Fig. 4

Viability of cell clusters in the circulatory system. A Trypan blue staining of single MCF-7 cells and cell clusters. The black arrow indicates single cell, whereas the white arrows point to cell cluster. B Western blot analysis of the protein levels of PD-L1 in MCF-7 and MDA-MB-231 cells. C Immunofluorescence analysis of the protein levels of PD-L1 in MDA-MB-231 cells. DAPI (nuclei, blue), PD-L1 (green). D Immunofluorescence analysis of the cytoskeleton localization in 4T1 cells. DAPI (nuclei, blue), F-actin (green). E Design and operation of the cluster-chip. F The cluster-chip captures MCF-7 cell clusters. Scale bars,20/50 µm. Pan-CK (green), DAPI (nuclei, blue). G Left: lung of mice with breast cancer in situ, the black arrow points to lung metastases. Right: the cluster-chip captures CTC clusters in bright field. Blood was collected from a mouse model of breast cancer in situ. Scale bars,20 µm. H Representative images of spontaneously shed CTC cluster in vivo. Pan-CK (green), CD45 (red) and DAPI (nuclei, blue). Scale bars, 20 µm. I The cluster-chip captures CTC clusters. Blood was collected three days after 4T1-GFP cell clusters were injected into the tail vein of mice. DAPI (nuclei, blue), GFP (green). Scale bars, 50 µm