung tissue was embedded in paraffin and stained with H&E. 2 representative images per group are presented to show microscopic lung metastasis; scale bar corresponds to 100 μm. g Metastatic area per lung was quantified and is expressed as % area of lung tissue. Dots indicate individual animals; mean ± SEM (n = 7 (Scr) and 9 (Mthf1l KD)); unpaired, two-tailed t-test with Welch’s correction. Source data are provided as a Source Data file.