IADR Abstract Archives
Stromal Configuration by Human Carcinoma Transplantation in Mouse Tongue
Objectives: Specific microenvironment of oral squamous cell carcinoma (OSCC) is established by the communication between cancer cells and host stroma. The present study aimed to assess the spatial reconfiguration of stromal components such as blood/lymphatic vessels and peripheral nerves induced by transplanted human OSCC cell lines in mouse tongue.
Methods: OSCC cell lines that represented high-metastatic potential (OSC19 and OSC20) and low-metastatic potential (KOSC2 and HO-1-u-1) were used. Each cell line grown in culture was transplanted into a BALB/c nude-mouse tongue. Whole tongue tissues were dissected from the animals at periodical intervals after inoculation, followed by preparation of paraffin-embedded serial sections. Cancer cells, blood/lymphatic vessels, and nerve fibers were immunolabeled step-wisely with cytokeratin, PECAM-1, Lyve-1, and NFP antibodies, respectively. For three-dimensional (3D) reconstruction using 100 serial sections (4 μm thick), RGB color images of immunostained sections were acquired using a virtual microscope (NanoZoomer, Hamamatsu Photonics), and image registration and segmentation were automated with the aid of Image-J/Fiji software (NIH).
Results: The 3D reconstruction highlighted the vascular enrichment in each tumor microenvironment, and the volumetric analysis proved the higher densities of blood/lymphatic endothelial cells in the intra-tumoral stroma than in the peripheral environment. Our 3D survey also enabled delineation of tumor-host border and quantitative assessment of blood/lymphatic vessels penetrating through the border zone. The average size of these tumor-penetrating spots suggested that blood/lymphatic vessels were dilated in high-metastatic OSC19 and OSC20 tumor stroma in comparison to those in low-metastatic KOSC2. We also achieved the direct visualization of the unique invasive properties among cell lines, in particular, prominent infiltration of HO-1-u-1 cells around peripheral nerves.
Conclusions: The 3D morphometric analyses, which cannot be extrapolated from 2D histology, provide a new insight in our understanding of stromal remodeling with OSCC development and invasion.
Methods: OSCC cell lines that represented high-metastatic potential (OSC19 and OSC20) and low-metastatic potential (KOSC2 and HO-1-u-1) were used. Each cell line grown in culture was transplanted into a BALB/c nude-mouse tongue. Whole tongue tissues were dissected from the animals at periodical intervals after inoculation, followed by preparation of paraffin-embedded serial sections. Cancer cells, blood/lymphatic vessels, and nerve fibers were immunolabeled step-wisely with cytokeratin, PECAM-1, Lyve-1, and NFP antibodies, respectively. For three-dimensional (3D) reconstruction using 100 serial sections (4 μm thick), RGB color images of immunostained sections were acquired using a virtual microscope (NanoZoomer, Hamamatsu Photonics), and image registration and segmentation were automated with the aid of Image-J/Fiji software (NIH).
Results: The 3D reconstruction highlighted the vascular enrichment in each tumor microenvironment, and the volumetric analysis proved the higher densities of blood/lymphatic endothelial cells in the intra-tumoral stroma than in the peripheral environment. Our 3D survey also enabled delineation of tumor-host border and quantitative assessment of blood/lymphatic vessels penetrating through the border zone. The average size of these tumor-penetrating spots suggested that blood/lymphatic vessels were dilated in high-metastatic OSC19 and OSC20 tumor stroma in comparison to those in low-metastatic KOSC2. We also achieved the direct visualization of the unique invasive properties among cell lines, in particular, prominent infiltration of HO-1-u-1 cells around peripheral nerves.
Conclusions: The 3D morphometric analyses, which cannot be extrapolated from 2D histology, provide a new insight in our understanding of stromal remodeling with OSCC development and invasion.