Abstract 1960: Invasive potential of colorectal cancer through tumor budding in 3D
Authors: Haddad Tariq , van den Dobbelsteen Luuk , Lugli Alessandro , van Lent-van Vliet Shannon , Simmer Femke , Zlobec Inti , Nagtegaal Iris 
Published: June 2022

Abstract
Background and Objectives: Tumor budding (TB) is defined as isolated single cancer cells or clusters of up to four cells located at the invasive tumor front of colorectal cancer (CRC) and other solid cancers. Despite the growing body of evidence establishing TB as a strong independent prognostic marker in CRC, further investigation is needed to elucidate the biological mechanisms behind TB and its invasive potential. Pseudobudding (PsB), in contrast to TB, is defined as single cancer cells or clusters of up to four cells that occur due to gland disruption and external influences such as inflammation. We hypothesize that the invasive potential of a tumor is therefore better reflected by the active process of TB rather than through the reactive process of PsB. By improved visualization of the invasive tumor front, we can characterize TB and PsB, elucidate their underlying biological processes, and improve future TB scoring accuracy in routine diagnostic practice.
Methods: Cases containing TB and PsB were selected from the pathology archives. Thick sections (80-90µm) of formalin-fixed paraffin-embedded CRC tissue were taken using a microtome and stained using a free-floating immunostaining protocol for Pan-cytokeratin (epithelium) and DAPI (nuclei). Thick sections were imaged via confocal microscopy. Three-dimensional z-stacks were processed and analyzed using FIJI software. Tissue microarrays (TMAs) of regions with TB and PsB were created and RNA spatial transcriptomics was conducted on thin TMA sections (5µm) using the Nanostring GeoMx Digital Spatial Profiling Platform.
Results: For sections where the invasive margin contained TB, both single cells and cell clusters appear isolated from the core tumor in 3D. These single cells and cell clusters exhibit migratory and invasive features associated with epithelial-mesenchymal transition (EMT), as evident by complete loss of cell-to-cell adhesions and shape shifting through elongation of the entire cell. In contrast, PsB is characterized by cellular debris surrounding a discontinuous epithelial lining. Undivided cells and cell clusters within the damaged area are observed as involuntarily disassociated with lagging remnants of cell-to-cell adhesions. This may be due to inflammatory cells disrupting the extracellular matrix and pulling the cells apart from one another. The identification of differing morphologies allows for further study of the RNA spatial transcriptome (of which the data will be presented during the meeting).
Conclusion: Our findings show that TB and PsB are independent features within the invasive tumor margin which represent two separate biological processes. Considering the strong predictive capacity of TB as a marker of poor prognosis in CRC, TB scoring within routine diagnostic practice benefits from avoiding areas with a strong inflammatory reaction and PsB.
Citation Format: Tariq Haddad, Luuk van den Dobbelsteen, Alessandro Lugli, Shannon van Lent-van Vliet, Femke Simmer, Inti Zlobec, Iris Nagtegaal. Invasive potential of colorectal cancer through tumor budding in 3D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1960.