Single-Dose Neoadjuvant AKT Pathway Inhibitor Reduces Growth of Hepatocellular Carcinoma after Laser Thermal Ablation in Small-Animal Model.

BackgroundLocal recurrence following thermal ablation of hepatocellular carcinoma (HCC) larger than 2-3 cm remains a challenging clinical problem. Prior studies suggest that phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)-dependent protein kinase B (AKT) signaling mediates HCC cell survival caused by moderate heat stress in vitro, but these findings need in vivo validation.PurposeTo test the hypothesis that neoadjuvant inhibition of PI3K/mTOR/AKT signaling reduces HCC tumor growth in vivo after laser ablation and to evaluate the effects of moderate heat stress on molecular signaling and cellular function in HCC cells in vitro.Materials and MethodsHCC tumor-bearing mice were randomized to neoadjuvant PI3K/mTOR inhibitor (BEZ235) or control groups followed by an intentional partial laser ablation or sham ablation; there were at least nine mice per group. Postablation tumor growth was monitored up to 7 days. Tumor volumes were compared for drug or ablation groups by using two-way analysis of variance. N1S1 HCC cells pretreated with BEZ235 or control and subjected to moderate heat stress (45°C for 10 minutes) or control (37°C for 10 minutes) were analyzed by using mass spectrometry. Protein interaction networks were derived from protein expression analysis software, and cellular function activation state (Z-score) and fold-change in AKT phosphorylation were calculated.ResultsThere was a 37%-75% reduction in HCC tumor volume by day 7 after ablation in the BEZ235 plus ablation group (713 mm3 ± 417) compared with vehicle plus sham (1559 mm3 ± 552), vehicle plus ablation (1041 mm3 ± 591), and BEZ235 plus sham (1108 mm3 ± 523) groups (P < .001, P = .04, and P = .005, respectively). PI3K/mTOR inhibition prevented moderate heat stress-induced AKT signaling (Z-score, -0.2; P < .001) and isoform-specific AKT phosphorylation compared with the vehicle plus heat stress group. PI3K/mTOR inhibition prevented moderate heat stress-induced global effects on HCC molecular signaling and cellular function, including decreased cell survival, growth, and proliferation (Z-score, -0.3 to -3.2; P < .001) and increased apoptosis and cell death (Z-score, 0.4-1.1; P < .001).ConclusionModerate heat stress induces PI3K/mTOR/AKT-dependent global effects on hepatocellular carcinoma (HCC) cell survival, function, and death. Neoadjuvant PI3K/mTOR/AKT inhibition reduces postablation HCC tumor growth.© RSNA, 2019Online supplemental material is available for this article.See also the editorial by White in this issue.

Heat Stress and Thermal Ablation Induce Local Expression of Nerve Growth Factor Inducible (VGF) in Hepatocytes and Hepatocellular Carcinoma: Preclinical and Clinical Studies.

The purposes of this study were to test the hypothesis that heat stress and hepatic thermal ablation induce nerve growth factor inducible (VGF) and to determine intrahepatic versus systemic VGF expression induced by thermal ablation in vivo and in patients. Hepatocytes and HCC cells were subjected to moderate (45°C) or physiologic (37°C) heat stress for 10 min and assessed for VGF expression at 0-72 h post-heat stress (n ≥ 3 experiments). Orthotopic N1S1 HCC-bearing rats were randomized to sham or laser thermal ablation (3 W × 90 s), and liver/serum was harvested at 0-7 days postablation for analysis of VGF expression (n ≥ 6 per group). Serum was collected from patients undergoing thermal ablation for HCC (n = 16) at baseline, 3-6, and 18-24 h postablation and analyzed for VGF expression. Data were analyzed using ordinary or repeated-measures one-way analysis of variance and post hoc pairwise comparison with Dunnett's test. Moderate heat stress induced time-dependent VGF mRNA (3- to 15-fold; p < 0.04) and protein expression and secretion (3.1- to 3.3-fold; p < 0.05). Thermal ablation induced VGF expression at the hepatic ablation margin at 1 and 3 days postablation but not remote from the ablation zone or distant intrahepatic lobe. There was no detectable serum VGF following hepatic thermal ablation in rats and no increase in serum VGF following HCC thermal ablation in patients at 3-6 and 18-24 h postablation compared to baseline (0.71- and 0.63-fold; p = 0.27 and p = 0.16, respectively). Moderate heat stress induces expression and secretion of VGF in HCC cells and hepatocytes in vitro, and thermal ablation induces local intrahepatic but not distant intrahepatic or systemic VGF expression in vivo.

Heat Stress and Hepatic Laser Thermal Ablation Induce Hepatocellular Carcinoma Growth: Role of PI3K/mTOR/AKT Signaling.

Purpose To determine if heat stress and hepatic laser thermal ablation induce hepatocellular carcinoma (HCC) growth and to identify growth factors induced by heat stress. Materials and Methods Non-heat-stressed HCC cells were cocultured with HCC cells or hepatocytes that were heat stressed at 37°C (physiologic), 45°C (moderate), or 50°C (severe) for 10 minutes and proliferation monitored with bioluminescence imaging for up to 6 days after heat stress (three experiments). Rats bearing orthotopic N1S1 HCC were randomly assigned to undergo immediate sham or laser thermal (3 W for 60 or 90 seconds; hereafter, 3W×60s and 3W×90s, respectively) ablation of the median (local) or left (distant) hepatic lobe, and tumor growth was monitored with magnetic resonance imaging for up to 18 days after ablation (six or more rats per group). Experiments were repeated with rats randomly assigned to receive either the adjuvant phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor (NVP-BEZ235) or the vehicle control. Heat-stressed HCC cells and hepatocytes were analyzed by using microarray or quantitative real-time polymerase chain reaction analysis for growth factor expression (three or more experiments). Groups were compared by using one- or two-way analysis of variance, and post hoc pairwise comparison was performed with the Dunnett test. Results There were more non-heat-stressed HCC cells when cells were cocultured with cells subjected to moderate but not physiologic or severe heat stress (P < .001 for both). Local intrahepatic N1S1 tumors were larger at day 18 in the 3W×60s (mean, 3102 mm3 ± 463 [standard error]; P = .004) and 3W×90s (mean, 3538 mm3 ± 667; P < .001) groups than in the sham group (mean, 1363 mm3 ± 361) but not in distant intrahepatic tumors (P = .31). Adjuvant BEZ235 resulted in smaller N1S1 tumors in the BEZ235 and laser thermal ablation group than in the vehicle control and laser thermal ablation group (mean, 1731 mm3 ± 1457 vs 3844 mm3 ± 2400, P < .001). Moderate heat stress induced expression of growth factors in HCC cells and hepatocytes, including heparin-binding growth factor, fibroblast growth factor 21, and nerve growth factor (range, 2.9-66.9-fold; P < .05). Conclusion Moderate heat stress and laser thermal ablation induce hepatocellular carcinoma growth, which is prevented with adjuvant PI3K/mTOR/protein kinase B inhibition.

Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma.

PURPOSE: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling. MATERIALS AND METHODS: MET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining. RESULTS: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling. CONCLUSIONS: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

Blockade of CCR2 reduces macrophage influx and development of chronic renal damage in murine renovascular hypertension.

Renovascular hypertension (RVH) is a common cause of both cardiovascular and renal morbidity and mortality. In renal artery stenosis (RAS), atrophy in the stenotic kidney is associated with an influx of macrophages and other mononuclear cells. We tested the hypothesis that chemokine receptor 2 (CCR2) inhibition would reduce chronic renal injury by reducing macrophage influx in the stenotic kidney of mice with RAS. We employed a well-established murine model of RVH to define the relationship between macrophage infiltration and development of renal atrophy in the stenotic kidney. To determine the role of chemokine ligand 2 (CCL2)/CCR2 signaling in the development of renal atrophy, mice were treated with the CCR2 inhibitor RS-102895 at the time of RAS surgery and followed for 4 wk. Renal tubular epithelial cells expressed CCL2 by 3 days following surgery, a time at which no significant light microscopic alterations, including interstitial inflammation, were identified. Macrophage influx increased with time following surgery. At 4 wk, the development of severe renal atrophy was accompanied by an influx of inducible nitric oxide synthase (iNOS)+ and CD206+ macrophages that coexpressed F4/80, with a modest increase in macrophages coexpressing arginase 1 and F4/80. The CCR2 inhibitor RS-102895 attenuated renal atrophy and significantly reduced the number of dual-stained F4/80+ iNOS+ and F4/80+ CD206+ but not F4/80+ arginase 1+ macrophages. CCR2 inhibition reduces iNOS+ and CD206+ macrophage accumulation that coexpress F4/80 and renal atrophy in experimental renal artery stenosis. CCR2 blockade may provide a novel therapeutic approach to humans with RVH.

Methodological differences account for inconsistencies in reported free VEGF concentrations in pregnant rats.

Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8-29 pg/ml) and high (527-1,030 pg/ml) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Plasma VEGF in nonpregnant, day 7 pregnant, and day 19 pregnant rats was measured using rat and mouse ELISAs (R&D Systems). The rat ELISA detected VEGF in plasma from nonpregnant rats but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (median: 2,273 vs. 0 pg/ml) than in nonpregnant (97 vs. 20 pg/ml) and day 7 pregnant (66 vs. 2 pg/ml) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from nonpregnant and day 7 pregnant rats was high for the rat ELISA (82-105%) but low for the mouse ELISA (17-22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. The use of the rat versus mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations in nonpregnant and pregnant rats are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, these data should be interpreted cautiously.

Evolution of cardiac and renal impairment detected by high-field cardiovascular magnetic resonance in mice with renal artery stenosis.

BACKGROUND: Renal artery stenosis (RAS) promotes hypertension and cardiac dysfunction. The 2-kidney, 1-clip mouse model in many ways resembles RAS in humans and is amenable for genetic manipulation, but difficult to evaluate noninvasively. We hypothesized that cardiovascular magnetic resonance (CMR) is capable of detecting progressive cardiac and renal dysfunction in mice with RAS and monitoring the progression of the disease longitudinally. METHODS: RAS was induced at baseline in eighteen mice by constricting the renal artery. Nine additional animals served as normal controls. CMR scans (16.4 T) were performed in all mice one week before and 2 and 4 weeks after baseline. Renal volumes and hemodynamics were assessed using 3D fast imaging with steady-state precession and arterial spin labelling, and cardiac function using CMR cine. Renal hypoxia was investigated using blood oxygen-level dependent (BOLD) MR. RESULTS: Two weeks after surgery, mean arterial pressure was elevated in RAS mice. The stenotic kidney (STK) showed atrophy, while the contra-lateral kidney (CLK) showed hypertrophy. Renal blood flow (RBF) and cortical oxygenation level declined in the STK but remained unchanged in CLK. Moreover, cardiac end-diastolic and stroke volumes decreased and myocardial mass increased. At 4 weeks, STK RBF remained declined and the STK cortex and medulla showed development of hypoxia. Additionally, BOLD detected a mild hypoxia in CLK cortex. Cardiac end-diastolic and stroke volumes remained reduced and left ventricular hypertrophy worsened. Left ventricular filling velocities (E/A) indicated progression of cardiac dysfunction towards restrictive filling. CONCLUSIONS: CMR detected longitudinal progression of cardiac and renal dysfunction in 2K, 1C mice. These observations support the use of high-field CMR to obtain useful information regarding chronic cardiac and renal dysfunction in small animals.

Evaluation of strategies to reduce radiation dose in perfusion CT imaging using a reproducible biologic phantom.

OBJECTIVE: The purpose of this study is to evaluate radiation dose reduction strategies in perfusion CT by using a biologic phantom. MATERIALS AND METHODS: A formalin-preserved porcine liver was submerged in a 32-cm-wide acrylic phantom filled with water. The portal vein was connected to a continuous flow pump. The phantom was scanned with a perfusion CT protocol using 80 kVp and 400 mAs, every 1 second, for 50 seconds. This was repeated using 100 and 20 mAs. It was also repeated again using 400 mAs to assess reproducibility. A sparser scan frequency was simulated retrospectively. Blood flow was determined for each dataset using the maximum slope and deconvolution methods. RESULTS: Measurements of the mean blood flow values in identical regions of interest had a percent difference of 7% for repeated perfusion CT protocols using the same settings regardless of perfusion model used. The 100 mAs scans agreed with 400 mAs scans, with percent differences of 21% and 31% for the maximum slope and deconvolution methods, respectively. At a simulated frequency of one scan every 4 seconds, blood flow values differed up to 17% and 60% from the reference scan for the maximum slope and deconvolution methods, respectively. At 20 mAs and one scan every 1 second, or 400 mAs and a simulated frequency of one scan every 8 seconds, both computation methods failed to provide accurate blood flow estimates. CONCLUSION: The biologic phantom showed reproducible measurements that can help in optimizing perfusion CT protocols by determining both the acquisition parameters that affect radiation dose and the accuracy of estimates from different perfusion models.

Molecular bioluminescence imaging as a noninvasive tool for monitoring tumor growth and therapeutic response to MRI-guided laser ablation in a rat model of hepatocellular carcinoma.

OBJECTIVES: The objective of this study was to quantitatively compare tumor imaging by magnetic resonance imaging (MRI) and molecular bioluminescence imaging (BLI) and test the feasibility of monitoring the effect of MRI-guided laser ablation on tumor viability by 2-dimensional BLI and 3-dimensional diffuse luminescence tomography (3D DLIT) in an orthotopic rat model of hepatocellular carcinoma. MATERIALS AND METHODS: This study was approved by the animal care committee. Rats underwent injection of N1S1 cells stably transfected with an empty vector (n = 3) or a heat shock element luciferase reporter (HSE-luc; n = 4) into the liver. All rats underwent MRI to assess tumor establishment and volume and 2-dimensional BLI to assess tumor luminescence at day 7 with subsequent MRI and 2D BLI and 3D DLIT in select animals at days 14 and 21. Magnetic resonance imaging-guided laser ablation of the tumor was performed with preablation and postablation 2D BLI and/or 3D DLIT (n = 2). The tumors underwent histopathologic analysis to assess tumor viability. RESULTS: The MRI scans demonstrated hyperintense T2-weighted lesions at 3 of 3 and 4 of 4 sites in the empty vector and HSE-luc rats, respectively. Two-dimensional BLI quantitation demonstrated 23.0-fold higher radiance in the HSE-luc group compared with the empty vector group at day 7 (P < 0.01) and a significant correlation with tumor volume by MRI (r = 0.86; P < 0.03). Tumor dimensions by 3D DLIT and MRI demonstrated good agreement. Three-dimensional DLIT quantitation demonstrated better agreement with the percentage of nonviable tumor by histopathology than did 2D BLI quantitation after the MRI-guided laser ablation. CONCLUSIONS: Bioluminescence imaging is feasible as a noninvasive, quantitative tool for monitoring tumor growth and therapeutic response to thermal ablation in a rat model of hepatocellular carcinoma.

Disparate roles of marrow- and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammation.

Systemic inflammatory response syndrome (SIRS) occurs in a range of infectious and non-infectious disease processes. Toll-like receptors (TLRs) initiate such responses. We have shown that parenchymal cell TLR4 activation drives LPS-induced systemic inflammation; SIRS does not develop in mice lacking TLR4 expression on parenchymal cells. The parenchymal cell types whose TLR4 activation directs this process have not been identified. Employing a bone marrow transplant model to compartmentalize TLR4 signaling, we characterized blood neutrophil and cytokine responses, NF-κB1 activation, and Tnf-α, Il6, and Ccl2 induction in several organs (spleen, aorta, liver, lung) near the time of LPS-induced symptom onset. Aorta, liver, and lung gene responses corresponded with both LPS-induced symptom onset patterns and plasma cytokine/chemokine levels. Parenchymal cells in aorta, liver, and lung bearing TLR4 responded to LPS with chemokine generation and were associated with increased plasma chemokine levels. We propose that parenchymal cells direct SIRS in response to LPS.

Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension.

Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-ß signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-ß/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-ß/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.

LPS-induced murine systemic inflammation is driven by parenchymal cell activation and exclusively predicted by early MCP-1 plasma levels.

Systemic inflammation remains a major cause of morbidity and mortality in the United States, across many disease processes. One classic murine model to study this syndrome is lipopolysaccharide (LPS)-induced Toll-like receptor 4 (TLR4)-dependent systemic inflammation. Although most studies have focused on inflammatory cell TLR4 responses, parenchymal cells also express TLR4. Our objective was to define the in vivo role of parenchymal- versus marrow-derived cell activation via TLR4 during LPS-induced inflammation. Mice bearing TLR4 on parenchymal cells only, marrow-derived cells only, both, or neither were generated using bone marrow transplantation. Mortality occurred only in mice that had TLR4 expression on their parenchymal cells. Before onset, virtually all major plasma cytokines and blood neutrophil responses were related to marrow-derived cell activation via TLR4. The only cytokine predictive of oncoming systemic inflammation was the chemokine monocyte chemoattractant protein-1. Late blood neutrophil responses were related to the presence of TLR4 on either parenchymal or marrow cells, whereas plasma cytokine elevations late in LPS-induced systemic inflammation were dependent on mice having TLR4 in both cell compartments. Parenchymal cell activation via TLR4 is a key component of LPS-induced systemic inflammation and mortality, although most plasma cytokine levels and blood neutrophil responses were not key components. Given its unique role, future studies into monocyte chemoattractant protein-1's exact role during systemic inflammation are warranted.

Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension.

Unilateral renal artery stenosis (RAS) leads to atrophy of the stenotic kidney and compensatory enlargement of the contralateral kidney. Although the two-kidney, one-clip (2K1C) model has been extensively used to model human RAS, the cellular responses in the stenotic and contralateral kidneys, particularly in the murine model, have received relatively little attention. We studied mice 2, 5, and 11 wk after unilateral RAS. These mice became hypertensive within 1 wk. The contralateral kidney increased in size within 2 wk after surgery. This enlargement was associated with a transient increase in expression of phospho-extracellular signal-regulated kinase (p-ERK), the proliferation markers proliferating cell nuclear antigen and Ki-67, the cell cycle inhibitors p21 and p27, and transforming growth factor-beta, with return to baseline levels by 11 wk. The size of the stenotic kidney was unchanged at 2 wk but progressively decreased between 5 and 11 wk. Unlike the contralateral kidney, which showed minimal histopathological alterations, the stenotic kidney developed progressive interstitial fibrosis, tubular atrophy, and interstitial inflammation. Surprisingly, the stenotic kidney showed a proliferative response, which involved largely tubular epithelial cells. The atrophic kidney had little evidence of apoptosis, despite persistent upregulation of p53; expression of cell cycle regulatory proteins in the stenotic kidney was persistently increased through 11 wk. These studies indicate that in the 2K1C model, the stenotic kidney and contralateral, enlarged kidney exhibit a distinct temporal expression of proteins involved in cell growth, cell survival, apoptosis, inflammation, and fibrosis. Notably, an unexpected proliferative response occurs in the stenotic kidney that undergoes atrophy.

Toward development and production of human T cells in swine for potential use in adoptive T cell immunotherapy.

Immunotherapy and vaccination for cancer or infection are generally approached by administration of antigen or stimulation of antigen-presenting cells or both. These measures may fail if the treated individual lacks T cells specific for the immunogen(s). We tested another strategy-the generation of new T cells from hematopoietic stem cells that might be used for adoptive immunotherapy. To test this concept, we introduced T cell-depleted human bone marrow cells into fetal swine and tested the swine for human T cells at various times after birth. Human T cells were detected in the thymus and blood of the treated swine. These cells were generated de novo as they contained human T cell receptor excision circles not present in the T cell-depleted bone marrow. The human T cells were highly diverse and included novel specificities capable of responding to antigen presented by human antigen-presenting cells. Our findings constitute a first step in a new promising approach to immunotherapy in which tumor- or virus-specific T cell clones lacking in an individual might be generated in a surrogate host from hematopoietic stem cells of the individual to be treated.

Inhibition of RAS-mediated transformation and tumorigenesis by targeting the downstream E3 ubiquitin ligase seven in absentia homologue.

Constitutively active RAS small GTPases promote the genesis of human cancers. An important goal in cancer biology is to identify means of countervailing activated RAS signaling to reverse malignant transformation. Oncogenic K-RAS mutations are found in virtually all pancreatic adenocarcinomas, making the RAS pathway an ideal target for therapeutic intervention. How to best contravene hyperactivated RAS signaling has remained elusive in human pancreatic cancers. Guided by the Drosophila studies, we reasoned that a downstream mediator of RAS signals might be a suitable anti-RAS target. The E3 ubiquitin ligase seven in absentia (SINA) is an essential downstream component of the Drosophila RAS signal transduction pathway. Thus, we determined the roles of the conserved human homologues of SINA, SIAHs, in mammalian RAS signaling and RAS-mediated tumorigenesis. We report that similar to its Drosophila counterpart, human SIAH is also required for oncogenic RAS signaling in pancreatic cancer. Inhibiting SIAH-dependent proteolysis blocked RAS-mediated focus formation in fibroblasts and abolished the tumor growth of human pancreatic cancer cells in soft agar as well as in athymic nude mice. Given the high level of conservation of RAS and SIAH function, our study provides useful insights into altered proteolysis in the RAS pathway in tumor initiation, progression, and oncogenesis. By targeting SIAH, we have found a novel means to contravene oncogenic RAS signaling and block RAS-mediated transformation/tumorigenesis. Thus, SIAH may offer a novel therapeutic target to halt tumor growth and ameliorate RAS-mediated pancreatic cancer.

Intrinsic resistance of hepatocytes to complement-mediated injury.

When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.

Survival of Human Neurofibroma in Immunodeficient Mice and Initial Results of Therapy With Pirfenidone.

Neurofibromatosis type I is a common tumor predisposing disease in humans. Surgical therapy can be applied only in selected patients with resectable masses. Hence, development of new therapies for this disease is urgent. We used human neurofibroma implants in mice with severe combined immunodeficiency (SCID) as a model to test the toxicity and potential efficacy of pirfenidone, a new therapeutic agent. Two hundred twelve human neurofibromas were transplanted into various locations in 59 experimental animals, and 30 mice with implants received oral pirfenidone for up to six weeks. Survival of neurofibromas in animals treated with pirfenidone was lower than in the control group $(P=.02)$. Tumors did not change histologic appearance or vascularization in response to pirfenidone. Treatment with pirfenidone, a new antifibrotic agent, inhibits survival of some tumors without causing toxicity in animals.

Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo.

Human cells can fuse with damaged or diseased somatic cells in vivo. Whether human cells fuse in vivo in the absence of disease and with cells of disparate species is unknown. Such a question is of current interest because blood exchanges between species through direct physical contact, via insect vectors or parasitism, are thought to underlie the transmission of zoonotic agents. In a model of human-pig chimerism, we show that some human hematopoietic stem cells engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens. * differentiation * fusion * retrovirus