Christophe M Raynaud ¹ Ayesha Jabeen ¹ Eiman I Ahmed ^1,2 Satanay Zuhair Hubrack ³ Apryl Sanchez ¹ Shimaa Sherif ¹ Ahmad A Al-Shaibi ³ Bernice Lo ³ Jessica Roelands ⁴ Davide Bedognetti ^5,6 Wouter R.L. Hendrickx ^1,5*

• ¹ Tumor biology and immunology laboratory, Research Branch, Sidra Medicine, Doha, Qatar
• ² Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar
• ³ Laboratory of immunoregulation, Research Branch, Sidra Medicine, Doha, Qatar
• ⁴ Department of Pathology, Leiden University Medical Center (LUMC), Leiden, Netherlands
• ⁵ College of Health & Life Science, Hamad Bin Khalifa University, Doha, Qatar
• ⁶ Clinical and Experimental Oncology and Hematology, Ospedale Policlinico San Martino, Genova, Italy

Introduction: Colorectal cancer (CRC) is a prevalent malignancy with significant morbidity and mortality worldwide. A deeper understanding of the interaction of cancer cells with other cells in the tumor microenvironment is crucial for devising effective therapeutic strategies. MUC2, a major component of the protective mucus layer in the gastrointestinal tract, has been implicated in CRC progression and immune response regulation. Method: In this study, we sought to elucidate the relationship between MUC2 expression and immune infiltration within CRC, using in vitro models involving two well-established cell lines, HT-29 and LS-174T. By employing CRISPR-mediated MUC2 knockout, we investigated the influence of MUC2 on tumor immune infiltration and its interplay with T cells and NK cells enriched peripheral blood mononuclear cells (PBMCs) in 3D spheroid cultures. Results: While MUC2 was more abundant in LS-174T cell line compared to HT-29, its knockout resulted in increased immune infiltration solely in the HT-29 cell line, but not in the LS-174T cell line. We revealed that the removal of MUC2 protein was compensated in LS-174T by the expression of other gel forming mucin proteins (MUC6, MUC5B) commonly expressed in gastrointestinal epithelium, while this was not observed in HT-29 cell line.Our study is the first to demonstrate that MUC2 functions as a physical barrier to immune infiltration in colorectal cancer (CRC) in vitro. In HT-29 cells, MUC2 knockout increased immune infiltration, while in LS-174T cells, compensatory expression of other mucins (MUC6, MUC5B) maintained the barrier. These findings reveal the complexity of mucin biology in CRC and suggest that targeting mucin pathways could be a novel therapeutic approach.