Extended Data Fig. 1: Human SkM from a metastatic breast cancer patient harbours panCK⁺/ER⁺/PR⁺ breast cancer cells.

a) Representative multiplex IHC images from an ER⁺/PR⁺ primary breast tumour specimen, used as a positive staining control. ER/PR (breast cell marker) staining in purple, pan-cytokeratin (panCK, epithelial cell marker) staining in yellow. Scale bar: 1 mm (left), 100 μm (centre) and 10 μm (right). b) Representative multiplex IHC images from human SkM, used as a negative staining control. ER/PR staining in purple, panCK staining in yellow. Scale bar, 10 μm. c) Representative IHC image of the three human SkM sites sampled (tibialis anterior, quadriceps and gastrocnemius) from a patient with metastatic breast cancer (MBC). Scale bar, 1 cm. d) Multiplex IHC panels of three panCK⁺/ER⁺/PR⁺ cells located inside the quadriceps muscle. ER/PR staining in purple, pan-cytokeratin staining in yellow. Scale bar 100 μm, Inset 10 μm. e-f) Two micropictographs of serial sections (4 µm) of FFPE SkM tissue from an individual with metastatic breast cancer. The slides were stained with hematoxylin and eosin or sequentially stained with a multiplex IHC panel consisting of ER/PR (purple) and panCK (yellow). Two ER⁺/PR^+/panCK⁺ breast cancer cells were identified in a vessel located within the quadriceps muscle. Scale bar, 10 μm.