Thermochemical Ablation (TCA) is a multiplexed, minimally invasive, image-guided method of tissue destruction, which combines hyperosmotic stress with hyperthermic ablation. It is anticipated that at the margins of a TCA-treated lesion, the concentration of salt would be lower than in the central region, and the hyperthermia would be both mild and relatively short compared to the core of the lesion. The aim of this study was to assess the generality of the effects of combining mild hyperosmotic and hyperthermic stresses on 4 hepatocellular cancer (HCC) cell lines. Viability and clonogenicity of 4 HCC cell lines with different genomic landscapes, namely Hep3B, HepG2, SNU449, and SNU398 were assessed in triplicate after in vitro exposure to increasing concentrations (0-400 mM) of sodium acetate (NaOAc), and sodium chloride (NaCl) for 24h at 37°C. A subset was also exposed to a concurrent sublethal heat shock of 43°C for 3h at the onset of treatment. All cell lines tested proved very sensitive to combined mild stresses. Hep3B and SNU449 cells maintained limited viability (below 50%) whereas HepG2 and SNU398 were nearly undetectable (below 1% viability). A potent additive effect was particularly evident for the combination of NaCl (over 100 mM) and heat. In addition, osmotic stress by NaCl alone induced a greater loss of viability than NaOAc alone at concentrations over 100 mM. Hep3B and SNU449 cells were relatively resistant to mild thermal stress alone (below 20% loss) compared to HepG2 and SNU398 lines, in which viability after the heat shock alone decreased considerably (over 70% loss). Clonogenic assays likewise demonstrated added toxic effects with combined stresses. Simultaneous thermal and osmotic stresses even at low levels show additive cytotoxicity, and the effect was observed across all HCC cell lines tested. The magnitude of the effect in each case likely reflects a combination of effects including the genomic landscape of the tumor cells and their microenvironment. These results suggest that in the marginal zone of a TCA-treated tumor cells would experience persistent cytotoxic stress. TCA may prove effective in lowering local recurrence rates in HCC.

To evaluate the distribution and embolization effect of ultrasmall 40-60 µm doxorubicin eluting beads (DEBs) versus small 70-150 µm DEBs after intra-arterial administration in the N1S1 rat model of hepatocellular carcinoma (HCC). All studies were approved by our institutional animal care and use committee and were performed in accordance with institutional guidelines. Twenty adult male Sprague-Dawley rats underwent hepatic arterial chemoembolization using a left transcarotid approach and were randomly assigned to receive a fixed dose of 4x10*4 of ultrasmall or small DEBs. The embolization endpoint was the injection of the entire dose. After 3 or 7 days, five animals per group were euthanized. Each tumor was sectioned in three equidistant regions along axial planes (cranial, middle and caudal). Bead number, spatial distribution, and percentage of tumor necrosis were measured on histology section. Mann-Whitney and Wilcoxon test were used to evaluate differences between groups. The overall percentage of necrosis was 33% and 56% for the ultrasmall DEBs at 3 and 7 days, and 10% and 28% for the small group, respectively (P<0.001). The effect of the treatment with ultrasmall DEBs was similar across different areas of the same tumor (p>0.05). Small DEBs showed necrosis predominantly in the cranial (34%) and central (25%) regions at day 7 (p = 0.004). No significant differences in the total number of beads were observed between the groups at any of the time points (p>0.05). Significant differences in bead distribution were observed in the ultrasmall size group, at both time points, by comparing tumor versus normal parenchyma regions (p<0.05). Small beads aggregate more frequently within the tumor, whereas the ultrasmall beads were equally distributed across normal liver and tumor. Higher rates of tumor necrosis were observed after ultrasmall DEB chemoembolization using equivalent bead dose. Small beads demonstrated a more heterogeneous tumor necrosis pattern with certain tumor regions consistently spared after embolization. Ultrasmall beads tend to be evenly distributed across both normal liver and tumor, whereas small beads distribute preferentially within tumor.

To investigate factors that impact technical success rate of transcatheter arterial chemoembolization in the most commonly used rodent model in interventional radiology research. All studies were approved by our institutional animal care and use committee and were performed in accordance with institutional guidelines. Ninety-one adult male Sprague-Dawley rats underwent hepatic arterial catheterization using a left transcarotid approach and were divided into N1S1 tumor-bearing (n = 50) and non-tumor bearing (n = 41) groups. Diameter of the proper (PH), left (LH) and right hepatic (RH) arteries, presence of anatomic variants, and animal weight were recorded at the time of angiography. Technical success of the procedure was defined as the stable placement of the microcatheter tip into the PHA. Spearman’s rank correlation coefficient (r) was calculated to determine the relationship between size of hepatic artery and weight of rats. Mann-Whitney and Wilcoxon tests were used to evaluate the differences between groups. There was a strong positive correlation between diameter of the PH and the weight of rats in non-tumor bearing group (r = 0.65; P<0.0001) and a similar moderate association was observed in the tumor bearing group (r = 0.40, P = 0.004).The overall success rate was 81%. There was a significant difference in both the PH diameter and the weight of rats between successful and suboptimal PH catheterization (0.59 vs 0.44mm; P<0.0001, 337.9 vs 252.6 g; P<0.0001). The left hepatic artery was significantly larger in diameter compared with the right hepatic artery (P<0.0001).Variant of anatomy was observed in one case. Rat weight positively correlates with proper hepatic artery diameter. Technical success rate is greater with PH diameters over 0.6 mm, typically present in animals weighing over 338 g. Familiarity with these factors can increase technical success rates of TACE in this rodent model.

The purpose of our study was to examine the effects of the metabolic modulator sodium dichloroacetate (DCA), a known inhibitor of mitochondrial pyruvate dehydrogenase kinase (PDK),on liver cancer cells when combined with thermal stress. DCA effectively blocks glycolysis and sensitizes cancer cells to other triggers of mitochondrial-mediated apoptosis. Thermochemical Ablation (TCA) is a new and flexible platform technology harnessing exothermic chemistry in situ. One of the TCA modalities we have previously used is based on the neutralization reaction of acetic acid and sodium hydroxide, which introduces a hot, concentrated solution of sodium acetate, a salt naturally present in the tissues. In addition to the hyperthermia associated with the reaction, the local persistence of the salt represents an additional opportunity to achieve a multiplexed anti-cancer treatment. Accordingly, we predict that by using a salt that is itself a drug (i.e. DCA), a sustained, high, local concentration of an antineoplastic compound can be attained through TCA. Viability and clonogenicity of human liver cancer cell lines Hep3B and HepG2, were evaluated after exposure to increasing concentrations of DCA (0-100mM), for 24h, with or without a mild thermal stress (43C) of 1h. Triplicate samples were run in each case and experiments were repeated three times. Without thermal stress, the loss of metabolic activity in HepG2 was apparent 3h after removal of DCA. However, the effect was transient, as full activity was restored 24h after DCA removal. Hep3B cells displayed a similar trend. Interestingly, the addition of the thermal stress sensitized the cells to the metabolic effects of DCA, and cell viability was permanently impaired in a dose-dependent fashion. Clonogenic assays likewise demonstrated added toxic effects of combined metabolic modulation and thermal stress. Metabolic modulation from glycolysis to glucose oxidation by DCA potently sensitizes liver cancer cells to mild hyperthermia. Further work to dissect the molecular mechanisms of this synergy and in vivo effects is warranted.

Thermochemical ablation (TCA) is a multiplexed, method of tissue destruction, which combines thermal and osmotic stress. At the margins of a TCA-treated lesion, the concentration of salt would be low, and the hyperthermia would be both mild and relatively short compared to the core of the lesion. These milder conditions may allow for cancer cell survival and lead to tumor recurrence. To purpose of this study was to assess the effects of such mild osmotic and hyperthermic stress on liver cancer cells, and to identify the pathways implicated in the response mechanisms. Viability of human Hep3B, HepG2 and rat McA-RH7777 cancer cell lines was assessed after exposure to sodium acetate (NaOAc, 0-800mM) at 37 or 43˚C for time periods of 1-3h. Triplicates were done and each experiment repeated 3 times. Clonogenicity and protein expression in HepG2 cells (200mM NaOAc at 37 or 43˚C) was evaluated through colony formation assay and mass spectrometry proteomics. Loss of viability was observed without thermal stress at 200-400mM NaOAc and toxicity was much greater with combined thermal and osmotic stresses. In both HepG2 and McA-RH7777 cells toxicity was observed at 100 mM NaOAc for 1h a 37˚C. Hep3B cells were most robust, although at 3h and 43°C all but controls fared poorly. Clonogenic assays likewise demonstrated added toxic effects with combined stresses in all cases tested. LC-MS/MS proteomics revealed a greater increase in Heat Shock protein-60 (HSP60) expression in HepG2 cells subject to combination treatment compared to either stress alone. 22 additional proteins were up-regulated >2-fold in combination treatment. Several of these implicate the unfolded protein response and fatty acid oxidation pathways. Simultaneous thermal and osmotic stresses show synergistic effects on liver cancer. Thus TCA may prove effective in lowering local recurrences rates in solid tumors. Furthermore, our proteomic screening show that the unfolded protein response and fatty acid oxidation pathways seem to be implicated in the response mechanisms to hyperosmotic and hyperosmolar stresses, suggesting they are potential targets to enhance the efficiency of our TCA platform.