Patients with a presumptive diagnosis of lung cancer for whom surgical resection or sampling will be clinically indicated according to the standard practices of the Cardiothoracic Department of AHEPA University Hospital of Thessaloniki, will be enrolled in this prospective study once they give written informed consent for specimen imaging. Surgical resections will be performed per standard of care and there will be no difference in patients' clinical management depending on the acquisition or not of surgical specimens. Patients with altered mental status and those who are unable or unwilling to provide informed consent will be excluded from this study.
Sample preparation Following surgical resection, human lung tissue specimens will be placed in a sterile container by the surgeon in the operating room. Choice of container will be based on specimen size. Right after the end of the operation, each specimen will be fixed in neutral buffered formalin for 48 hours in tissue cassettes and will be embedded in paraffin wax following a standardized protocol.
Imaging protocol After the collection of the formalin-fixed and paraffin-embedded (FFPE) surgically resected lung cancer specimens, these will be transported to the μ-VIS X-ray Imaging Centre at the University of Southampton in accordance with particular biological material transfer agreement. The FFPE lung samples will be scanned at the μ-VIS X-ray Imaging Centre using a custom-built Nikon Metrology micro-CT scanner according to standardized protocol.
Histological assessment Following non-destructive micro-CT imaging, the scanned FFPE specimens will be transported to the Pathology Laboratory of the First Pathology Department of the National and Kapodistrian University of Athens in accordance with particular biological material transfer agreement. The specimens will be set in paraffin blocks for sectioning. After sectioning, sections will be de-paraffinized and stained using Movat's pentachrome stain. The sections will be imaged and assessed histologically by an experienced and "blinded" pulmonary pathologist (S.T.). The pathologist will also assess a number of non-scanned FFPE lung cancer specimens to confirm the non-destructive nature of micro-CT imaging for the resected specimens. Identification of radiation-induced or ischemic alterations, deviant necrosis or cellular degeneration could be indicative outcomes of a destructive imaging method.
Statistical analysis The generated imaging data will be statistically analyzed using Bland-Altman plots to determine intra- and inter-observer variability in the interpretation of micro-CT imaging data. Wilcoxon's rank sum test or Kruskal Wallis's test will be utilized to evaluate any differences within specific morphometric measurements (percent object volume, object surface/volume ratio, object surface density, structure model index, structure thickness, structure linear density, structure separation, connectivity, connectivity density) among different growth patterns. All statistical analyses will be performed with SPSS (version 27) and a p-value of less than 0.05 will be considered as the threshold of statistical significance.
