Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy

Abstract

Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor–stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver.

1 Video Captions

3D reconstitution of liver metastasis. Melanoma cells (B16F10) were labeled in vitro with lipophilic membrane dye DiI (red) and intrasplenically transplanted into genetically engineered LysM-eGFP mice. Neutrophils (rounded cell morphology) are depicted in bright green and reside inside the vasculature (AF647-albumin, blue). Kupffer cells are located in the vasculature and labeled with anti-F4/80 (white). Hepatic stellate cells (bright green) can be distinguished from neutrophils by their elongated cell shape situated outside of hepatic vasculature. Autofluorescent hepatocytes are represented in dark green. 5 days after melanoma cell injection confocal z planes (20× lens) were recorded every 1 μm and reconstructed into a movie. Scale bar: 200 μm. Images were corrected for contrast and brightness (AVI 533220 kb)

Visualizing immune cells in liver vasculature by spinning disk confocal microscopy. NKT cells (bright green) in the liver are visualized in CXCR6-GFP transgenic mice. Kupffer cells (red) are labeled with anti-F4/80. Autofluorescent hepatocytes are shown in dark green. Representative picture (20× lens) of a 30 min long image sequence with 10 s between frames. Scale bar: 68 μm. Images were corrected for contrast and brightness (AVI 41512 kb)

Visualizing NKT cell dynamics in the liver tumor microenvironment. CXCR6-GFP transgenic mice were injected with colon adenocarcinoma cells (C26 cells labeled with DID membrane dye). 3 days later, metastases (blue) and NKT cells (bright green) are pictured in the liver. Autofluorescent hepatocytes are shown in dark green. The 1 h image sequence was recorded with 15 s between frames. Scale bar: 140 μm. Images were corrected for contrast and brightness (AVI 68992 kb)

Long-term imaging of liver tumors. Neutrophils are visualized in LysM-eGFP transgenic mice. Image acquisition was performed right after tumor cells (B16F10 labeled with DID membrane dye (blue)) were intrasplenically injected. Dying tumor cells were detected with propidium iodide (red). Autofluorescent hepatocytes are shown in dark green. The movie over 3.5 h was recorded with 30 s between frames. Scale bar: 140 μm (MP4 26155 kb)