Correlations between cancer stemness and the tumor immune microenvironment, as well as ICIs-related biomarkers, including ICP expression levels, tumor mutational burden (TMB), and microsatellite instability (MSI), were subsequently analyzed.<h4>Results</h4>Spheroid cultures exhibited a significant elevation in the expression of stemness markers (e.g., ALDH, NANOG, and SOX9), confirming the successful enrichment of CSC subpopulations. This evidence concerns the gene NANOG and cancer.