Lenvatinib Inhibits The Effect Of Cancer-associated Fibroblasts On Driving Hepatocellular Carcinoma Metastasis By Downregulating CEMIP

Objective:Hepatocellular carcinoma(HCC,hepatocellular carcinoma)is the leading pathological type of primary liver cancer and the third leading cause of cancer death,with high recurrence and metastasis rates resulting in still suboptimal long-term survival rates for patients.As a first-line drug in the treatment of HCC,Lenvatinib is a multi-targeted tyrosine kinase inhibitor used.In recent years,the exploration focus of tumor development mechanism and treatment has gradually shifted from the tumor itself to the tumor microenvironment(TME).CAFs(cancer-associated fibroblasts)are the most important stromal cells in the TME and can contribute to HCC metastasis through a variety of pathways and are also potential targets for tumour therapy.The direct mechanism of action of lenvatinib on hepatocellular carcinoma cells is well established,but no studies have reported whether lenvatinib can affect the biological behaviour of CAFs.Our study aims to fill this gap and provide new ideas for therapy targeting the tumour microenvironment.Methods:At the tissue level,we examined the expression of CEMIP of CAFs in paraffin-embedded tissues of 104 HCC patients by immunohistochemistry for correlation of clinicopathological features,and collected tissues from 6 patients each with and without lenvatinib-treated HCC to observe H&E stained images and perform multicolor immunofluorescence staining.At the cellular level,we cultured primary PTFs(Paracancer tissue fibroblasts)and CAFs and further investigated their role in HCC metastasis and the effect of lenvatinib on them using scanning electron microscopy,immunofluorescence,wound-healing,and transwell.At the molecular level,we analysed data from transcriptomic sequencing and drug target proteomics to explore the effects of lenvatinib on CAFs and the possible mechanisms of action.At the animal level,we validated the effect of CAFs on intrahepatic metastasis of HCC by establish the in situ implantation model of hepatocellular carcinoma in nude mice.Results:1.The proportion of fibroblasts in cancer nests was higher than in normal liver tissue adjacent to the cancer.Patients with HCC with a high proportion of CAFs had a poor prognosis,and the proportion of CAFs was significantly lower in HCC patients treated with lenvatinib than in untreated patients.Patients with HCC with high CEMIP expression in CAFs had a worse prognosis.There were significant differences in tumor number,alcoholism,and death between the high and low CEMIP expression groups.Patients treated with lenvatinib had fewer CAFs and lower CEMIP expression.2.CAFs have stronger collagen contraction and migration ability than PTFs and can be inhibited by lenvatinib.Huh7 co-cultured with CAFs had stronger migratory ability,while lenvatinib-pretreated CAFs had diminished chemotaxis towards Huh7.lenvatinib-pretreated CAFs showed significantly reduced physical connection with Huh7.3.CEMIP(Cell Migration Inducible Protein),which was more highly expressed in CAFs than PTFs and down-regulated by lenvatinib,was screened by transcriptome sequencing.Lenvatinib inhibit the migration,collagen contraction and the collective migration of CAFs by down-regulating CEMIP.Lenvatinib has a wide range of targets in CAFs that are enriched in the regulation of actin cytoskeleton,cell proliferation,growth,and cancer pathways.4.In the nude mouse in situ implantation model,the mixed CAFs group had more tumor nodules than the pure Huh7 group and the mixed PTFs group,and the mixed 1/3CAFs group had more liver nodules than the mixed 1/6 CAFs group.Conclusion:We found that lenvatinib inhibited the effect of CAFs on driving HCC metastasis through downregulation of CEMIP.Patients with HCC with a high proportion of CAFs had a poor prognosis,and the proportion of CAFs was significantly lower in HCC patients treated with lenvatinib than in untreated patients.CAFs had a stronger capacity for migration and collagen contraction than PTFs,and lenvatinib inhibited these malignant phenotypes.Lenvatinib reduced the physical connection between CAFs and HCC cells,thereby inhibiting the effect of CAFs on driving HCC migration.We further explored the treatment of hepatocellular carcinoma by targeting the tumour microenvironment.