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

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 immunohisto-chemical 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.

Increased matrix stiffness promotes tumor progression of residual hepatocellular carcinoma after insufficient heat treatment

“…(16,17) In this study, we showed that increased matrix stiffness promotes the progression of heat-treated residual HCC cells, which will help to explain accelerated disease progression after incomplete RFA. Different from the previous reports that focused on the response of HCC cells themselves to sublethal heat, (9,10,(30)(31)(32)(33)(34) we propose a new mechanism of post-RFA increased stiffnessdependent residual tumor progression, namely, heat-treated residual HCC cells can sense the increased matrix stiffness after RFA to accelerate the tumor progression.…”

“…Multifaceted factors have been reported to be implicated in the acceleration of residual tumor or local recurrence after incomplete RFA. Residual HCC cells after RFA display the sarcomatous transformation, or the elevated expression of invasive and stem cell‐like markers . Concurrently, activated inflammatory responses in the microenvironment adjacent to the ablated site may also account for the aggressive progression of residual tumor after RFA, including enhanced expression of angiogenesis‐related genes, cytokines, growth factors, and chemokines and the enrichment of inflammatory cells .…”

“…Residual HCC cells after RFA display the sarcomatous transformation, (8) or the elevated expression of invasive and stem cell-like markers. (9,10) Concurrently, activated inflammatory responses in the microenvironment adjacent to the ablated site may also account for the aggressive progression of residual tumor after RFA, including enhanced expression of angiogenesis-related genes, cytokines, growth factors, and chemokines and the enrichment of inflammatory cells. (11)(12)(13)(14) However, RFA not only destroys the tumor, but also alters the biomechanical environment drastically.…”

Activated hepatic stellate cells secrete periostin to induce stem cell-like phenotype of residual hepatocellular carcinoma cells after heat treatment

“…Even more, some studies have shown that incomplete RFA accelerates the tumor progression by inducing epithelial-mesenehymal transition or expression of the stem cell markers in residual HCC cells 11, 12 . However, whether tumor environment provides supports to residual HCC cells after thermal ablation remains unknown.…”

Hepatic Radiofrequency Ablation–induced Stimulation of Distant Tumor Growth Is Suppressed by c-Met Inhibition

“…Therefore, for cases the mandatory ablation of normal tissue necessary in nearly all clinical cases to achieve an effective periablational margin) on distant extrahepatic tumor growth remains poorly characterized to our knowledge. Indeed, previous reports of pro-oncogenic effects of liver ablation have been based on models where incomplete ablation of liver tumors was performed, and off-target pro-oncogenic effects were attributed to secondary reactions within the partially injured tumor cells (4,10,28,30). Thus, much of the previous literature does not directly address the very common clinical scenario of complete local ablation, where normal liver in an adequate ablative margin comprises approximately 75% of ablated tissue volume (31,32).…”