Role for Putative Hepatocellular Carcinoma Stem Cell Subpopulations in Biological Response to Incomplete Thermal Ablation: In Vitro and In Vivo Pilot Study

Purpose: To investigate the potential role for CD44⁺ and CD90⁺ hepatocellular carcinoma (HCC) cellular subpopulations in biological response to thermal ablation-induced heat stress.

Methods: This study was approved by the institutional animal care committee. The N1S1 rat HCC cell line was subjected to sublethal heat stress (45 °C) or control (37 °C) for 10 min, costained with fluorescent-labeled antibodies against CD44, CD90, and 7-AAD after a 48-h recovery and analyzed by flow cytometry to assess the percentage of live CD44⁺ and CD90⁺ HCC cells (n = 4). Experiments were repeated with pretreatment of N1S1 cells with a dose titration of the dual PI3K-mTOR inhibitor BEZ235 or vehicle control (n = 3). Rats bearing orthotopic N1S1 tumors were subjected to ultrasound-guided partial laser ablation (n = 5) or sham ablation (n = 3), euthanized 24 h after ablation, and liver/tumor analyzed for immunohistochemical staining of CD44 and CD90. Differences between groups were compared with an unpaired t test.

Conclusion: These studies provide experimental evidence supporting a role for HCC cells expressing the putative stem cell marker CD44 in HCC response to heat stress.

Results: Sublethal heat stress induced a significant increase in the relative proportion of live CD44⁺ and CD90⁺ HCC cells compared to the control group: CD44⁺CD90⁻ (5.3-fold; p = 0.0001), CD44⁻CD90⁺ (2.4-fold; p = 0.003), and CD44⁺CD90⁺ (22.0-fold; p < 0.03). Inhibition of PI3K-mTOR prevented heat stress-induced enrichment of the population of live CD44⁺ HCC cells (p < 0.01), but not CD90⁺ cells (p > 0.10). Immunohistochemical analysis demonstrated preferential localization of clusters of CD44⁺ cells at both the tumor margin and ablation margin.