Background Colorectal cancer (CRC) is among the most frequently diagnosed solid tumors and a leading cause of cancer-related deaths worldwide, and particularly in Qatar. Despite advances in the therapeutic landscape of CRC over the last decade, a significant proportion of patients show primary or acquired resistance. It becomes increasingly accepted that the presence of a rare subpopulation of cells within tumors endowed with self-renewal capacity and stemness properties, denominated as cancer stem cells (CSCs), is responsible for tumor formation, metastasis, recurrence and resistance to standard therapy. Moreover, these cells were found to display immunomodulatory properties and, thus, can escape immune recognition. However, the detailed mechanisms behind their tumorigenicity, immune evasion and therapy resistance are not fully understood. Particularly, the exact role of immune checkpoint (ICP) molecules in colorectal CSC development and resistance to immunotherapy remains unclear. This study is aimed at a) obtaining detailed molecular profiling of CSCs isolated from CRC cell lines with a focus on differential expression of immunomodulatory markers and b) identifying novel druggable targets for eliminating CSCs.

Methods Four colorectal cancer cell lines (HCT-116, HT-29, SW480, SW620) and normal colorectal cells (CCD841) were used in this study. Spheroid culture in serum-free media and low attachment flasks was utilized to enrich CSCs. The expression of stemness markers and inhibitory immune checkpoints were assessed by real-time PCR and Western blot analyses. Besides, a comparative proteomic analysis of the whole colorectal CSC proteome and their differentiated tumor counterparts was performed using a mass spectrometry-based label-free shotgun proteomics approach.

Results Cells grown in spheroid culture exhibited significantly elevated expression of stemness markers (e.g. ALDH, Nanog, CD133, Sox-2, Sox-9), confirming the enrichment of CSCs subpopulation. Notably, the upregulation of the stemness markers was associated with a proportional increase in the expression of a wide range of inhibitory checkpoints, such as PD-L1, PD-L2, B7H3, CD47, and CD155 as compared to their differentiated counterparts, indicating the potential role of these immune checkpoints in CRC CSC features.

Conclusions In summary, the unique immunological phenotype of the colorectal CSCs reported in our study suggests that CSCs might play a crucial role in tumor immune evasion and may serve as a preferential target for checkpoint blockade immunotherapy.

Acknowledgements This study was supported by Qatar University Internal grant number QUCG-CPH-23/24-154 to HMK and Qatar Research, Development and Innovation (QRDI) Graduate Studies Research Assistantship (GSRA) grant number GSRA8-L-1-0506-21033 to OJH.