Visualization of vasculature in transplanted mouse models. Immunofluorescence and immunohistochemical staining of forma-lin-fixed mouse lung samples to enable differentiation between vessel co-option and angiogenesis in tumors. a, b Vessel co-opting tumors are observed in spontaneously formed lung metastases from mice with orthotopically implanted then surgically resected MDA-MB-231/LM2–4 breast tumors. In a sections are stained for alveolar cell marker podoplanin, EC marker CD34 and nuclei marker DAPI. Tumor cells can be seen filling alveolar spaces along the border and incorporating alveolar capillaries into the tumor core. In b is the corresponding section stained for HLA human cell marker and hematoxylin to show the presence of tumor cells with respect to host stroma and lung parenchyma. A bronchiole is also seen to be taken into the tumor and gradually filled with tumor cells. The tumor border is irregular. c, d Angiogenic growth is observed in spontaneously formed lung metastases from mice bearing intra-renal implanted RENCA tumor cells that later underwent nephrectomy. Sections are stained for alveolar and bronchial epithelium cell marker cytokeratin 7, EC marker CD34 and nuclei marker DAPI. RENCA tumors grow in “cannonball” shape, compressing lung tissue and excluding them. The lung–tumor interface of another nodule is shown at high magnification in d. Lung tissue is compressed or “pushed” aside to allow tumor expansion. The tumor border is smooth, and microvessels are not associated with alveolar epithelium within the tumor. Scale bar represents 200 μm. Regions in dashed boxes are expanded on the right. “T” = tumor. Arrow = columnar bronchial epithelium
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