To construct hepatocellular carcinoma (HCC) cells lines that can mimic sub-lethal heat shock (HS) in vitro and orthotopic HCC mouse model that received incomplete thermal ablation.

The suspension H22 cells were grown in 6-well plates, and the medium was replaced with fresh complete medium 8 hours before heat treatment. Then, the cells were divided into 4 groups for constant temperature water bath at 37°C, 42°C, 47°C, 50°C for 15 min, respectively. After the heat treatment was completed, the cells were maintained in a 37℃ incubator until detection. The phenotype of epithelial-mesenchymal transition (EMT) was detected by Western blot 48 hours later, and the cell viability was detected by Cell Counting Kit-8 (CCK-8) kit. The liver of mice was exposed by laparotomy along the abdomen midline and H22 cells (5×10⁶) were injected through the hepatic capsule to conduct orthotopic HCC model. When the longest diameter of the tumor reached approximately 8 mm, the mice were randomly divided into complete ablation group (cMWA), incomplete ablation group (iMWA) and Control group treated with sham operation. Microwave ablation along the long axis of tumor was performed to establish the incomplete thermal ablation model of orthotopic HCC. MRI was performed for tumor measurement and liver specimens were collected for histopathological assessment at day 14 after microwave ablation.

14 daysafter inoculation of H22 cells, the tumors reached average tumor diameter of 9.32 ± 0.83 mm which was predetermined to be suitable for microwave ablation. 14 days after microwave ablation, the average tumor diameter reached 12.93 ± 1.51mm in iMWA group, which were significantly bigger than preoperative period. In addition, immunofluorescence staining showed a large number of infiltrated neutrophils in the residual tumor of iMWA group. Western blot was used to observe the change in EMT-phenotype, results showed that the expressions of Vimentin and α-SMA were up-regulated and the expressions of E-cadherin were down-regulated.

HCC cells lines that can mimic Sub-lethal heat shock (HS) in vitro and orthotopic HCC mouse model that received incomplete thermal ablation were successfully constructed. There was substantial staining for neutrophils infiltration after incomplete ablation. Incomplete ablation may allow HCC cells to become more prone to migrate and to become more invasive.