Fig. 5: Re-expression of EHF and CDX1 promotes differentiation in poorly-differentiated CRC cells and inhibits tumour growth and metastasis in vitro and in vivo.

A, B Effect of EHF and CDX1 re-expression in poorly-differentiated HCT116 cells on A colony formation and B cell proliferation determined by MTS assay. C, D Effect of EHF and CDX1 knockdown alone and in combination in moderately-differentiated SW948 cells on C colony formation and D cell proliferation determined by MTS assay. Colony formation was assessed 14 days after seeding. Values shown for MTS assay (B, D) are mean ± SEM of a representative experiment performed in technical quadruplicate. E, F Effect of EHF and CDX1 re-expression alone and in combination in HCT116 cells on cell migration and cell invasion. F Effect of EHF and CDX1 knockdown, alone and in combination, in moderately-differentiated SW948 cells on cell migration and invasion. Values shown in E, F are mean ± SEM of the number of migrated and invaded cells, respectively, after 24 h, experiments performed in biological triplicates. G–I HCT116 stably re-expressing EHF and CDX1, alone or in combination were grown as xenografts in immune compromised mice. G Representative tumours at endpoint of HCT116^EV and HCT116^EHF+CDX1 cells. H Tumour volume was measured every second day for 15 days, and (I) tumour weight was determined at the experimental endpoint on day 15. Values shown are mean ± SEM of n = 5 mice, with 2 tumours injected per mouse (right and left flanks). J Histopathological assessment of differentiation grade in HCT116 xenografts following re-expression of EHF and CDX1 alone and in combination. K Histopathological assessment of differentiation grade in SW948 cells transfected with siRNAs targeting EHF and CDX1 alone and in combination. Mice were culled and xenografts removed 14 days post injection. L, M Effect of EHF and CDX1 re-expression on metastasis. HCT116 stably re-expressing EHF and CDX1, alone and in combination, were injected via the tail vein into NSG mice (n = 8 mice per isogenic line). Metastasis formation in the lung was determined after 8 weeks by L H&E staining of whole lungs or M quantitation of tumour burden by measuring human Vimentin DNA levels in the whole lung by q-RT-PCR. Values shown are mean ± SEM of whole lungs collected from n = 8 mice. *p < 0.05; **p < 0.01; ****p < 0.0001. Student’s t test in all cases.