Significance of CD90+ cancer stem cells in human liver cancer.

This study characterized cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) cell lines, tumor specimens, and blood samples. The CD90+ cells, but not the CD90(-) cells, from HCC cell lines displayed tumorigenic capacity. All the tumor specimens and 91.6% of blood samples from liver cancer patients bore the CD45(-)CD90+ population, which could generate tumor nodules in immunodeficient mice. The CD90+CD44+ cells demonstrated a more aggressive phenotype than the CD90+CD44(-) counterpart and formed metastatic lesions in the lung of immunodeficient mice. CD44 blockade prevented the formation of local and metastatic tumor nodules by the CD90+ cells. Differential gene expression profiles were identified in the CD45(-)CD90+ and CD45(-)CD90(-) cells isolated from tissue and blood samples from liver cancer patients and controls.

Hematopoietic chimerism in liver transplantation patients and hematopoietic stem/progenitor cells in adult human liver.

UNLABELLED: Liver transplantation (LT) is a cure for many liver diseases. Blood chimerism of donor origin can develop after LT, which raises the possibility of the existence of hematopoietic stem/progenitor cells (HSPCs) in the liver. We characterized the blood chimerism in a large cohort of 249 LT patients and analyzed putative HSPCs in adult human livers. The overall incidence of chimerism was 6.43%, of which 11.11% was among short-term (1 day to 6 months) and 3.77% was among long-term (6 months to 8 years) LT patients. Hematopoietic Lin(-) CD34(+) CD38(-) CD90(+) populations have been demonstrated to generate long-term lymphomyeloid grafts in transplantations. In human adult livers, we detected Lin(-) CD34(+) CD38(-) CD90(+) populations accounting for 0.03% ± 0.017% of the total single liver cells and for 0.05% ± 0.012% of CD45(+) liver cells. Both Lin(-) CD34(+) and Lin(-) CD45(+) liver cells, from extensively perfused human liver grafts, were capable of forming hematopoietic myeloid-lineage and erythroid-lineage methylcellulose colonies. More importantly, Lin(-) CD45(+) or CD45(+) liver cells could be engrafted into hematopoietic cells in an immunodeficient mouse model. These results are the first evidence of the presence of putative HSPC populations in the adult human liver, where the liver is a good ectopic niche. The discovery of the existence of HSPCs in the adult liver have implications for the understanding of extramarrow hematopoiesis, liver regeneration, mechanisms of tolerance in organ transplantation, and de novo cancer recurrence in LT patients. CONCLUSION: The human adult liver contains a small population of HSPCs. In LT patients, there are two types of chimerisms: transient chimerism, resulting from mature leucocytes, and long-term chimerism, derived from putative HSPCs in the liver graft.

Prediction of posthepatectomy recurrence of hepatocellular carcinoma by circulating cancer stem cells: a prospective study.

OBJECTIVE: To investigate whether circulating cancer stem cells (CSCs) of hepatocellular carcinoma (HCC) can predict its recurrence after hepatectomy. BACKGROUND: HCC recurrence frequently occurs within the first year after hepatectomy, probably due to circulating tumor cells that have been shed from the primary tumor before hepatectomy. Because CSCs are more likely to initiate tumor growth than mature cancer cells, a high level of circulating CSCs may be a hint for HCC recurrence. METHODS: Multicolor flow cytometry was used to detect the number of circulating CSCs (CD45CD90CD44) in the peripheral circulation of 82 HCC patients 1 day before hepatectomy. The patients were monitored by CT or MRI for recurrence every 3 months. RESULTS: Forty-one (50%) patients had recurrence after a median follow-up period of 13.2 months (range, 1.3-57.1 months). Patients with recurrence had a higher median level of circulating CSCs than patients without recurrence (0.02% vs. 0.01%; P < 0.0001). Circulating CSCs > 0.01% predicted intrahepatic recurrence (relative risk 3.54; 95% CI, 1.41-8.88; P = 0.007) and extrahepatic recurrence (relative risk 10.15; 95% CI, 3-34.4; P = 0.0002). Patients with >0.01% circulating CSCs had a lower 2-year recurrence-free survival rate (22.7% vs. 64.2%; P < 0.0001) and overall survival rate (58.5% vs. 94.1%; P = 0.0005) than patients with ≤0.01% circulating CSCs. On multivariable analysis, circulating CSCs > 0.01%, tumor stage and tumor size were independent factors predicting recurrence-free survival. CONCLUSIONS: Circulating CSCs predicted posthepatectomy HCC recurrence with high accuracy. They may be the target of eradication in the prevention of posthepatectomy HCC metastasis and recurrence.

Octamer 4 (Oct4) mediates chemotherapeutic drug resistance in liver cancer cells through a potential Oct4-AKT-ATP-binding cassette G2 pathway.

UNLABELLED: Chemoresistance presents a major obstacle to the efficacy of chemotherapeutic treatment of cancers. Using chemotherapeutic drugs to select drug-resistant cancer cells in hepatocellular carcinoma (HCC) and several other cancer cell lines, we demonstrate that chemoresistant cells displayed cancer stem cell features, such as increased self-renewal ability, cell motility, multiple drug resistance, and tumorigenicity. Octamer 4 (Oct4) messenger RNA (mRNA) levels were dramatically increased in chemoresistant cancer cells due to DNA demethylation regulation of Oct4. By functional study, Oct4 overexpression enhanced whereas Oct4 knockdown reduced liver cancer cell resistance to chemotherapeutic drugs in vitro and in xenograft tumors. It is known that the Oct4-TCL1-AKT pathway acts on embryonic stem cells and cancer stem cells in cell proliferation through inhibition of apoptosis. We further demonstrate that Oct4 overexpression induced activation of TCL1, AKT, and ABCG2 to mediate chemoresistance, which can be overcome by addition of the PI3K/AKT inhibitor; therefore, a direct pathway of Oct4-TCL1-AKT-ABCG2 or a combination of Oct4-TCL1-AKT with the AKT-ABCG2 pathway could be a potential new mechanism involved in liver cancer cell chemoresistance. Moreover, the clinical significance of the Oct4-AKT-ABCG2 pathway can be demonstrated in HCC patients, with a strong correlation of expression patterns in human HCC tumors. The role of the Oct4-AKT-ABCG2 axis in cancer cell chemoresistant machinery suggests that AKT pathway inhibition (PI3K inhibitors) not only inhibits cancer cell proliferation, but may also enhance chemosensitivity by target potential chemoresistant cells. CONCLUSION: Oct4, a transcriptional factor of pluripotent cells, can mediate chemoresistance through a potential Oct4-AKT-ABCG2 pathway.

[Effect of electroacupuncture on expression of Kisspeptin protein in hypothalamus of rats with polycystic ovary syndrome].

OBJECTIVE: To investigate the effect of electroacupuncture (EA) on the expression of Kisspeptin protein and activities of the hypothalamic-pituitary-ovarian axis(HPOA) in rats with Letrozole-induced polycystic ovary syndrome (PCOS). METHODS: Female SD rats were randomly divided into normal control, PCOS model and EA groups (n=6 rats in each group). The PCOS model was established by continuous gavage of letrozole for 21 d. EA(2 Hz/100 Hz, 0.6-1.4 mA) was applied to bilateral "Daimai" (GB26) for 20 min, once every day for 15 d. Body mass was measured every 4 days. Serum follicular stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and testosterone (T) were detected by radioimmunoassay. Histopathological changes of the ovarian were observed after H.E. staining, and the expression level of Kisspeptin protein in the hypothalamus was detected by Western blot. RESULTS: Following modeling, the body mass, serum T and LH contents, hypothalamic Kisspeptin protein expression and the number of ovarian follicles were significantly increased (P<0.05, P<0.01), while the number of ovarian corpus luteum was apparently decreased in comparison with the normal group (P<0.01). After EA intervention, the serum T, LH and E2 contents, the expression of Kisspeptin protein and the number of ovarian follicles were notably down-regulated (P<0.05, P<0.01), and the number of corpus luteum was significantly increased (P<0.01) in comparison with the model group. CONCLUSION: EA can regulate the levels of sex hormones and HPOA of PCOS rats, which may be related to its effect in down-regulating the expression of Kisspeptin protein in the hypothalamus.

An Akt/hypoxia-inducible factor-1alpha/platelet-derived growth factor-BB autocrine loop mediates hypoxia-induced chemoresistance in liver cancer cells and tumorigenic hepatic progenitor cells.

PURPOSE: The goals of the present study were to investigate the mechanism of hypoxia-mediated chemoresistance in liver cancer cells and tumorigenic hepatic progenitor (oval) cells and to determine whether disrupting an Akt/hypoxia-inducible factor-1alpha (HIF-1alpha)/platelet-derived growth factor (PDGF)-BB autocrine loop can enhance chemotherapeutic efficacy in hypoxia. EXPERIMENTAL DESIGN: Five hepatocellular carcinoma (HCC) cell lines and two hepatic progenitor cell lines were treated in vitro with cisplatin under both normoxic and hypoxic conditions. To generate ischemic hypoxia for tumor cells in vivo, hepatic artery ligation was applied to an orthotopic HCC model. Cisplatin and YC1, which is a HIF-1alpha inhibitor, were administered by portal vein and intratumoral injections, respectively. RESULTS: Cell viability was higher under hypoxic than normoxic conditions. HIF-1alpha and Akt were up-regulated under hypoxic conditions, forming an autocrine signaling loop with PDGF-BB. Akt/HIF-1alpha/PDGF-BB signaling regulated Akt to confer cisplatin resistance to HCC cell lines in vitro. This autocrine signaling loop also contributed to chemoresistance in the tumorigenic hepatic progenitor cell line PIL2 under hypoxic conditions but not in the nontumorigenic cell line PIL4. In an orthotopic HCC model, combining blockade of HIF-1alpha activity with ischemic hypoxia significantly enhanced the efficacy of chemotherapy, leading to suppression of tumor growth and prolongation of animal survival. CONCLUSION: Blockade of Akt/HIF-1alpha/PDGF-BB autocrine signaling could enhance the chemosensitivity of liver cancer cells and tumorigenic hepatic progenitor cells under hypoxic conditions and thus provide an effective therapeutic strategy for HCC.

Identification of local and circulating cancer stem cells in human liver cancer.

UNLABELLED: Increasing evidence has revealed the importance of cancer stem cells (CSCs) in carcinogenesis. Although liver CSCs have been identified in hepatocellular carcinoma (HCC) cell lines, no data have shown the presence of these cells in human settings. The present study was designed to delineate CSCs serially from HCC cell lines, human liver cancer specimens to blood samples, using CD90 as a potential marker. The number of CD90(+) cells increased with the tumorigenicity of HCC cell lines. CD45(-)CD90(+) cells were detected in all the tumor specimens, but not in the normal, cirrhotic, and parallel nontumorous livers. In addition, CD45(-)CD90(+) cells were detectable in 90% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis. A significant positive correlation between the number of CD45(-)CD90(+) cells in the tumor tissues and the number of CD45(-)CD90(+) cells in the blood samples was identified. CD90(+) cells sorted from cell lines and CD45(-)CD90(+) cells from the tumor tissues and blood samples of liver cancer patients generated tumor nodules in immunodeficient mice. Serial transplantation of CD90(+) cells from tumor xenografts generated tumor nodules in a second and subsequently third batch of immunodeficient mice. Treatment of CD90(+) CSCs with anti-human CD44 antibody induced cell apoptosis in a dose-dependent manner. CONCLUSION: Identification of CD45(-)CD90(+) CSCs in both tumor tissues and circulation suggests that CD45(-)CD90(+) could be used as a marker for human liver cancer and as a target for the diagnosis and therapy of this malignancy.

Cardiotrophin-1 enhances regeneration of cirrhotic liver remnant after hepatectomy through promotion of angiogenesis and cell proliferation.

BACKGROUND/AIM: Hepatic resection is not applicable to a certain proportion of hepatocellular carcinoma patients owing to an insufficient liver function reserve. The present study was designed to investigate the effects of cardiotrophin-1 (CT-1) on improving the function of CCl(4)-induced cirrhotic liver remnant after major hepatectomy. METHODS: CT-1 was administered to rats after hepatectomy according to different protocols. RESULTS: A double-dose CT-1 protocol improved liver function, enlarged the volume of liver remnant, upregulated the expression of von Willebrand factor and increased the number of BrdU(+) or Ki-67(+) hepatocytes. Administration of CT-1 enhanced the expression of nuclear factor-kappaB (P65), vascular endothelial growth factor (VEGF), CyclinD1 and p42/44 in the liver remnant. However, the effects of CT-1 were blocked by a VEGF receptor blocker, PTK787. Although the expression of gp130, a receptor of CT-1, was downregulated in the diseased hepatocytes isolated from the cirrhotic liver, CT-1 could still stimulate the cell proliferation. CT-1 administration enhanced the expression of P65 and VEGF in the diseased hepatocytes, but the augmented P65 and VEGF expression was blocked by PTK787 administration. CONCLUSION: Short-term administration of CT-1 could improve the function of cirrhotic liver remnant and stimulate liver regeneration through promotion of angiogenesis and cell proliferation.

EGFR L792H and G796R: Two Novel Mutations Mediating Resistance to the Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib.

BACKGROUND: The third-generation EGFR tyrosine kinase inhibitor osimertinib has been approved in many countries to treat advanced NSCLC in patients with the EGFR T790M mutation. As the development of acquired resistance is inevitable, it is urgent that the mechanisms of such resistance be clarified. METHODS: DNA samples from a cohort of 340 patients with lung adenocarcinoma who were taking osimertinib were subjected to next-generation sequencing and screened in terms of the frequencies of the L792H and G796R mutations. Ba/F3 cells stably expressing the EGFR L858R/T790M mutations (in cis) with either the L792H or G796R mutation were created to investigate the impact of the two novel mutations on EGFR tyrosine kinase inhibitors and other potential drug combinations in vitro. Structural analyses were performed by using Schrödinger/Maestro software (version 11.1.012, Schrödinger LLC, Cambridge, MA). RESULTS: L792H and G796R were detected in 1.76% (six of 340) and 0.56% (two of 340) patients with lung adenocarcinoma treated with osimertinib, respectively. The introduction of L792H or G796R mutations against an L858R/T790M background caused dramatic reductions in osimertinib sensitivity. Structural modeling showed that mutations in cis with T790M either forced the ligand (osimertinib) to rotate out (breaking the binding) or pulled the hinge loop (breaking the hinge). Various other drug combinations. including cetuximab with EAI045, failed to inhibit either cis mutant effectively. CONCLUSIONS: The EGFR L858R/T790M/L792H and L858R/T790M/G796R mutations conferred resistance to osimertinib both in vitro and in silico. For patients in whom the two resistance mutations occur at low frequency, more precise treatment strategies and additional combinational approaches are required.

Rac activation is associated with hepatocellular carcinoma metastasis by up-regulation of vascular endothelial growth factor expression.

PURPOSE: Hepatocellular carcinoma (HCC) is associated with a propensity for vascular invasion and metastasis, which contribute to poor prognosis. Angiogenesis is a crucial process contributing to tumor growth and metastasis. Recently, Rac has been suggested to play a role in angiogenesis. However, the actual role of Rac in HCC angiogenesis remains unclear. Given that vascular endothelial growth factor (VEGF) is an important angiogenic factor in HCC, the purpose of this study was to evaluate the possible correlation between Rac activation and VEGF expression in HCC tumor samples, as well as the mechanism involved in Rac-induced HCC angiogenesis. EXPERIMENTAL DESIGN: We evaluated Rac and VEGF expression in the HCC tissue microarray of paired primary and metastatic HCC samples using immunohistochemical staining. The role of Rac-induced HCC angiogenesis was also evaluated in vitro in HCC cell lines. RESULTS: We first showed that activation of Rac was correlated with HCC metastasis (P<0.001), and its expression was significantly correlated with VEGF expression by tissue microarray. Ectopic Rac-dominant active transfection in Hep3B cells increased VEGF secretion, which induced the morphologic change and proliferation of human umbilical vein endothelial cells, resulting in the promotion of angiogenesis. Rac induced the transcriptional activation of VEGF by direct interaction with hypoxia-inducible factor-1alpha (HIF-1alpha) expression. In hypoxic conditions, Rac promoted angiogenesis through an increase in HIF-1alpha stabilization. CONCLUSION: This study shows that Rac is a novel angiogenic factor for HCC through the enhancement of HIF-1alpha protein stability, which provides a possible therapeutic target in the development of inhibitors of angiogenesis.

Identification of brain-derived neurotrophic factor as a novel functional protein in hepatocellular carcinoma.

This study aims to identify a novel molecule that may contribute to hepatocarcinogenesis in a rat orthotopic hepatocellular carcinoma model. The hepatocellular carcinoma model was generated by injection of tumor cells into the left lobe of the liver. Proteomic approaches, including ProteinChip and two-dimensional electrophoresis, were used to identify proteins from serially collected rat serum samples. By both ProteinChip and two-dimensional electrophoresis techniques, the level of a 27-kDa protein was found to be augmented in serum samples during tumor development, decreased after left lobectomy, and reincreased at the time of tumor recurrence. The protein was identified to be brain-derived neurotrophic factor (BDNF). By using specific primers and monoclonal antibody, the expression pattern of BDNF was confirmed in tumor tissue but not in the adjacent nontumorous liver tissue. In addition, the truncated isoform of BDNF receptor-tyrosine protein kinase receptor B was only found in tumor tissue. An in vitro study showed that exogenous BDNF could induce tumor cell proliferation predominantly in relatively small numbers of inoculated cells. Administration of BDNF to tumor cell lines induced significantly increased expression of heat shock protein 90 (Hsp90) and cyclin D1, and blocking the activity of Hsp90 could reverse the up-regulation of cyclin D1 induced by BDNF. The present study revealed that BDNF and its receptor were uniquely expressed in tumor tissue and cell lines of hepatocellular carcimona but not in nontumorous liver tissue and normal cell line. BDNF could stimulate tumor cell proliferation in a Hsp90-dependent manner.

High doses of tyrosine kinase inhibitor PTK787 enhance the efficacy of ischemic hypoxia for the treatment of hepatocellular carcinoma: dual effects on cancer cell and angiogenesis.

The present study aimed to investigate the therapeutic efficacy of combining vascular endothelial growth factor (VEGF) receptor blockade using tyrosine kinase inhibitor PTK787 with hypoxia for the treatment of hepatocellular carcinoma (HCC). The in vivo effects of the treatments were determined in a rat orthotopic HCC model, in which hypoxia was generated by hepatic artery ligation (HAL). Compared with HAL alone, PTK787 combined with HAL significantly prolonged the animal survival, reduced the tumor size, induced more tumor tissue necrosis and apoptosis, and down-regulated the expression of von Willebrand factor. The mechanism was explored in vitro using murine HCC and endothelial cell lines, respectively. PTK787 combined with hypoxia decreased the expression of VEGF and VEGF receptors in both cell lines and suppressed the cell viability by induction of cell cycle arrest and promotion of apoptosis. Up-regulation of cleaved form caspase-9 and down-regulation of Bcl-2 and cyclin D1 were detected with the combined treatment. Hypoxia sensitized endothelial cells to the inhibitory effect of PTK787 on forming tubular-like structure. The motility of tumor cells was inhibited by hypoxia and the combined approach, with down-regulation of Rac1, Rho, and phosphorylated Akt expression. However, in the endothelial cells, the combined treatment inhibited the hypoxia-enhanced cell motility, with suppressed Rac1, Rho, and phosphorylated Akt expression. In conclusion, PTK787 combined with hypoxia achieved a better therapeutic efficacy than hypoxia alone through enhancing hypoxia-induced antitumor cell effect and preventing the activation of angiogenic process.

Therapeutic potential of cardiotrophin 1 in fulminant hepatic failure: dual roles in antiapoptosis and cell repair.

HYPOTHESIS: Administration of cardiotrophin 1 (CT-1) can treat experimental fulminant hepatic failure (FHF). DESIGN: Rat model with FHF induced by D-galactosamine (D-gal). SETTING: Fulminant hepatic failure is a rapidly progressive disease that lacks effective nonsurgical treatment. Cardiotrophin 1 is a member of the interleukin 6 family that can protect cells from damage in some animal disease models. ANIMALS: A rat model of FHF was induced by an intraperitoneal injection of D-gal (1.4 g/kg of body weight). Cardiotrophin 1 was administered at different time points after D-gal injection. RESULTS: Administration of CT-1 at 12 and 18 hours had a survival rate of 80% (12/15) and 70% (7/10), respectively, which was significantly higher than that of nontreatment (28% [5/18]). In addition, improvement of liver histologic findings, shortening of activated clotting time, and decrease in serum levels of total bilirubin and alanine aminotransferase were detected with CT-1 treatment. Administration of CT-1 decreased apoptotic cells and increased Ki-67 cells in the liver tissues. In vitro, CT-1 administration significantly decreased apoptotic cells and sequentially down-regulated the expression of proapoptotic molecules and up-regulated the expression of antiapoptotic molecules at different culture periods. D-galactosamine culture induced morphologic damage in a hepatocyte cell line, which was greatly improved by CT-1 administration. In addition, CT-1-treated cells demonstrated increased expression of glycoprotein 130 and up-regulation of cyclin D1 and heat shock protein 90. CONCLUSION: Cardiotrophin 1 may improve the outcome of D-gal-induced FHF through its effects on antiapoptosis and cell repair.

Significance of the serum brain-derived neurotrophic factor and platelets in hepatocellular carcinoma.

The present study investigated the significance of the serum brain-derived neurotrophic factor (BDNF) and platelets in relation to the clinicopathological features of hepatocellular carcinoma (HCC) patients. Localization of the BDNF expression in human HCC tissues was performed by immunohistochemistry. The measurement of soluble BDNF in the serum was performed by enzyme-linked immunosorbent assay. BDNF was expressed in the cytoplasm of the tumor cells. A positive correlation between the tissue and serum levels of BDNF was identified in the HCC patients. The serum levels of BDNF were positively correlated with the platelet counts in the HCC patients. A higher level of serum BDNF was significantly correlated with a tumor size >5 cm, poorly differentiated HCC, the presence of microsatellite tumor nodules, and the absence of cirrhosis in the non-tumorous tissues. A higher level of the serum BDNF/platelet ratio was associated with a poorer disease-free survival after hepatic resection. This study suggested that the tumor cell was a source of serum BDNF in HCC. A higher serum BDNF level was associated with a more advanced tumor status in the HCC patients. The interaction between serum BDNF and platelets might play an important role in HCC tumor progression.

The potential role of hypoxia inducible factor 1alpha in tumor progression after hypoxia and chemotherapy in hepatocellular carcinoma.

This study investigates the possible molecular basis leading to failure in a treatment that is composed of hypoxia and chemotherapy in a rat orthotopic hepatoma model. Hypoxia was induced by hepatic artery ligation, whereas chemotherapeutic effect was achieved by intraportal injection of cisplatin. High-dose sodium salicylate was administered to achieve transcriptional blockade. Significant prolongation of animal survival was observed in the groups receiving hepatic artery ligation with cisplatin or sodium salicylate. Massive tumor cell necrosis and apoptosis were found in the ligation and all of the combined treatment groups. Up-regulation of hypoxia inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) at both mRNA and protein levels were detected in the groups receiving ligation and ligation with cisplatin, whereas a decreased level of von Hippel-Lindau tumor suppressor protein was identified in the group receiving ligation with cisplatin. Sodium salicylate enhanced expression of von Hippel-Lindau tumor suppressor protein but down-regulated HIF-1alpha and VEGF levels after ligation with or without cisplatin. An increased number of activated hepatic stellate cells in the tumors were observed in the ligation and ligation with cisplatin groups, whereas they were greatly reduced by sodium salicylate. In vitro study revealed that under hypoxic condition, both cisplatin and sodium salicylate could remarkably augment P53 and caspase 3 levels. Cisplatin stimulated HIF-1alpha up-regulation, whereas sodium salicylate suppressed HIF-1alpha expression. In conclusion, tumor progression after hypoxia and chemotherapy might be related to up-regulation of HIF-1alpha and subsequent VEGF production, and transcriptional blockade by sodium salicylate could enhance the therapeutic efficacy of hypoxia and chemotherapy.

Tumor microvessel density as a predictor of recurrence after resection of hepatocellular carcinoma: a prospective study.

PURPOSE: This study prospectively evaluated the correlation of tumor microvessel density (MVD) with clinicopathologic features and postoperative recurrence in patients undergoing resection of hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Tumor MVD was assessed in 100 patients with resection of HCC using a computer image analyzer after immunostaining for CD34 (MVD-CD34) and von Willebrand factor (MVD-vWF), respectively. Patients were prospectively followed for recurrence. RESULTS: Mean tumor MVD-CD34 (236/0.74 mm(2)) was higher than mean tumor MVD-vWF (87/0.74 mm(2)) (P <.001). By multiple regression analysis, tumor size was the only pathologic feature significantly related to tumor MVD-CD34. The median MVD-CD34 was 316/0.74 mm(2) in HCCs < or = 5 cm (n = 46) and 146/0.74 mm(2) in HCCs more than 5 cm (n = 54) (P <.001). Among patients with HCCs < or = 5 cm, those with higher than median MVD-CD34 had worse disease-free survival (at 3 years, 13%) than those with a lower MVD-CD34 (at 3 year, 74%) (P =.002). Multivariate analysis showed that tumor MVD-CD34 was the only significant factor predictive of disease-free survival in patients with HCC < or = 5 cm. For HCCs more than 5 cm, MVD-CD34 did not have a significant prognostic influence. MVD-vWF did not have a significant prognostic influence on disease-free survival in either HCCs < or = 5 cm or more than 5 cm. CONCLUSION: This study shows that a high MVD-CD34 was predictive of early postresection recurrence in patients with HCCs < or = 5 cm and, therefore, may be a novel prognostic marker in this subset of patients.

Discovery and Evaluation of Clinical Candidate AZD3759, a Potent, Oral Active, Central Nervous System-Penetrant, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor.

Recent reports suggest that an increasing number of patients with lung cancer, especially those with activating mutations of the epidermal growth factor receptor (EGFR), also present with brain metastases and leptomeningeal metastases. These patients have poor prognosis as there are no approved drugs for these indications. Available agents have poor efficacy for these patients even at well above their standard dose. Herein, we report the discovery of (4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl (2R)-2,4-dimethylpiperazine-1-carboxylate 1m (AZD3759), an investigational drug currently in Phase 1 clinical trial, which has excellent central nervous system penetration and which induces profound regression of brain metastases in a mouse model.

Preparation of 66Ga- and 68Ga-labeled Ga(III)-deferoxamine-folate as potential folate-receptor-targeted PET radiopharmaceuticals.

A folate-receptor-targeting radiopharmaceutical, Ga(III)-deferoxamine-folate (Ga-DF-Folate), was radiolabeled with two positron-emitting isotopes of gallium, cyclotron-produced (66)Ga (9.5 hour half-life) and generator-produced (68)Ga (68 minute half-life). The [(66)Ga]Ga-DF-Folate was administered to athymic mice with folate-receptor-positive human KB cell tumor xenografts to demonstrate that microPET mouse tumor imaging is feasible with (66)Ga, despite the relatively high positron energy of this radionuclide. Using the athymic mouse KB tumor xenograft model, dual-isotope autoradiography was also performed following i.v. co-administration of [(18)F]-FDG, a marker of regional metabolic activity, and folate-receptor-targeted [(111)In]In-DTPA-Folate. The autoradiographic images of 1 mm tumor sections demonstrate the gross heterogeneity of the KB cell tumor xenograft, as well as subtle disparity in the regional accumulation of the two radiotracers.

Gene expression profiling of liver cancer stem cells by RNA-sequencing.

BACKGROUND: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90(+) liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90(+) cells sorted from tumor (CD90(+)CSCs) with parallel non-tumorous liver tissues (CD90(+)NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CD90(+) cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90(+) cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90(+)CSCs and CD90(+)NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90(+)CSCs and CD90(+)NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90(+)CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90(+)CSCs compared to CD90(+)NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90(+)CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90(+)CSCs in liver tumor tissues. CONCLUSIONS/SIGNIFICANCE: The identified genes, such as GPC3 that are distinctly expressed in liver CD90(+)CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells.

Role of stem cells in normal liver and cancer.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality in the world because current treatments, including both surgical and non-surgical ones, cannot effectively cure this disease. Increasing evidence has revealed the importance of cancer stem cells (CSCs) in hepatocarcinogenesis, and the idea of targeting CSCs sheds light on more effective therapeutic strategies for HCC. In this review, normal stem cells and putative CSCs in the liver are briefly introduced. Studies about signaling pathways that regulate pathophysiological activities of liver CSCs and the therapeutic potential by targeting CSCs are also summarized and discussed.