Role for putative hepatocellular carcinoma stem cell subpopulations in biological response to incomplete thermal ablation: in vitro and in vivo pilot study.
OBJECTIVE
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.


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.


CONCLUSIONS
These studies provide experimental evidence supporting a role for HCC cells expressing the putative stem cell marker CD44 in HCC response to heat stress.
Medical Scientist Training Program, College of Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN, 55905, USA, thompson.scott@mayo.edu.
Scott M
SM
Thompson
Matthew R
MR
Callstrom
Kim A
KA
Butters
Shari L
SL
Sutor
Bruce
B
Knudsen
Joseph P
JP
Grande
Lewis R
LR
Roberts
David A
DA
Woodrum
D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000601:surgery*; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005434:Flow Cytometry / Q000379:methods; D006358:Hot Temperature; D066298:In Vitro Techniques; D053685:Laser Therapy / Q000379:methods*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000601:surgery*; D010865:Pilot Projects; D051381:Rats; D013234:Stem Cells / Q000378:metabolism*
D014408:Biomarkers, Tumor