High expression of SLIT2 in intrahepatic cholangiocarcinoma stroma

Introduction Intrahepatic cholangiocarcinoma is the second most common primary liver cancer with very poor prognosis. SLIT2 is the ligand of ROBO-receptors, which were shown to be deleted exclusively in invasive CCA (Goeppert et al., 2022). Project aim is to investigate SLIT2-ligand in iCCA tumor-microenvironment. [1]

Methods To detect expression of SLIT2 in iCCA microenvironment spatially, RNA-Scope was applied with vimentin co-detection in healthy liver, fibrotic liver and iCCA tissue samples. For in-vitro functional assays SLIT2 was knocked-down in LX-2, a hepatic stellate cell line, using siPOOL. The effects were investigated in colony-formation assay and cell-adhesion assay. Knockdown confirmation and changes in target genes were checked by qPCR.

Results We have optimized and validated RNA-scope assay. The results revealed the localization in the tumor stroma, which was then confirmed with vimentin co-detection. Low SLIT2 was observed in healthy liver; fibrotic liver showed an increase in SLIT2 signal and the strongest signal was detected in the tumor stroma in iCCA. In LX-2, clonogenicity and adhesion of the cells were increased with knockdown of SLIT2. alpha-SMA and Fibronectin were significantly downregulated, whereas ICAM was upregulated.

Conclusion RNAScope showed iCCA tumor stroma has enriched SLIT2 levels. SLIT2-knockdown in LX-2 cells showed downregulation at fibrosis genes. Further investigation of SLIT2 related effects on CCA cell lines will be done.

Publication History

Article published online:
20 January 2025

• References

• 1 Goeppert B. 1 et al. 2022 “Integrative Analysis Reveals Early and Distinct Genetic and Epigenetic Changes in Intraductal Papillary and Tubulopapillary Cholangiocarcinogenesis.”. Gut.