Bacterial toxins as alternative anticancer agents exploiting liposome nanoparticles (LNP): How LNP-encapsulated recombinant C. jejuni CDTB acts against Caco-2 Colorectal Cancer Cells

Colon cancer (CC) is among the most prevalent cancers and a significant cause of death globally, with 153,020 new cases reported in 2023 [1, 2]. The defining characteristics of cancer include uncontrolled and unregulated cell growth, invasive, immortalization, proliferative, and metastasis [3]. The conventional treatment options include radiotherapy, chemotherapy and surgery, which have significant side effects [4]. Therefore, several studies have focused on developing further innovative therapeutic strategies, finding that microorganisms, although associated with the pathogenesis of CC, can act as highly selective therapeutic agents for the treatment of CC [5]. Cytolethal distending toxins (CDTs) are a group of bacterial protein toxins produced by several Gram negative bacteria, including C. jejuni These toxins induce apoptosis and cause double-strand breaks, leading to cell cycle arrest at the G2/M phase, primarily through the action of the catalytic subunit CdtB. This research focuses on the promising use of Cj-CdtB as a possible, useful therapy in the treatment of CC. Two different liposomal formulations were employed: undecorated and fucose-decorated liposome, both containing recombinant and purified Cj-CdtB to treat CC cells (Caco-2), besides the same formulations containing CDT-lysates. Our data demonstrate that the two different liposomes, Lipo CdtB and Fuc CdtB, can rapidly enter epithelial intestinal tumor cells and exert an antiproliferative effect, with differences related to the specific formulation. We found that after 48 h, Caco-2 tumor cells treated with Lipo-CdtB and Fuc-CdtB showed a significant reduction in cell number, and an increase in the percentage of dead cells was found for Fuc-CdtB. At 72h, this trend becomes even more evident compared to CdtB-treated cells. Treatment with FUC CdtB showed a significant increase in cells blocked in the G2/M phase compared to Control and CdtB-treated cells, confirming that the CdtB subunit alone is not sufficient to cause blockade in G2/M in the absence of the CdtA and CdtC subunits, which act as intracellular carriers. Furthermore, analysis of the CD133 molecule, a typical marker of cancer stem cells, revealed that treatment with Fuc-CdtB induces a reduction in CD133 surface levels after 48 hours. Further experiments are in progress to clarify the mechanistic effect on Caco-2 cancer stem cells. This work has been funded by the European Union - NextGenerationEU, Mission 4, Component 1, under the Italian Ministry of University and Research (MUR) National Innovation Ecosystem grant ECS00000041 - VITALITY - CUP H33C22000430006