ASO Treatment in CTCs of Human HCC

CTC enrichment and detection in whole-blood samples of HCC patients were conducted following the method described previously.37 Cells that were CD45 negative, ASGR positive, and DAPI stained that met morphologic features of malignant cells (large cellular size, high nucleus-to-cytoplasm ratio, and visible nucleoli) were scored as HCC CTCs. ASO activity was assessed using a 3D culture condition that could culture and expand a very limited number of CTCs.38 Briefly, CTCs isolated were re-suspended in 150 μL DMEM containing unconjugated or GalNAc-conjugated MALAT1 ASO. Matrigel (Becton Dickinson, Franklin Lakes, NJ) was thawed and mixed equally with the CTC-containing DMEM. The prepared mixture was then incubated in a 24-well plate for 30 min at 37°C. Then, 500 μL DMEM containing the ASO was added to give specified final concentrations of ASO. The medium was replenished every 48 h. Spheroids were collected after 14 days of culture to get the sufficient amount of RNA for qRT-PCR.