Three-Dimensional Histologic, Immunohistochemical, and Multiplex Immunofluorescence Analyses of Dynamic Vessel Co-Option of Spread Through Air Spaces in Lung Adenocarcinoma

Spread through air spaces (STAS) is a method of invasion in lung adenocarcinoma and is associated with tumor recurrence and poor survival. The spatial orientation of STAS cells in the lung alveolar parenchyma is not known. The aim of this study was to use high-resolution and high-quality three-dimensional (3D) reconstruction of images from immunohistochemical (IHC) and multiplex immunofluorescence (IF) experiments to understand the spatial architecture of tumor cell clusters by STAS in the lung parenchyma.Four lung adenocarcinomas, three micropapillary-predominant and one solid predominant adenocarcinoma subtypes, were investigated. A 3D reconstruction image was created from formalin-fixed, paraffin-embedded blocks. A total of 350 serial sections were obtained and subjected to hematoxylin and eosin (100 slides), IHC (200 slides), and multiplex IF staining (50 slides) with the following antibodies: cluster of differentiation 31, collagen type IV, thyroid transcription factor-1, and E-cadherin. Whole slide images were reconstructed into 3D images for evaluation.Serial 3D image analysis by hematoxylin and eosin, IHC, and IF staining revealed that the micropapillary clusters and solid nests of STAS are focally attached to the alveolar walls, away from the main tumor.Our 3D reconstructions found that STAS tumor cells can attach to the alveolar walls rather than appearing free floating, as seen on the two-dimensional sections. This suggests that the tumor cells detach from the main tumor, migrate through air spaces, and reattach to the alveolar walls through vessel co-option, allowing them to survive and grow. This may explain the higher recurrence rate and worse survival of patients with STAS-positive tumors who undergo limited resection than those who undergo lobectomy.