Hepatology, VOL. 72, NO. 4, 2020  Bespoken Nanoceria: An Effective Treatment in Experimental Hepatocellular Carcinoma Guillermo Fernández-Varo ,1,2 Meritxell Perramón ,1 Silvia Carvajal,1 Denise Oró,1 Eudald Casals,3 Loreto Boix,4 Laura Oller,1 Laura Macías-Muñoz,1 Santi Marfà,1 Gregori Casals,1,5 Manuel Morales-Ruiz,1,2,5 Pedro Casado ,6 Pedro R. Cutillas, Jordi Bruix, Miquel Navasa, Josep Fuster, Juan Carlos Garcia-Valdecasas, Mihai C. Pavel, Víctor Puntes,8-10 6 4 7 7 7 7 1,2 and Wladimiro Jiménez BACKGROUND AND AIMS: Despite the availability of of proteins mainly related to cell adhesion and RNA splicing. new-generation drugs, hepatocellular carcinoma (HCC) is still CeO2NPs decreased phosphatidylcholine-derived arachidonic the third most frequent cause of cancer-related deaths world- acid and reverted the HCC-induced increase of linoleic acid wide. Cerium oxide nanoparticles (CeO2NPs) have emerged in several lipid components. Furthermore, CeO2NPs reduced as an antioxidant agent in experimental liver disease because serum alpha-protein levels and improved the survival of HCC of their antioxidant, anti-inflammatory, and antisteatotic prop- rats. Nanoceria uptake by ex vivo perfused human livers and erties. In the present study, we aimed to elucidate the poten- in vitro human hepatocytes was also demonstrated. tial of CeO2NPs as therapeutic agents in HCC. CONCLUSIONS: These data indicate that CeO2NPs par- APPROACH AND RESULTS: HCC was induced in 110 tially revert the cellular mechanisms involved in tumor pro- Wistar rats by intraperitoneal administration of diethylni- gression and significantly increase survival in HCC rats, trosamine for 16 weeks. Animals were treated with vehicle suggesting that they could be effective in patients with HCC. or CeO2NPs at weeks 16 and 17. At the eighteenth week, (Hepatology 2020;72:1267-1282). nanoceria biodistribution was assessed by mass spectrometry (MS). The effect of CeO2NPs on tumor progression and H animal survival was investigated. Hepatic tissue MS-based epatocellular carcinoma (HCC) is the third phosphoproteomics as well as analysis of principal lipid com- leading cause of death by cancer worldwide.(1) ponents were performed. The intracellular uptake of CeO2NPs HCC commonly arises in patients with by human ex vivo perfused livers and human hepatocytes was underlying chronic liver disease and is considered a typ- analyzed. Nanoceria was mainly accumulated in the liver, where it reduced macrophage infiltration and inflammatory ical inflammation-associated tumor.(2) The appearance gene expression. Nanoceria treatment increased liver apoptotic of cirrhosis greatly favors the onset of HCC through activity, while proliferation was attenuated. Phosphoproteomic mechanisms not yet well known that began to be elu- analysis revealed that CeO2NPs affected the phosphorylation cidated in recent years.(3) In the last decade, complex Abbreviations: AA, arachidonic acid; AFP, alpha-fetoprotein; CD, cluster of differentiation; CE, cholesterol ester; CeO2NP, cerium oxide nanoparticle; DEN, diethylnitrosamine; Δ-6D, Δ-6 desaturase; EDX, energy-dispersive X-ray spectroscopy; ERK, extracellular signal–regulated kinase; FA, fatty acid; HAADF, high-angle annular dark-field; HCC, hepatocellular carcinoma; ICP-MS, inductively coupled plasma–mass spectrometry; Itgb4, integrin beta 4; LA, linoleic acid; MAPK, mitogen-activated protein kinase; MS, mass spectrometry; NEFA, nonesterified FA; NMP, normothermic machine perfusion; NP, nanoparticle; PC, phosphatidylcholine; PE, phosphatidylethanolamine; P-ERK, phosphorylated ERK; PUFA, polyunsaturated FA; ROS, reactive oxygen species; STEM, scanning transmission electron microscope; TEM, transmission electron microscopy; TG, triglycerides; TMAOH, tetramethylammonium hydroxide; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. Received August 26, 2019; accepted December 20, 2019. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.31139/suppinfo. Supported by Dirección General de Investigación Científica y Técnica, Ministerio de Ciencia, Innovación y Universidades (SAF15-64126-R and RTI2018-094734-B-C21, to W.J.; SAF2016-75358-R, to M.M.-R.), Agència de Gestió d’Ajuts Universitaris i de Recerca (SGR 2017/2019, to W.J.), Instituto de Salud Carlos III (FIS PI15-00077 and FIS PI19-00774, to G.C. and G.F.-V.; PI18/00763, to J.B. and L.B.), AECC (PI044031, to J.B. and L.B.), and WCR (AICR) (16-0026, to J.B. and L.B.). Co-funded by European Regional Development Fund/ European Social Fund (ERDF/ESF) "A way to make Europe"/ "Investing in your future". Cofinanced by Agència de Gestió d’Ajuts Universitaris i de Recerca and ERDF/ESF "A way to make Europe"/ "Investing in your future" under the Operational Program of Catalonia 2014-2020 (grant 2018 PROD 00187). The Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III. 1267 FERNÁNDEZ-VARO ET AL. Hepatology, October 2020 genetic alterations, epigenetic chromosomal aberrations, Antioxidant agents have demonstrated their efficacy and cellular signaling pathways triggering tumor devel- in chronic liver diseases equilibrating hepatic ROS opment, progression, and metastasis have been charac- metabolism, thereby improving liver functionality.(13) terized.(4) The current systemic treatments of HCC are Recently, cerium oxide nanoparticles (CeO2NPs) based on molecularly targeted therapies. Sorafenib, a have emerged as an antioxidant and anti-inflammatory multikinase inhibitor, was the first compound for first- agent. Superoxide dismutase activity(14) (conversion of line treatment of patients with advanced-stage HCC.(5) superoxide anion into hydrogen peroxide and finally However, clinical trials have found only modest oxygen), catalase activity(15,16) (hydrogen peroxide into improvement in overall survival,(6) and the emergence oxygen and water), and peroxidase activity(17) (hydro- of resistance episodes reveals the need to develop effec- gen peroxide into hydroxyl radicals), among others, tive therapies for HCC.(7) Although the available drugs have been attributed to CeO2NPs. Consequently, improve clinical outcomes, the median overall survival the wide spectrum of antioxidant enzyme-mimetic continues to be approximately 1 year.(8,9) activities of CeO2NPs has been explored in the treat- Of particular importance is the role of reactive ment of many diseases related to the overproduction oxygen species (ROS) in the onset and progression of ROS. Thus, the ability of CeO2NPs to modu- of HCC.(10) Mechanistic studies show that ROS late oxidative stress in diseases ranging from retinal induce alterations in DNA and modify key cellular degeneration,(18) neurodegenerative diseases,(19) dia- processes such as cell proliferation and apoptosis.(11) betes,(20) ischemia,(21) cardiopathies,(22) gastrointesti- Thus, it has been hypothesized that HCC develops nal inflammation,(23) and especially cancer has been because chronic oxidative stress exerts a selective pres- described.(24-27) As well, the therapeutic possibilities sure that favors the outgrowth of cells from progenitor of CeO2NPs have been shown in the case of exper- clones that are more resistant to oxidative damage.(12) imental liver disease.(28-32) © 2020 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creat​ive Commo​ns Attri​butio​n-NonCo​mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.31139 Potential conflict of interest: Dr. Bruix consults for, advises, is on the speakers’ bureau of, and received grants from Bayer-Shering and BTG. He consults for and advises MSD. He consults for and is on the speakers’ bureau for Sirtex. He consults for and received grants from Arqule and Ipsen. He consults for Novartis, Bristol-Myers Squibb, Eisai, Kowa, Terumo, Gilead, Bio Alliance, Roche, AbbVie, Merck, AstraZeneca, Incyte, Quirem, Adaptimmune, and Lilly. ARTICLE INFORMATION: From the 1 Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 2 Departament of Biomedicine, University of Barcelona, Barcelona, Spain; 3 School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, China; 4 Barcelona-Clinic Liver Cancer Group, Liver Unit, Hospital Clinic de Barcelona, CIBERehd, IDIBAPS, University of Barcelona, Barcelona, Spain; 5 Working Group for the Biochemical Assessment of Hepatic Disease-SEQCML, Barcelona, Spain; 6 Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK; 7 Liver Surgery and Transplant Unit, Digestive and Metabolic Diseases Institute, Hospital Clínic of Barcelona, CIBERehd, IDIBAPS, University of Barcelona, Barcelona, Spain; 8 Institut Català de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; 9 Institut Català de Nanociència i Nanotecnologia (ICN2), Bellaterra, Spain; 10 Vall d’Hebron Institute of Research (VHIR), Barcelona, Spain. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Wladimiro Jiménez, Ph.D. or Servicio de Bioquímica y Genética Molecular Victor Puntes, Ph.D. Hospital Clinic Universitario Institut Català de Nanociència i Nanotecnología, Campus UAB Villarroel 170 Barcelona 08193, Spain Barcelona 08036, Spain E-mail: victor.puntes@icn2.cat E-mail: wjimenez@clinic.cat Tel.: +34 93 7374622 Tel.: +34 93 2275400 ext. 3091 1268 Hepatology, Vol. 72, No. 4, 2020 FERNÁNDEZ-VARO ET AL. We considered the potential therapeutic value HCC INDUCTION IN RATS of CeO2NPs in an experimental model of HCC in rats by chronic administration of diethylnitrosamine Experimental studies were performed in 118 male (DEN). We assessed the impact of CeO2NPs on Wistar rats weighing 200 g (Charles-River, Saint tumor progression and survival, the accumulation in Aubin les Elseuf, France). HCC was chemically isolated human liver, and their intracellular adsorption induced in 110 rats by an intraperitoneal administra- by human liver-derived cancer cells. tion of DEN (50 mg/kg body weight; Sigma-Aldrich) once a week for 16 weeks, and eight healthy rats were included as a control group. Material and Methods ACCUMULATION OF Ce IN SYNTHESIS AND ISOLATED HUMAN LIVERS CHARACTERIZATION OF RAT To assess whether the human liver parallels the kinetic SERUM ALBUMIN STABILIZED adsorption pattern shown by CeO2NPs in the rat liver, CeO2NPs human livers from three donors, designated subjects H1, H2, and H3, were used in the present study. Livers CeO2NPs of 4-5 nm were synthesized by the chem- were procured in the Hospital Clinic (Barcelona, Spain), ical precipitation of cerium (III) nitrate hexahydrate rejected for transplantation, and approved for research. (Sigma-Aldrich, St. Louis, MO) in a basic aqueous solution.(14,15,17,28) Cerium (III), 10 mM, was dissolved in 100 mL of Milli-Q water at room temperature. CeO2NPs ADSORPTION BY HUMAN To this, 3 mL of tetramethylammonium hydroxide HEPATOCYTE CANCER CELLS (TMAOH) solution (1 M) was added slowly at room To investigate whether human hepatocytes are able temperature under vigorous stirring (final concentra- to intracellularly take up CeO2NPs, subsequent stud- tion of 10 mM), and the mixture was allowed to age ies were performed in HepG2 cells, a human cell line under mild stirring overnight. During the first minutes, derived from a liver HCC (ATCC, Manassas, VA). the solution is colorless, and then it turns progressively brownish. Afterward, nanoparticles (NPs) were puri- fied by centrifugation (10,000g, 10 minutes, at room ETHICAL APPROVAL temperature), and the resultant pellet was resuspended The study was approved and performed according to in 100 mL aqueous solution of 1 mM TMAOH. The the criteria of the Investigation and Ethics Committee xylenol orange test and inductively coupled plasma– of the Hospital Clínic Universitari (Barcelona, Spain). mass spectrometry (ICP-MS) indicated a full con- Animals received humane care according to the cri- version of Ce to CeO2NPs. Thus, the final CeO2 teria outlined in the “Guide for the Care and Use of concentration for this synthesis was determined to be Laboratory Animals”. The study of the human livers was 1.72 mg/mL (6.7•1015 NPs/mL) and then diluted to conducted in the Hospital Clínic de Barcelona, which the 1 mg/mL employed solution. Further details on is part of the European Union–funded Consortium for CeO2NPs synthesis and characterization can be found Organ Preservation in Europe (http://www.ope-eu.com). in the Supporting Information. For further information, please refer to the Supporting Information. CeO2NPs ADMINISTRATION CeO2NPs or vehicle were dispersed in saline solu- tion and intravenously given as a bolus (500 µL) Results through the tail vein. CeO2NPs (0.1 mg/kg body weight) or vehicle (saline solution containing CHARACTERIZATION OF CeO2NPs TMAOH ammonium salts 0.8 mM) were injected A description of the characterization of the twice a week for 2 consecutive weeks starting at the CeO2NPs used in this study has been published.(32) sixteenth week after beginning DEN administration. High-resolution transmission electron microscopic 1269 FERNÁNDEZ-VARO ET AL. Hepatology, October 2020 (TEM) analysis indicated that the NPs had a spherical in the liver and the spleen, respectively, whereas these morphology and were predominantly within the size figures were 77.3% and 21.7% 3 weeks after the com- range of 4-20 nm. The X-ray diffraction pattern showed pletion of the CeO2NPs dosing schedule (Fig. 1C). pure CeO2NPs with the typical peak broadening char- acteristic of nanosized particles. Initially, the employed SERUM BIOCHEMICAL NPs are positively charged, and the colloidal stability PARAMETERS AND CIRCULATING is mediated by electrostatic repulsion (zeta potential LEVELS OF ALPHA-FETOPROTEIN +43.0 ± 1.3 mV, conductivity 0.303 ± 0.006 mS/cm, and pH 4.3). Albumin conjugation in the surface of HCC rats showed significant alterations of liver the CeO2NPs has been characterized by the increase function tests such as decreased levels of albumin and in dynamic light scattering (from 5 to 20 nm) and glucose, higher levels of total bilirubin and total cho- decrease in zeta potential (from +42.8 to −10 mV, lesterol, and increased activity of markers of hepatocyte which is the average zeta potential value of proteins in injury such as aspartate aminotransferase, alanine ami- serum) (Supporting Fig. S1). Indeed, the solubility of notransferase, and gamma-glutamyl transferase. We did CeO2NPs in physiological media is challenging, and not observe significant differences between HCC rats a strong tendency to aggregate and sediment impedes receiving or not receiving CeO2NPs (Supporting Table their medical use even more, modifying their protec- S2). However, nanoceria administration did significantly tive effects toward the proinflammatory (https://www. reduce the circulating levels of alpha-fetoprotein (AFP), front​iersin.org/artic​les/10.3389/fimmu.2017.00970/​ a tumor-associated marker for HCC (Fig. 1B). full). To evaluate the electronic structure of the CeO2NPs and the influence on Ce valence state due EFFECT OF CeO2NPs ON to the presence of rat serum albumin, we performed COLLAGEN CONTENT AND X-ray photoelectron spectroscopy measurements. Ce three-dimensional spectra of all samples are shown in CELLULAR APOPTOSIS IN LIVER Supporting Fig. S2, where it can be clearly observed TISSUE that the Ce3+/Ce4+ ratio is not affected by the presence Long-term administration of DEN promoted of bovine serum albumin in the CeO2NPs surface. incipient formation of fibrotic septa and periportal accumulation of collagen as a consequence of continu- THE RAT MODEL OF ous hepatic injury. However, no significant differences DEN-INDUCED LIVER INJURY in hepatic collagen content were noted between treated and nontreated rats with HCC (Fig. 2A). EXHIBITS CHARACTERISTICS The terminal deoxynucleotidyl transferase–mediated OF MULTIFOCAL HCC deoxyuridine triphosphate nick-end labeling (TUNEL) Macroscopic examination of the liver specimens and assay showed a significant increase in positive cells in the histological examination confirmed the development of liver sections of DEN-injured rats treated with CeO2NPs multifocal HCC nodules with a dysmorphic or dyschro- (Fig. 2B). We also observed a significantly increased mic appearance (Fig. 1A) that was markedly attenuated protein expression of activated caspase-3 in HCC rats in the animals receiving CeO2NPs. Interestingly, DEN- treated with CeO2NPs (Fig. 2C). These results indicate injured rats treated with CeO2NPs showed a signifi- that treatment with CeO2NPs results in acceleration of cantly lower liver/body weight ratio (Fig. 1B). apoptosis, thereby inhibiting HCC growth. LIVER AND SPLEEN ARE THE CeO2NPs DECREASE MAJOR TARGETS OF CeO2NPs IN MACROPHAGE INFILTRATION HCC RATS AND REDUCE INFLAMMATORY GENE OVEREXPRESSION IN LIVER Following intravenous CeO2NPs administration, tis- TISSUE sue Ce accumulation was analyzed by ICP-MS. Two weeks after the last administration of CeO2NPs 90.0% Macrophage infiltration measured by positive and 7.7% of the total dose of Ce collected was located cluster of differentiation 68 (CD68) staining was 1270 Hepatology, Vol. 72, No. 4, 2020 FERNÁNDEZ-VARO ET AL. A +&&9HKLFOH C 2UJDQDFFXPXODWLRQ   ZHHNV ZHHNV  &HULXPFROOHFWHG PJ&H[      /LYHU 6SOHHQ /XQJ .LGQH\ +HDUW 6HUXP &HFROOHFWHG        B %RG\ZHLJKW /LYHUERG\ZHLJKWUDWLR $OSKDIHWRSURWHLQ   [ñ  &RQFHQWUDWLRQ SJPO   [ñ :HLJKW J  5DWLR      [ñ      &RQWURO &RQWURO &RQWURO +&& +&& +&& FIG. 1. DEN-induced model of HCC and biodistribution of CeO2NPs. (A) Representative photographs of the liver and hematoxylin and eosin staining (magnification, 40) from control and DEN-injured rats. DEN was administered for 16 weeks, followed by a 2-week washout period. Green arrows point at nodules present in HCC rats. (B) Body weight, liver/body weight ratio, and serum levels of AFP in control rats and HCC rats. Serum samples were collected 1 week after the last dose of CeO2NPs. (C) ICP-MS quantification of cerium. Major organs and serum from HCC rats receiving CeO2NPs collected at two different time points: 2 (n = 3) and 3 (n = 4) weeks after the last administration of CeO2NPs. This corresponds to the nineteenth and twentieth weeks after starting DEN administration. **P < 0.01 and ***P < 0.001 versus control; #P < 0.05 versus HCC + vehicle. One-way analysis of variance and Newman-Keuls post hoc test. observed in intratumoral and peritumoral areas, CeO2NPs DECREASE HEPATIC being significantly lower in HCC rats receiving CELLULAR PROLIFERATION CeO2NPs (Fig. 3A). mRNA expression of inflam- matory, macrophage phenotype, cell growth and We assessed the effect of CeO2NPs on cell pro- differentiation genes was analyzed in liver biopsies liferation and the phosphorylation levels of proteins of control and HCC rats. Chronic administration involved in tumor progression. Immunohistochemistry of DEN induced higher gene expression of mac- revealed abundant cellular proliferation in liver sec- rophage M1 markers, such as interleukin 1 beta, tions from DEN-injured animals. The cell prolifer- tumor necrosis factor alpha, inducible nitric oxide ation rate, measured as the percent of Ki67-positive synthase, and cyclooxygenase-2 (Supporting Table hepatocyte nuclei, was markedly lower in CeO2NPs- S3). Interestingly, administration of CeO2NPs sig- treated rats (Fig. 3B). To ascertain the potential effect nificantly down-regulated M1 genes involved in of CeO2NPs on interfering with the Ras/mitogen- proinflammatory function. activated protein kinase (MAPK) signaling pathway, 1271 FERNÁNDEZ-VARO ET AL. Hepatology, October 2020 A +&&9HKLFOH &ROODJHQFRQWHQW  )LEURVLVDUHD      &RQWURO +&& B 781(/TXDQWLILFDWLRQ +&&9HKLFOH   3RVLWLYHFHOOVILHOG    &RQWURO +&& &DVSDVH C &RQWURO +&&9HKLFOH   DUELWUDU\XQLWV 'HQVLWRPHWU\ $FWLYH  FDVSDVH .'D  *$3'+ .'D  &RQWURO +&& FIG. 2. Effect of CeO2NPs on hepatic fibrosis and cellular apoptosis. (A) Sirius red staining of representative liver sections obtained from control and DEN-injured rats (magnification, 100). (B) Representative TUNEL assay in liver sections of control rats and HCC rats (magnification, 200). White arrows point to apoptotic (red) cells. (C) Representative western blot for activated caspase-3 in liver tissue of control rats and HCC rats. *P < 0.05 and ***P < 0.001 versus control group; #P < 0.05 and ##P < 0.01 versus HCC + vehicle. One-way analysis of variance and Newman-Keuls post hoc test, Kruskal-Wallis test, and Dunn’s post hoc test, with unpaired Student t test when appropriate. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase. we assessed protein expression of total extracellular phosphoproteome profile by mass spectrometry signal–regulated kinase 1/2 (ERK1/2) and phosphor- (MS). We identified and quantified a total of 5,048 ylated ERK1/2 (P-ERK1/2) in liver samples of HCC phosphopeptides in six independent biological rep- rats by western blot. It was of note that CeO2NPs licates that were run twice. Principal component treatment resulted in a significant reduction of analysis showed that CeO2NPs-treated samples sep- P-ERK1/2 (Fig. 3C). These results suggest that the arate from vehicle-treated samples in principal com- antiproliferative action of CeO2NPs is related to ponent 1 (Fig. 4A). At arbitrary threshold values of interference with the Ras/MAPK signaling pathway ±0.8-fold change (log2) and P ˂ 0.05, the phos- in HCC rats. phorylation of 349 peptides was increased, while the phosphorylation of 133 was decreased after EFFECT OF CeO2NPs ON ALTERED CeO2NPs treatment (Fig. 4B; Supporting Table S4). This set of regulated phosphopeptides included 20 CELL SIGNALING PATHWAYS IN phosphorylation sites in kinases, of which 11 were LIVER TISSUE increased and nine were decreased (Fig. 4C). Gene To investigate the effects of CeO2NPs on ontology analysis showed that cell–cell adhesion kinase-driven signaling pathways, we evaluated the and RNA splicing were enriched in the set of genes 1272 Hepatology, Vol. 72, No. 4, 2020 FERNÁNDEZ-VARO ET AL. A +&&9HKLFOH 0$&523+$*(,1),/75$7,21  S &'3RVLWLYHFHOOV       +&& B +&&9HKLFOH &(//352/,)(5$7,21 .LSRVLWLYHFHOOV   S     C +&& +&& +&& &21752/ 9(+,&/(  .'D DUELWUDU\XQLWV  'HQVLWRPHWU\ S(5. S(5. .'D    (5. .'D  (5. .'D  *$3'+ .'D S(5. S(5. FIG. 3. Effect of CeO2NPs on infiltrating cells, cellular proliferation, and levels of P-ERK1/2 in tumoral tissue. (A) CD68 immunostaining of representative liver sections from HCC rats (magnification, 200). (B) Ki67 immunostaining of representative liver sections from HCC rats (magnification, 200). Red arrows mark nuclei positive for Ki67 staining. (C) Representative western blots of total ERK1/2 and P-ERK1/2 in liver homogenates of control rats and HCC rats. Forty micrograms of protein extracts were loaded per lane. *P < 0.05 and **P < 0.01 versus control; ##P < 0.01 versus HCC + vehicle. One-way analysis of variance and Newman-Keuls post hoc test, with unpaired Student t test when appropriate. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase. that code for the regulated phosphopeptides (Fig. after CeO2NPs treatment (Fig. 4F). Finally, the set 4D). The set of regulated phosphopeptides linked of regulated phosphopeptides linked to RNA splic- to cell–cell adhesion included 43 sites in 27 proteins ing included 36 sites in 18 proteins that presented that presented an increased phosphorylation after increased phosphorylation and four sites in three CeO2NPs treatment and 16 sites in 11 proteins that proteins that showed decreased phosphorylation presented a decreased phosphorylation after NP (Fig. 4G). These data suggest that CeO2NPs have a treatment (Fig. 4E). In addition, proteins linked to global effect on the phosphorylation pattern of liver cell-matrix adhesion including CD44 and integrin cells from rats with HCC that mainly affects pro- beta 4 (Itgb4) showed a decreased phosphorylation teins related to cell adhesion and RNA splicing. 1273 FERNÁNDEZ-VARO ET AL. Hepatology, October 2020 A B C 3NQ 6 (HIN 6 SDN  :QN 6 9+  $UDI   /DWV 6 3UNFL 7  3UNFL 7 ORJ SYDOXH  0DSN  5RFN 6 3&   0LQN 7 13 13 13 9+ 9+ 13  /DWV 7 9+ 9+ 13  3UNFK  13  9+ 1HN 7       3WN 6 3&  $DN 7    7DRN  )ROG 'FON 6 1HN 6 0DUN 6 D E             ORJ SYDOXH       9DVS 7 3KOGE 6 6Q[ 6 3DFVLQ 6 (SV 6 3DFVLQ 6 3NQ 6 /UUILS 6 +QUQSN 7 (UF 7 &WQQG 6 &GDS < %DLDSO  &UNO < &UNO 6 )QESO 6 )QESO 6 )QESO 7 6HUES  ,GK  %DLDSO  6KURRP < &WQQG 6 )QESO 6 6HUES 6 &KPSE 6 &WQQG < )QESO < &WQQG 6 +QUQSN 6 (LIJ 6 1FN < (LIJ 7 6KWQ 6 (LIK 6 6SWDQ 6 6KURRP < &UNO < 8VR  6HUES  3KOGE 6 3DUG 7 9FO 6 0DUN 6 'EQO  %DJ 6 -XS 6 &JQ 6 &WQQG 6 &WQQE 7 7OQ 6 +FIF 6 6SWDQ 7 &WQQE 7 &WQQE 6 3NS 6 7OQ 7 7OQ 6 (LIJ 6       1XPEHURI+LWV F G                  ,WJE  &G   7UDE 6 7UDE 6 7UDE 6 6UP 6 6UP 6 6UUP 6 ,ZV 6 6UUP 6 +QUQSN 7 6UUP 6 0.8 (increased) or <−0.8 (decreased). Statistical significance was assessed using an unpaired two-tailed Student t test. (C) Chart showing phosphorylation sites in kinases regulated by CeO2NPs treatment. (D) Volcano plot showing gene ontologies enriched in proteins whose phosphorylation was regulated by CeO2NPs. Statistical significance of the enrichment was assessed using a modified Fisher’s exact test. (E) Phosphorylation sites linked to cell–cell adhesion and regulated by CeO2NPs treatment. (F) Effect of CeO2NPs treatment over the phosphorylation of CD44 and Itgb4. (G) Phosphorylation sites linked to RNA splicing and regulated by CeO2NPs treatment. EFFECT OF CeO2NPs ON HEPATIC phosphatidylcholine (PC)–derived FAs, which are by far LIPID METABOLISM the most abundant lipid component in the liver. In fact, we observed a significant decrease in polyunsaturated Analysis of total fatty acids (FAs) of principal lipid FAs (PUFAs), which was exclusively due to a marked components in hepatic tissue of HCC animals indi- diminution in arachidonic acid (AA; C20:4n6; Fig. 5A). cates a dysregulation of FA metabolism mainly occur- We also observed significant changes in C14:0, C16:1, ring in cholesterol ester (CE)– and nonesterified FA and C17:0, although the differences were quantitatively (NEFA)– derived FAs (Supporting Table S5). The most much less important. Moreover, in NEFA-derived, important effects induced by CeO2NPs were found in triglyceride (TG)-derived, and CE-derived FAs, we 1274 Hepatology, Vol. 72, No. 4, 2020 FERNÁNDEZ-VARO ET AL. A B C FIG. 5. Representative chromatogram of PC-derived FAs in rats with HCC. (A) Superposition of a representative gas chromatography MS total ion current chromatograms showing the analysis of PC-derived FAs obtained from HCC rats treated with vehicle (red) or CeO2NPs (blue). The represented peaks are the FAs which showed significant differences between groups. Gas chromatography peaks are labeled with the corresponding FA identification. For each peak there is a graph with the quantification of the FA. (B) Effect of nanoceria treatment in linoleic FAs in different lipid components (NEFA, TG, and CE). (C) Effect of CeO2NPs in PE-derived docosahexaenoic acid (C22:6n3). *P < 0.05 and **P < 0.01 versus control; #P < 0.05 versus HCC + vehicle. Unpaired Student t test and Mann-Whitney test when appropriate. observed that HCC rats showed significantly increased NPs on the survival of two groups of HCC rats. The liver content of linoleic acid (LA; C18:2n6) than median survival reached by CeO2NPs-treated rats was healthy rats, a phenomenon that was reversed in HCC significantly higher than that in rats receiving vehicle animals treated with CeO2NPs (Fig. 5B). Of note, on (P < 0.05) (Fig. 6A). Next, we were interested in com- analyzing phosphatidylethanolamine (PE)–derived FAs, paring the effect of CeO2NPs to that of sorafenib. Four we observed that CeO2NPs administration was associ- groups of rats receiving vehicle, CeO2NPs, sorafenib, ated with a significant reduction in the very long chain and CeO2NPs plus sorafenib were investigated. The PUFA docosahexaenoic acid (C22:6n3) (Fig. 5C). median survival was markedly lower in HCC rats receiving vehicle (15.5 days, P < 0.05) than in those CeO2NPs IMPROVE SURVIVAL IN animals receiving CeO2NPs (31 days), sorafenib (33.5 days), or CeO2NPs plus sorafenib (33.5 days) DEN-INJURED RATS (Fig. 6B). The combined therapy showed the longest To assess how the above changes translate into survival, although differences did not reach statistical clinical outcome, we investigated the impact of these significance. 1275 FERNÁNDEZ-VARO ET AL. Hepatology, October 2020 A  Q   S 9HKLFOH Q  6XUYLYDO                  'D\VDIWHUODVWGRVH B S 6RUDIHQLE Q  S 6RUDIHQLE 6XUYLYDO  Q  S Q  9HKLFOH Q              'D\VDIWHUODVWGRVH FIG. 6. Effect of CeO2NPs on survival. (A) HCC rats randomly received two weekly doses of CeO2NPs or vehicle through the tail vein at the sixteenth and seventeenth weeks, and their survival was analyzed. (B) HCC rats were randomly distributed into four groups receiving vehicle, CeO2NPs, sorafenib, or the combination CeO2NPs and sorafenib. Sorafenib was delivered daily by intragastric administration (10 mg/kg) for 14 days. P < 0.05 and P < 0.01 versus HCC + vehicle, log-rank (Mantel-Cox) test. CeO2NPs RETENTION BY HUMAN least 50% when compared to the previous time point LIVER and further decreased at 60 minutes. In the case of donor H3, at 60 minutes of NMP, the Ce concentra- To determine whether CeO2NPs can be internal- tion in the perfusate was still around 45% due to the ized by the human liver, three experimentally viable fact that in this particular organ the injected dose of human livers, declined for transplantation, were per- NPs was almost 100 times higher than in H1 and H2 fused with CeO2NPs under normothermic machine livers. perfusion (NMP) (Supporting Information). Liver Ce subcellular location was examined using con- function tests and hemodynamic parameters were ventional bright and enhanced dark field TEM. Liver monitored during perfusion (data not shown) to tissue from all donors was morphologically well pre- ensure organ viability and proper device functioning. served, and the cells presented a viable morphology. To study the cellular uptake and intracellular local- Liver biopsies obtained from donor H3 were exam- ization of CeO2NPs in human livers, ICP-MS and ined under conventional TEM and energy-dispersive TEM imaging were performed. The concentration of X-ray spectroscopy (EDX). In conventional TEM, Ce in the serum leaving the liver through the hepatic CeO2NPs appeared as small, dense, black structures veins reached the highest levels 15 minutes after NPs in the form of agglomerates of different sizes inside administration (Fig. 7A). At 30 minutes of perfusion, blood vessels, the space of Disse, endothelial cells, the amount of Ce in the perfusate was reduced by at and some blood circulating cells (Fig. 7B). CeO2NPs 1276 Hepatology, Vol. 72, No. 4, 2020 FERNÁNDEZ-VARO ET AL. A B  + + &HLQWKHSHUIXVDWH   +         7LPH PLQ C —P —P FIG. 7. Accumulation of Ce in isolated human livers. (A) Amount of Ce in the perfusate 0, 15, 30, and 60 minutes after NP administration. ICP-MS data are expressed as percent of Ce present in the perfusate compared to the highest Ce amount present in the serum of each liver. (B) Assessment of CeO2NPs subcellular location in human hepatic tissue after 1 hour of NP infusion; (1,2,3) TEM image of a blood vessel containing NPs surrounded by hepatocytes. In bright field (1), aggregates of CeO2NPs (white arrows) appear as dark, electrodense spots. In dark field (2), nanoparticles are seen bright. (3) Magnified view of the boxed area depicting the nanoparticles in the space of Disse. (4,5) TEM image of a hepatocyte in the proximity of an endothelial cell containing CeO2NPs, bright and dark field, respectively. (6) High-magnification micrograph of the boxed area showing in detail the NPs inside the endothelial cell. *Apoptotic cell. (C) (1) STEM-HAADF image of endosome-like vesicles of an endothelial cell. (2) STEM-EDX element-distribution map showing the spatial distribution of Ce in the region outlined. Four adjacent pixel/spectra binned for each point. (3) EDX sum spectrum (Y = counts, X = keV) showing the average elemental composition of the image. Abbreviations: E, endothelial cell; ER, endoplasmic reticulum; H, hepatocyte; LD, lipid droplet; M, mitochondria; N, nucleus; P, platelet; RBC, red blood cell; S, sinusoid; SD, space of Disse. were observed both free and within intracellular, sin- EDX spectrum (right panel) confirms the elemental gle-membrane, endosome-like organelles. In dark composition. In addition to Os from the postfixation, field TEM, given its high atomic number relative to Cu from the TEM grid, and Pb from the staining, Ce the elements typically found in organic tissues, Ce is is the only element detected by using EDX analysis. expected to appear as very bright dots, as shown in Fig. 7B. The presence of Ce was further confirmed CeO2NPs ADSORPTION BY using high-angle annular dark-field scanning trans- HUMAN HEPATOCYTE CANCER mission electron microscopy (HAADF-STEM), CELLS which provides structural and chemical information with atomic resolution. Figure 7C shows a representa- To assess whether human hepatocytes can inter- tive region in the set of analyzed images. The elemen- nalize CeO2NPs, HepG2 cells, a human derived cell tal map (central panel) shows the spatial distribution cancer line, were exposed to CeO2NPs (10 µg/mL) of Ce in the region outlined in the left panel, and the for 24 hours and subjected to TEM analysis. NPs were 1277 FERNÁNDEZ-VARO ET AL. Hepatology, October 2020 FIG. 8. CeO2NPs adsorption in human hepatocyte cancer cells. (A) TEMs of HepG2 cells showing the uptake of NPs: (1) hepatocyte exposed to CeO2NPs (white arrows) for 24 hours, with some NPs adhering to the cell membrane, others confined to vesicles, and a few free in the cytoplasm; (2) formation of pseudopodia with NPs in the center; (3,4) vesicle loaded with CeO2NPs in bright and dark fields, respectively. (B) (1) TEM image displaying NPs in the cytoplasm of a hepatocyte; (2) STEM-HAADF image from the NPs; (3) Ce elemental mapping with four adjacent pixel/spectra binned for each point; (4) EDX sum spectrum (Y = counts, X = keV) showing the average elemental composition of the image. Abbreviations: ER, endoplasmic reticulum; LD, lipid droplet; M, mitochondria; N, nucleus. strongly attached to the outer leaflet of the plasmatic industries and catalytic exhaust converters for membrane, free in the cytoplasm, and mostly inside decades.(33) The use of these NPs as a therapeutic numerous endosome-like bodies of diverse morphology tool is still a matter of concern. This is mainly due (Fig. 8A). The mitochondria, the endoplasmic retic- to the tendency of CeO2NPs to evolve when in con- ulum, and the nucleus of these hepatocytes appeared tact with physiological media.(34) Variability in the normal. To verify that the electron-dense granules were response and loss of antioxidant activity are the out- indeed CeO2NPs, we performed dark field microscopy comes of these alterations. In the current investiga- followed by HAADF-STEM. In both dark field TEM tion, some of these difficulties have been overcome and STEM, NPs appeared as bright spots. The Ce ele- by preparing albumin-coated CeO2NPs with high mental map and EDX spectrum confirmed the presence monodispersity and high stability in the physiologi- of Ce in the preparations (Fig. 8B). cal media. This coating prevents the development of large NP aggregates and protein corona formation, resulting in a stable colloidal solution with sustained Discussion and more intense effects. Unfortunately, HCC is still rather orphan in terms The ability of nanoceria to catalyze redox reac- of highly effective systemic treatment. In this con- tions has been widely used in petrochemical text, oxidative stress, mainly contributed by ROS, has 1278 Hepatology, Vol. 72, No. 4, 2020 FERNÁNDEZ-VARO ET AL. been implicated in the pathogenesis of several diseases of hepatocytes that is associated with the devel- including cancer. It is well known that ROS can drive opment of HCC. This occurred in the frame of the initial development and progression of cancer as attenuated macrophage M1 proinflammatory gene well as down-regulate antioxidant enzymes that nor- expression in the liver tissue of HCC-treated rats. mally combat free radical production.(10) Consequently, Tumor-associated macrophages are well known for many antioxidant compounds, enzymes, and inhib- their trophic abilities and for providing immuno- itors of reduced nicotinamide adenine dinucleotide suppressive tumor microenvironment and therefore phosphate oxidase have been studied for treating facilitating tumor progression.(35) In that sense, low- chronic inflammation and cancer. However, results to ering macrophage numbers in liver tissue by means date have been suboptimal, mainly due to their low of chemotaxis inhibition or cell death could partially systemic bioavailability and insufficient levels at the explain the antitumor effects of nanoceria. target sites. Evidence supports that CeO2NPs have a specific Here we consider that CeO2NPs could be an antitumorigenic effect in HCC rats. First, following NP-based therapy platform in HCC, which would be NPs administration, we observed increased liver apop- able to induce ROS degradation and tumor recession totic activity. This is consistent with previous studies by virtue of their great self-regenerating antioxidant describing that after exposure to antioxidant cuprous capacity. Because the antioxidant effect of CeO2NPs oxide NPs, lung melanoma cells activate caspase-3 and is catalytic and consequently permanent, this would caspase-9, inducing apoptosis of tumor cells.(36) On the represent a clear competitive advantage over other other hand, CeO2NPs also resulted in decreased levels antioxidant therapies needing permanent application. of P-ERK1/2, an essential component of the Ras/Raf/ In addition, CeO2NPs are only active as a catalyst MAPK kinase/ERK signaling pathway. This is among when there is an excess of ROS; otherwise, they are the principal routes controlling cell survival, differen- inert and appear innocuous. tiation, proliferation, growth, angiogenesis, regulation The carcinogenic effect of DEN is due to an of glucose and lipid metabolism, and inflammation.(37) enhancement of hepatocyte proliferation mainly The impact of CeO2NPs on cell phosphorylation in the centrilobular hepatocytes. DEN is bioacti- in HCC has not been systematically investigated vated following hydroxylation by the cytochrome using untargeted MS-based proteomics. Our initial P450 (CYP) system; then, the hydroxylated DEN principal component analysis suggests a global effect is oxidized by CYP2E1 to reactive products in rat of CeO2NPs over protein phosphorylation in the liver liver liposomes. DEN-treated rats displayed macro- of HCC rats that significantly affected 9.5% of all scopically distorted liver, with altered liver weight detected phosphorylation sites. The effect of CeO2NPs and anomalous microscopic architecture of the liver comprised both increased and decreased phosphoryla- parenchyma showing diffuse dysplasia and fibrotic tion. The administration of CeO2NPs affected kinases tracts. This prompted us to assess whether the HCC involved in signaling pathways related to apoptosis, liver, as indeed occurs in the normal liver, is also a cell proliferation, migration, and survival such as p21 main target for CeO2NPs. Our results further con- (RAC1) activated kinase 2, eukaryotic elongation fac- firm that, even in a liver with intense tumorigenic tor 2 kinase, protein tyrosine kinase 2/focal adhesion activity, CeO2NPs maintain their high selective tar- kinase 2, and NIMA-related kinase 9. Interestingly, geting on the hepatic tissue. a gene ontology analysis showed an enrichment of Parameters indicating altered tissue growth or proteins linked to RNA splicing and cell–cell adhe- proliferation and ongoing proinflammatory pro- sion in the subset of proteins whose phosphorylation cesses were significantly less activated in rats receiv- were significantly regulated after CeO2NPs treatment. ing CeO2NPs. Treated rats showed increased liver/ Splicing is a process frequently deregulated in can- body weight ratio, decreased macrophage infiltration, cer cells because it can regulate the function of key and lower amount of Ki67-positive cells. Ki67 is a proteins involved in apoptosis, proliferation, angio- nuclear antigen extensively used as a proliferation genesis, and migration.(38) In this regard, CeO2NPs marker and as a prognostic indicator for cancer. We treatment that reduced cell proliferation caused both also observed decreased serum concentration of AFP, an increase and a decrease in the phosphorylation of the main serological biomarker of dedifferentiation proteins involved in splicing. Cell adhesion is also a 1279 FERNÁNDEZ-VARO ET AL. Hepatology, October 2020 process heavily deregulated in cancer cells with mul- which are likely due to an abnormal essential FA tiple proteins involved in cell–cell adhesion consid- metabolism involving Δ-6 desaturase (Δ-6D).(43) The ered as tumor suppression or oncogenes.(39) Our gene activity and expression of this desaturase are regulated ontology analysis indicates that CeO2NPs produce a by the intracellular redox state.(45) This suggests that large effect on the phosphorylation pattern of proteins the restoration of normal hepatic levels of LA in the involved in cell–cell adhesion with proteins presenting HCC rats treated with CeO2NPs could result from both overphosphorylation and downphosphorylation, the reactivation of Δ-6D activity due to the reduction suggesting an alteration in this biological process. We in oxidative stress. observed a reduced phosphorylation of two other cell The translation of the antitumorigenic effects surface proteins involved in cell adhesion, CD44 and induced by CeO2NPs into a clinically significant Itgb4. improvement was assessed by investigating the A hallmark of cancer cells is dysregulation of FA effect of CeO2NPs on survival. Treated HCC rats metabolism to support proliferation.(40) Accordingly, showed a clear amelioration in this parameter. To total serum TG and cholesterol were found to be sig- date, tyrosine kinase inhibitors, such as sorafenib, nificantly decreased in HCC rats. Highly proliferative lenvatinib, cabozatinib, or regorafenib, as well as the cancer cells have strong lipid and cholesterol avidity; antiangiogenic antibody ramucirumab, are consid- consequently, these cells either increase the uptake ered effective therapies in patients with advanced of exogenous lipids or overactivate their endogenous HCC.(46) The effect of CeO2NPs on overall survival synthesis.(41) Excessive lipids and cholesterol in cancer was similar to that observed with sorafenib, which cells are stored in lipid droplets as cholesteryl esters,(37) indicates that these NPs are at least as effective as which is in agreement with our findings of increased sorafenib under the conditions studied. The com- CE-derived FAs in hepatic HCC tissue. The analysis bination of both treatments did not result in an of principal lipid components also revealed an increase additional improvement in survival in comparison in NEFA probably due to an increased generation to to each treatment administered alone. CeO2NPs support tumor growth. and sorafenib likely interfere with common signal- The most important effects induced by CeO2NPs ing pathways, such as angiogenesis through vascular are found in PC-derived FAs. The decrease in endothelial growth factor signaling, which would PC-PUFAs resulting from CeO2NPs administration explain why the combination of both compounds was mostly due to a decrease in AA.(42) Phospholipases did not result in any additional effect. The effects A2, C, and D can mediate the release of esterified AA of CeO2NPs on the ERK1/2 signaling pathway, the from cellular phospholipids, a process which already modulation of the phosphorylation state of a high seems incremented in HCC. Free AA can be metab- number of peptides, and their manifestations on cell olized through enzymatic reactions or act as a second proliferation and apoptosis mirror some the abun- messenger in signal transduction pathways(43); some dant data reported on sorafenib effects.(47) of these pathways were demonstrated to be signifi- For a comprehensive understanding of whether cantly up-regulated in our HCC rats. After nanoceria the behavior of CeO2NPs in the human liver resem- treatment, the decrease in esterified PC-AA was more bles that observed in rats with HCC, we admin- pronounced. This phenomenon seems to be related to istered nanoceria to human livers under ex vivo the increase of apoptosis in these rats because free AA normothermic perfusion. The ex vivo experiments is able to promote the activation of sphingomyelinase confirmed that CeO2NPs have high avidity for and the apoptotic process.(44) human liver because they accumulate in the target CeO2NPs treatment reversed the increase in tissue readily after administration. The NPs were NEFA-derived, TG-derived, and CE-derived LA in found both free and within intracellular, single- HCC rats. LA, one of the most abundant FAs in all membrane, endosome-like organelles. The elemen- lipid components, has been reported to change the tal analysis combined with the STEM helped us to metabolism of intrahepatic CD4+ T cells, leading confirm their presence and distinguish them from them to apoptosis and, thus, contributing to HCC endogenous structures and artifacts in the tissue. development.(44) Neoplastic hepatocyte lesions have Moreover, in vitro experiments with the HepG2 been associated with changes in the PUFA profile, cell line confirmed the uptake and retention of 1280 Hepatology, Vol. 72, No. 4, 2020 FERNÁNDEZ-VARO ET AL. CeO2NPs by human hepatocyte cancer cells mostly epithelial-to-mesenchymal transition, increased invasion and risk in endosome-like bodies. of rebound growth. Cancer Lett 2013;329:74-83. In conclusion, these results indicate that the anti- 8) Bruix J, Qin S, Merle P, Granito A, Huang YH, Bodoky G, et al.; RESORCE Investigators. 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