Background and purpose: Both regorafenib (REG) and TAS102 are novel anti-tumor drugs in the treatment of the digestive cancers. REG in combination with 5-fluorouracil (5-FU)

a pyrimidine analogue

could inhibit the progression of multi- drug resistant metastatic colorectal cancer. Cancer stem cells (CSCs) determine tumor self-renewal

heterogeneity and therapeutic resistance

and targeting CSCs provides a curable approach to multiple malignancies. This study aimed to investigate the therapeutic effect and potential mechanism of REG in combination with TAS102 against hepatocellular carcinoma (HCC). Methods: A panel of human liver cancer cell lines HepG2

Huh7 and SK-Hep1 were used in this study. Liver cancer cells and xenograft tumor animals were treated with REG and TAS102 monotherapy or combining regimen. Cell counting kit-8 (CCK-8) was used to assess HCC cell viability. The subpopulations of CD133 positive cells were detected and analyzed using flow cytometry. Western blot was used to evaluate these protein contents in HCC cells with diverse drug treatments. HCC spheres were cultured in growing sphere medium with diverse drugs. Xenograft tumor animals were used to assess the therapeutic effect in vivo. Results: REG or TAS102 monotherapy significantly inhibited HCC cell viability. TAS102 in combination therapy reduced the increase of CD133 + cell subpopulation

SRY-related high mobility group box protein-2 (SOX2) and aldehyde dehydrogenase 1A (ALDH1A) expressions induced by REG alone. Moreover

REG attenuated TAS102-induced myeloid cell leukemia-1 (MCL-1) expression. REG in combination with TAS102 ameliorated the formation of HCC sphere. Animal experiment demonstrated that REG in combination with TAS102 significantly eliminated HCC tumor growth in xenograft animals. Conclusion: REG in combination with TAS102 regulates HCC stemness and MCL-1 signaling and improves the therapeutic effect against HCC. The combination therapy may provide a novel strategy for refractory HCC patients.