Three-dimensional Visualization and Morphometry of Oral Squamous Cell Carcinoma Architecture

Objective: Recent advance in 3D reconstruction technology allows us to inspect the details of tumor architecture. The present study aimed at gaining 3D quantitative information about cell proliferation and atypia, and invasion into surrounding stroma of oral squamous cell carcinoma (SCC). Materials and methods: SCC cases of tongue in Japanese patients were collected from our University Hospital. Based on the pathodiagnostic survey on HE-stained sections, tissue core biopsies of 3 mm in diameter were dissected using a paraffin tissue microarray. Serially-cut sections (4 µm thick, 250-300 sections per individual biopsies) were dual-immunostained using cytokeratin antibodies for discrimination of parenchyma cytoplasm and Ki67 for cell proliferation assessment. Digital views were captured at 4 to 10 magnifications and reconstruction of 3D cancer architectures was achieved by alignment and superposition of a set of sectional views with the aid of RATOC TRI-SRF2 software. Results: In the current 3D reconstruction, cytokeratin positive parenchyma (yellow) and Ki67-positive nuclei (blue) were differentially labeled with Vector stain so that the labeled components were discriminated from stroma by means of RGB color segmentation procedures. Ki67 negative nuclei of cancer cells were also captured as an open space demarcated by cytokeratin-positive cytoplasm. The reconstructed 3D allowed us to conduct morphometry on 6-7×10⁷ µm³ in total tissue volume and 10⁴ nuclei, making it possible to accumulate quantitative information regarding parenchyma volume, nucleus number, Ki67-positive/negative ratio, N/C volume ratio, and distribution profile of nucleus size. Most interestingly, the 3D morphometry proved the continuous architecture of cancer parenchyma even at the infiltration front where 2D histology showed scattering and invasion of small cancer foci into tongue muscle. Conclusion: The present results support the potency and advantage of histology-based 3D reconstruction in the understanding of oral SCC biology and prognosis. Supported by Grants-in-Aid from MEXT/JSPS of Japan.