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

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Abstract

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.

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.

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.

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Acknowledgments

This publication was supported by CTSA Grant TL1 TR000137 from the National Center for Advancing Translational Science (NCATS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Additional research support provided in part by a SIR Foundation allied scientist training Grant. Infrastructure support was provided by NIH construction grant NIH C06 RR018898.

Conflict of interest

Mr. Scott Thompson received a research grant from the SIR Foundation—Allied Scientist Training Grant. Dr. Lewis Roberts received research grants from Bristol Myers-Squibb, Merck, Nordion, and Bayer. Dr. Matthew Callstrom, Ms. Kim Butters, Ms. Shari Sutor, Mr. Bruce Knudsen, Dr. Joseph Grande, and Dr. David Woodrum declare that they have no conflict of interest.

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Authors and Affiliations

  1. Medical Scientist Training Program, College of Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN, 55905, USA

    Scott M. Thompson

  2. Department of Radiology, College of Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN, 55905, USA

    Matthew R. Callstrom, Kim A. Butters & David A. Woodrum

  3. Genomics Research Center, College of Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN, 55905, USA

    Shari L. Sutor

  4. Department of Laboratory Medicine and Pathology, College of Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN, 55905, USA

    Bruce Knudsen & Joseph P. Grande

  5. Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN, 55905, USA

    Lewis R. Roberts

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Correspondence to Scott M. Thompson.

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Thompson, S.M., Callstrom, M.R., Butters, K.A. et al. Role for Putative Hepatocellular Carcinoma Stem Cell Subpopulations in Biological Response to Incomplete Thermal Ablation: In Vitro and In Vivo Pilot Study. Cardiovasc Intervent Radiol 37, 1343–1351 (2014). https://doi.org/10.1007/s00270-013-0828-3

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