Fig. 4. Pharmacological inhibition of G9a HMTase activity is reducing tumor-initiating capacity in primary CRC patient samples.

A Schematic representation of serial organoid formation assay using primary human CRC tissues. Cancer stem cell (CSC) fraction is enriched in non-adherent spheroid cultures. B Flow cytometry profiling of patient-derived spheroid cultures for CCSC markers CD133 and CD44. Normal progenitor cells (HIEC) and CCSC-like HT29 were presented as controls. C Quantitative PCR assessment of CCSC markers LGR5, CD44, and CD113, as well as G9a expression in bulk primary organoids vs. CSC-enriched fractions (spheroid cultures) (Two patients tested, n = 3 per patient, ***: p ≤ 0.0004; **: p ≤ 0.0093; *: p ≤ 0.041). D Primary organoid formation frequency observed upon UNC0642 treatments (2.5 μM and 5 μM, 7 days). Organoid counts were normalized vs. DMSO controls (three patients, n ≥ 12, ***: p < 0.0001). E Organoid formation frequencies observed in secondary plating assays, for DMSO, UNC0642 2.5 μM and UNC0642 5 μM groups. Organoid counts were normalized vs. DMSO controls (three patients, n = 6, ***: p < 0.0001; **: p = 0.005). F Integrative analysis using ChIP-seq and RNA-seq transcriptome profiling to identify G9a and H3K9me2 co-enriched annotated genes in primary human CCSCs (GSE82131) that are silenced in HT29 (CCSC-like) vs. normal progenitors (HIEC) (p < 0.05). G Gene ontology analysis of G9a/H3K9me2 co-enriched genes and silenced in CCSC (vs. normal) reveals the implication of key pathways linked to tumorigenicity. H Diagram illustrating relative mRNA expression of G9a/H3K9me2 co-enriched genes in human CCSCs (vs. HIEC, p < 0.05) across key functional categories (GO:0030178, M18757, GO:0031012). I Recapitulative schematic highlighting the impact of G9a inhibition on the loss of pluripotent-like gene expression patterns and the onset of differentiation transcriptional programs in human CRC-initiating cells.