Combined Immune Deficiency Mice Research Articles

Detection of oesophageal cancer biomarkers by plasma proteomic profiling of human cell line xenografts in response to chemotherapy

Background:The incidence of oesophageal adenocarcinoma is increasing worldwide but survival remains poor. Neoadjuvant chemotherapy may improve survival, but targeting treatment to patients who respond to chemotherapy could be improved by the availability of markers of response. This study sought proteomic markers of therapeutic response using an adenocarcinoma xenograft model.Methods:Epirubicin, cisplatin or 5-fluorouracil was administered to severe combined immune-deficient mice bearing OE19 oesophageal adenocarcinoma xenografts. Murine plasma samples from treated and untreated xenografts were analysed by surface-enhanced laser desorption/ionisation time-of-flight mass spectroscopy, and panels of peaks were found using class prediction models that distinguished treatment groups. Proteins in these peaks were identified by mass spectroscopy in tryptic digests of purified fractions. Five paired samples from oesophageal cancer patients before and after chemotherapy were analysed using the same methodology.Results:Plasma protein peaks were identified that differed significantly (P<0.05, ANOVA) between the treated xenograft and control groups. Marker panels predicted treated vs untreated xenografts with sensitivities of 100%, specificities of 86–100% and test efficiencies of 89–100%. Three of the proteins identified in these panels, apolipoprotein A-I, serum amyloid A and transthyretin were confirmed in the clinical samples.Conclusion:Plasma protein markers can be detected in response to chemotherapy in oesophageal adenocarcinoma xenografts and in clinical samples, and have the potential to monitor response and guide chemotherapy in oesophageal adenocarcinoma.

Human CD34 + Cells in Experimental Myocardial Infarction

Human CD34(+) cells have been used in clinical trials for treatment of myocardial infarction (MI). However, it is unknown how long the CD34(+) cells persist in hearts, whether the improvement in cardiac function is sustained, or what are the underlying mechanisms. We sought to track the fate of injected human CD34(+) cells in the hearts of severe combined immune deficiency (SCID) mice after experimental MI and to determine the mechanisms of action. We used multimodality molecular imaging to track the fate of injected human CD34(+) cells in the hearts of SCID mice after experimental MI, and used selective antibody blocking to determine the mechanisms of action. Bioluminescence imaging showed that injected CD34(+) cells survived in the hearts for longer than 12 months. The PET signal from the injected cells was detected in the wall of the left ventricle. Cardiac MRI showed that left ventricular ejection fraction was significantly improved in the treated mice compared to the control mice for up to 52 weeks (P<0.05). Furthermore, treatment with anti-alpha4beta1 showed that generation of human-derived cardiomyocytes was inhibited, whereas anti-vascular endothelial growth factor (VEGF) treatment blocked the production of human-derived endothelial cells. However, the improvement in cardiac function was abolished only in the anti-VEGF, but not anti-alpha4beta1, treated group. Angiogenesis and/or paracrine effect, but not myogenesis, is responsible for functional improvement following CD34(+) cells therapy.

Establishment of a new Glivec-resistant chronic myeloid leukemia cell line, SNUCML-02, using an in vivo model

In this study, we report a newly established chronic myeloid leukemia (CML) cell line, SNUCML-02, which is resistant to imatinib and describe its biological characteristics. Mononuclear cells were obtained from the bone marrow of a CML patient in blast crisis and were cultured in Dulbecco's modified Eagle's medium/F12 containing 20% fetal bovine serum. After 2 months of primary culture, these cells were injected into nonobese diabetic/severe combined immune-deficient mice via tail vein. Eight weeks after injection, mice were sacrificed and ex vivo culture was performed from the bone marrow cells isolated from the mice. The established cell line was named as SNUCML-02 and the biological features were characterized by cytogenetic analysis, fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction, sequencing analysis, cell proliferation assay, and Western blot analysis. Cytogenetic studies using conventional G-banding and fluorescent in situ hybridization of SNUCML-02 demonstrated classical Philadelphia chromosome, (9;22)(q34;q11.2), and other abnormalities, such as add(11)(q23), +19 and +der(9;22). SNUCML-02 has the same BCR-ABL fusion transcript as was seen in K562 cells, but has no mutations in the ABL kinase domain. SNUCML-02 was more resistant to imatinib (STI571, Gleevec, Glivec) than other CML cell lines (K562, Kcl22, and BV173). SNUCML-02 has constitutive activation of extracellular signal-regulated kinase phosphorylation. In addition, interleukin-3 induced c-ABL phosphorylation and constitutively enhanced extracellular signal-regulated kinase phosphorylation was not inhibited by imatinib in SNUCML-02. SNUCML-02 is a new established cell line with a relatively high level of resistance to imatinib, which is useful for investigating the pathogenesis of CML progression, and will be useful in developing optimal therapeutic strategies for this ailment.

Effective Targeting of Quiescent Chronic Myelogenous Leukemia Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate

Imatinib mesylate (IM) induces remission in chronic myelogenous leukemia (CML) patients but does not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. Here we investigated the ability of HDAC inhibitors (HDACis) to target CML stem cells. Treatment with HDACis combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACis with IM markedly diminished LSCs in a transgenic mouse model of CML. The interaction of IM and HDACis inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents an effective strategy to target LSCs in CML patients receiving tyrosine kinase inhibitors.

The Lamina Propria of Adult Human Oral Mucosa Harbors a Novel Stem Cell Population

The highly regenerative capacity of the human adult oral mucosa suggests the existence of a robust stem cell (SC) population in its lamina propria (OMLP). The purpose of this study was to characterize the availability, growth, immunophenotype, and potency of this presumable SC population. Cells positive for the embryonic stem cell transcription factors Oct4 and Sox2 and for p75 formed distinct cord-like structure in the OMLP. Regardless of donor age, trillions of cells, termed human oral mucosa stem cells (hOMSC), 95% of which express mesenchymal stromal cell markers, were simply, and reproducibly produced from a biopsy of 3-4 x 2 x 1 mm(3). A total of 40-60% of these cells was positive for Oct4, Sox2, and Nanog and 60-80% expressed constitutively neural and neural crest SC markers. hOMSC differentiated in culture into mesodermal (osteoblastic, chondroblastic, and adipocytic), definitive endoderm and ectodermal (neuronal) lineages. Unexpectedly, hOMSC treated with dexamethasone formed tumors consisting of two germ layer-derived tissues when transplanted in severe combined immune deficiency mice. The tumors consisted of tissues produced by neural crest cells during embryogenesis-cartilage, bone, fat, striated muscle, and neural tissue. These results show that the adult OMLP harbors a primitive SC population with a distinct primitive neural-crest like phenotype and identifies the in vivo localization of putative ancestors for this population. This is the first report on ectodermal- and mesodermal-derived mixed tumors formation by a SC population derived from a nonmalignant somatic adult human tissue.

Meningeal Immunity, Learning and Memory, and IL-4: Do T cells Make You Smart? (87.35)

Abstract T cells were recently shown to support learning and memory, although the underlying mechanisms for this phenomenon are yet unknown. Here, we show that severe combined immune deficient (SCID) mice, shown to be impaired in assays of learning and memory, display a pronounced pro-inflammatory (M1) skew of meningeal myeloid cells. Acute lymphopenia induced by FTY720, a sphingosine-phosphate receptor agonist, or blockade of T cell migration into the meninges using anti-VLA4 antibodies, results in similar cognitive impairment and M1-skewed meningeal myeloid phenotype in wild type mice. The role of IL-4 is critical: we show accumulation of IL-4+ CD4+ T cells in the meninges of wild type mice after MWM training. Moreover, IL-4 knockout mice exhibit profound cognitive impairment and M1 skew. Transfer of IL-4 knockout bone marrow to wild type mice results in meningeal myeloid inflammation and impaired learning, while transfer of wild type T lymphocytes to IL-4 knockout mice is sufficient to rescue both behavioral phenotype and M1 skew. Our results point to a critical role for meningeal T cells, and for IL-4 in particular, in the regulation of meningeal myeloid cells and concomitant cognitive function. These results might lead to development of new immune-based therapies for cognitive conditions associated with immune decline, such as HIV- and age-associated dementia.

In vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP-labeled Myoblasts

In vivo fluorescence imaging (FLI) enables monitoring fluorescent protein (FP)-labeled cells and proteins in living organisms noninvasively. Here, we examined whether this modality could reach a sufficient sensitivity to allow evaluation of the regeneration process of enhanced green fluorescent protein (eGFP)-labeled muscle precursors (myoblasts). Using a basic FLI station, we were able to detect clear fluorescence signals generated by 40,000 labeled cells injected into a tibialis anterior (TA) muscle of mouse. We observed that the signal declined to approximately 25% on the 48 hours of cell injection followed by a recovery starting at the second day and reached a peak of approximately 45% of the original signal by the 7th day, suggesting that the survived population underwent a limited run of proliferation before differentiation. To assess whether transplanted myoblasts could form satellite cells, we injured the transplanted muscles repeatedly with cardiotoxin. We observed a recovery of fluorescence signal following a disappearance of the signal after each cardiotoxin injection. Histology results showed donor-derived cells located underneath basal membrane and expressing Pax7, confirming that the regeneration observed by imaging was indeed mediated by donor-derived satellite cells. Our results show that FLI is a powerful tool that can extend our ability to unveil complicated biological processes such as stem cell-mediated regeneration.

Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells

Adenoviral vector-mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC) when transient gene expression is preferred. Previous studies have demonstrated that fiber-retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we investigated the potential of bi-directional promoter-controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs. We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase promoter and minimal core promoter from human cytomegalovirus. The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types, including candidate HSCs, was compared to Ad5F35-DeltaLNGFR-IRES vector encoding phosphoglycerate kinase promoter-controlled bicistronic expression cassette for DeltaLNGFR and GFP. Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between repopulating progenitor cells. In nonobese diabetic severe combined immune deficient mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution. This study indicates that bi-directional promoter-controlled Ad5F35 vector, such as Ad5F35-DeltaLNGFR-BiDp, can be particularly useful for manipulation of myeloid progenitor cells and potentially in myeloid lineage leukemic cells as well.

FTY720 Demonstrates Promising in-Vitro and in-Vivo Pre-Clinical Activity by Down-Modulation of Cyclin D1 and Pakt in Mantle Cell Lymphoma.

Abstract 3728Poster Board III-664Over-expression of Cyclin D1 and constitutive phosphorylation of Akt has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Here we describe FTY720 (fingolimod), an immunosuppressive agent currently being explored in phase III studies in renal transplantation and multiple sclerosis patients, to mediate time- and dose-dependent cell death in primary MCL cells (6 patients) and MCL cell lines, Jeko and Mino. FTY720-induced apoptosis was associated with reactive oxygen species (ROS) generation, Bax up-regulation but not associated with caspase 3 activation in MCL. FTY720 treatment resulted in time-dependent down-modulation of Cyclin D1 and phospho Akt (p-Akt) protein level, two critical disease-relevant molecules in the pathogenesis of MCL. Consistent with the modulation of Cyclin D1, FTY720-induced cell cycle arrest with accumulation of cells in G0/G1 and G2/M phases of the cell cycle with concomitant decrease in S phase entry. Importantly, FTY720 treatment was also associated with a time-dependent phospho Erk (p-Erk) induction in Mino and Jeko cells. To determine the in vivo efficacy of FTY720, we developed a preclinical, in vivo xenograft model of human MCL where MCL cell lines (Jeko, Mino and SP53) were engrafted into severe combined immune deficient (SCID) mice. Cell dose titration trials identified 4 × 107 Mino or Jeko cells injected intravenously via tail vein to result in consistent engraftment and fatal tumor burden in all mice. All mice engrafted with 4 × 107 Jeko cells developed a disseminated disease within 3 weeks and had a median survival of 28 days (compared to 43 days for Mino and 51 days for SP53). Because the Jeko cell line was established from the peripheral blood of a patient with blastic variant MCL and demonstrated a more resistant phenotype to several immuno-chemoterapeutic compounds, this cell line was chosen to create a more stringent in vivo preclinical model. SCID mice were treated with the monoclonal antibody TMβ1 to deplete murine NK cells, engrafted with 4 × 107 Jeko cells and observed daily for signs of tumor burden. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal injection of 100 μl of saline or FTY720 (5 mg/kg resuspended in 100 μl of saline), every day, for two weeks. The median survival for FTY720-treated mice (N=10) was 38 days (95% CI:30-39) compared to 26.5 days (95% CI: 26-27 days) for the control group mice (N=10). The results from the log-rank test indicated an overall statistical significant difference in survival functions between the FTY720 treatment and the control group (p=0.001).These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant the further investigation of FTY720 in combination with other agents in clinical trials treating patients with MCL. Disclosures:No relevant conflicts of interest to declare.

Survivin is an essential mediator of arthritis interacting with urokinase signalling

Proto-oncogene survivin has recently been identified as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (RA). In the present material of 132 RA patients and 82 controls, the levels of survivin correlated to urokinase (uPA) (r= 0.46), a plasminogen activator over-expressed in inflamed joints and known to exhibit potent arthritogenic properties. Here we evaluate the functional relationship between these proteins using primary synovial fibroblasts and leucocytes of RA patients, human monocytic (THP-1) and fibroblast (MRC-5) cell lines. Using inhibitors of intracellular signalling, we show that uPA and survivin share common transduction pathways in synovial fibroblasts being dependent on the activity of tyrosine kinases, phosphatidylinositide 3 kinase and mitogen effector kinase. Moreover, uPA production is significantly reduced in fibroblasts if survivin synthesis has been silenced by siRNA. Importantly, silencing of survivin in fibroblasts prevented their invasive growth in knee joints of severe combined immune deficient mice. Interaction of uPA with receptor up-regulates survivin expression in leucocytes. In turn, survivin is required for the up-regulation of uPA receptor on the cell surface. These findings indicate that survivin is an essential mediator of arthritogenic properties of uPA regulating its synthesis in synovial fibroblasts and uPAR expression in leucocytes. Close correlation between survivin and uPA levels in patients with RA supports the importance of this connection for the pathogenesis of arthritis.

Testosterone Induces Porcine Primordial Follicle Development In Vitro.

Mammalian ovaries contain a large stock of oocytes enclosed in primordial follicles. A limited number of primordial follicles start to develop, while the remaining large numbers of them are quiescent. The mechanisms controlling the initiation of oocyte growth and primordial follicle development remain to be elucidated. Recent studies have shown that testosterone regulates early stages of folliculogenesis and follicle recruitment in vivo and in vitro. The objective of this study was to examine the effect of testosterone on initiation of primordial follicle development in cultured porcine ovarian tissues and subsequent xenotransplantation. Small pieces (1 mm × 1 mm × 1 mm) of ovarian cortex containing primordial follicles were dissected from ovaries of prepubertal pigs, and cultured in α-minimum essential medium supplemented with bovine plasma, insulin-transferrin-selenium, 2-mercaptoethanol, sodium pyruvate and different concentrations of testosterone (0, 10-7, 10-6, 10-5 and 10-4 M) or estradiol-17β (0, 10-7, 10-6 and 10-5 M) for 7 days. They were then fixed to examine the development of primordial follicles histologically. Ovarian tissues before culture contained primordial follicles (85 ± 6%) and small proportion of degenerated follicles (15 ± 6%). After 7 days of culture without hormone (control), no primordial follicle developed to the intermediate (early primary) or primary stage, and 56 ± 2% follicles were degenerated. On the other hand, in the tissues cultured with 10-6 M testosterone, higher percentages of intermediate follicles and small proportion of primary follicles were observed (17 ± 4% and 1 ± 1%, respectively). Percentages of degenerated follicles were significantly lower in 10-6 M (24 ± 1%) and 10-5 M (34 ± 1%) testosterone groups compared to the control. Estradiol-17β at any concentrations did not affect follicle development, although 10-6 M estradiol-17β diminished the percentage of degenerated follicles (40 ± 4%) compared to the control (61 ± 6%). Addition of an androgen receptor antagonist, cyproterone acetate (10-7 and 10-6 M) inhibited the stimulatory effect of testosterone (10-6 M) on primordial follicle development, suggesting the receptor-mediated action of testosterone. We next examined the presence of the androgen receptor in primordial follicles. Immunohistological examination of ovarian cortical sections revealed that androgen receptor signals were localized in oocytes in primordial follicles. In addition, in the Western blot analysis, anti-androgen receptor antibody recognized a band around 100 kDa in isolated oocytes from primordial follicles. Finally, the effect of testosterone on primordial follicle development was confirmed by xenotransplantation. Ovarian cortical pieces cultured for 7 days with and without 10-6 M testosterone were transplanted under the kidney capsule of female severe combined immune deficient mice for two months. Histological analysis of testosterone-treated xenografts showed that higher percentages of follicles developed to the primary, secondary and antral follicles compared to the control grafts in which no follicular development was observed. In conclusion, testosterone induces porcine primordial follicle development in vitro. (poster)

Cotransplantation of Placental Mesenchymal Stromal Cells Enhances Single and Double Cord Blood Engraftment in Nonobese Diabetic/Severe Combined Immune Deficient Mice

Limited cell numbers in a unit restricts cord blood transplantation (CBT) in adults. We evaluated whether cotransplantation of placental mesenchymal stromal cells (MSCs) would enhance engraftment. Plastic adherent cells from placenta demonstrated typical characteristics of MSCs. In six individual experiments, 4 cohorts of 24 nonobese diabetic/severe combined immune deficient (NOD/SCID) mice were evaluated. Cohort 1 received 5 x 10(4) CD34+ cells from unit (U) one (SCBT); cohort 2 received 5 x 10(4) CD34+ cells from U1 + 4 x 10(4) MSCs (SCBT+MSCs); cohort 3 received 2.5 x 10(4) CD34+ cells from U1 + 2.5 x 10(4) CD34+ cells from U2 (double cord blood transplant [DCBT]); cohort 4 received 2.5 x 10(4) CD34+ cells from U1 + 2.5 x 10(4) CD34+ cells from U2 + 4 x 10(4) MSCs (DCBT+MSCs). Hematopoietic engraftment evaluated after 6 to 8 weeks, was similar in recipients of SCBT and DCBT. MSC cotransplantation demonstrated enhanced engraftment in DCBT (51.8 +/- 6.8% versus 14.9 +/- 6.5%; p = .04) with an increased trend in SCBT (48.7 +/- 7.7% versus 17.5 +/- 6.1%; p = .07). In DCBT, cotransplantation of placental MSCs reduced single cord dominance. Self-renewal capacity was assessed by serial transplantation in secondary recipients infused with engrafted human cells from primary mice transplanted with or without MSCs. In secondary transplant experiments, 13 of 17 evaluable mice engrafted at levels of 1% to 6.5%. Despite enhanced engraftment in primary mice, long-term engraftment capacity was unaltered with MSC cotransplantation. Imaging studies showed MSCs migrated to pelvic region and improved cord blood (CB) CD34+ homing. Cotransplantation of placental MSCs enhanced cord blood engraftment and may act by improving homing of CD34+ cells.

P38 MAP Kinase Inhibits Neutrophil Development Through Phosphorylation of C/EBPα on Serine 21

Many extracellular stimuli regulate growth, survival, and differentiation responses through activation of the dual specificity mitogen activated protein kinase (MAPK) kinase three (MKK3) and its downstream effector p38 MAPK. Using CD34+ hematopoietic progenitor cells, here we describe a novel role for MKK3-p38MAPK in the regulation of myelopoiesis. Inhibition of p38MAPK utilizing the pharmacological inhibitor SB203580, enhanced neutrophil development ex vivo, but conversely reduced eosinophil differentiation. In contrast, constitutive activation of MKK3 dramatically inhibited neutrophil differentiation. Transplantation of beta2-microglobulin(-/-) nonobese diabetic/severe combined immune deficient (NOD/SCID) mice with CD34+ cells ectopically expressing constitutively active MKK3 resulted in reduced neutrophil differentiation in vivo, whereas eosinophil development was enhanced. Inhibitory phosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha) on serine 21 was induced upon activation of p38MAPK. Moreover, ectopic expression of a non-phosphorylatable C/EBPalpha mutant was sufficient to abrogate MKK3-induced inhibition of neutrophil development. Furthermore, treatment of CD34+ progenitors from patients with severe congenital neutropenia with SB203580 restored neutrophil development. These results establish a novel role for MKK3-p38MAPK in the regulation of lineage choices during myelopoiesis through modulation of C/EBPalpha activity. This signaling module may thus provide an important therapeutic target in the treatment of bone marrow failure.

Sam-Pointed Domain Containing Ets Transcription Factor in Luminal Breast Cancer Pathogenesis

We previously described frequent overexpression of Sam-pointed domain containing Ets transcription factor (SPDEF), also known as PDEF, in human breast cancer, and suggested a role for this transcription factor in breast tumor progression. To seek evidence in support of this hypothesis, the MCF-12A breast epithelial cell line was transfected with an SPDEF expression plasmid or with control vector plasmid and the transfected cells tested for their tumorigenic growth in vivo. The data showed that SPDEF expression in MCF-12A cells induced accelerated tumor growth in severe combined immune deficient mice compared with vector-transfected MCF-12A cells. Furthermore, Gene Expression Omnibus and Oncomine databases were mined to determine any correlation between SPDEF expression levels and clinical outcome. High SPDEF expression correlated with poor overall survival of patients with estrogen receptor+ breast cancer, in three independent data sets. In contrast, little correlation was observed between SPDEF expression and cancer relapse or remote metastases. SPDEF expression was further found to be restricted to tumors arising in the luminal epithelial lineage including estrogen receptor+ luminal subtype breast tumors, Her2/neu-positive tumors, and apocrine carcinomas. In contrast, little SPDEF expression was found in the basal subtype of breast tumors. Based on these results, we hypothesize that SPDEF has a function in the specification of the progenitor cells of the luminal epithelial lineage that become targets of oncogenesis in luminal breast cancer.

Effects of the Anti-VEGF Monoclonal Antibody Bevacizumab in a Preclinical Model and in Patients With Refractory and Multiple Relapsed Hodgkin Lymphoma

No curative treatment is currently available for refractory or relapsed Hodgkin lymphoma (HL) after high-dose chemotherapy. Thus, new drugs with different modes of action are needed. Vascular endothelial growth factor (VEGF), a key regulator of tumor-angiogenesis, is elevated in sera of patients with HL. Hodgkin and Reed-Sternberg cells also express the growth-stimulating VEGF-R2 receptor suggesting that VEGF could contribute to the pathophysiology of this malignancy. We investigated the effects of the humanized anti-VEGF monoclonal antibody bevacizumab (BV) against human HL xenografts in severe combined immune deficiency mice and in a compassionate use program in HL patients with multiple relapsed and progressive diseases. After a 4-week run-in phase of single agent BV, combined gemcitabine and BV therapy was administered. In the animal model, BV delayed the growth of HL tumors significantly (P=0.0004). Out of 5 patients included, BV alone had biologic effects as determined by tumor size, blood flow, fluorodeoxyglucose-uptake, and serum markers CCL17/thymus and activation-related chemokine, and sCD30 in 4 patients. The combination of BV and gemcitabine led to partial or complete remission in 3 of 5 patients. Accordingly, VEGF deprivation by the anti-VEGF antibody BV has antitumor activity in established HL tumors in a preclinical model. Furthermore, BV single agent therapy has biologic effects in HL patients indicating clinical activity. On the basis of these results, a prospective clinical study has been initiated to further investigate the impact of this antiangiogenic approach in HL.

Correlation of primary tumor engraftment in immune deficient mice and relapse rate in patients with early-stage non-small cell lung carcinoma (NSCLC)

11082 Background: Compared to cell lines, primary tumor xenografts potentially are more clinically relevant cancer models as they more closely reflect the phenotype and genotype of the original cancer. However, only a minority of tumors engraft successfully in severe combined immune deficient (scid) mice and can be passaged serially. Although xenograft models are used extensively, few studies have investigated whether tumors that engraft represent a distinct clinical subset. We hypothesized that NSCLC tumors with more aggressive clinical and histological features have greater engraftment capacity than those with a less aggressive phenotype. Methods: Fresh primary tumors were harvested from NSCLC patients who underwent curative resection. Tumor fragments were implanted into the subcutaneous tissue of non-obese diabetic-scid mice within 24 hrs of excision. Patient characteristics for tumors that engrafted (XG) and did not engraft (No-XG) were compared. Only tumors from patients with &gt;1-yr follow-up were evaluated for time to progression (TTP) and to correlate clinicopathological features with engraftment. Results: Between March 2005 and October 2008, 110 tumors were implanted. Of these, 45 (41%) engrafted and were passaged serially in vivo. The histological features of the primary were retained in 93% of XG tumors. Squamous cell carcinomas engrafted significantly more than adenocarcinomas (57% versus 26%, p=0.03). There were no significant differences in differentiation grade or clinical stage between the XG and No-XG groups. XG patients had significantly shorter TTP than the No-XG group (10.19 versus 18.64 months, p=0.003). In multivariate analysis the ability to form a xenograft was an independent predictor of relapse (HR 4.15 95% CI 1.152–14.94, p=0.03). Conclusions: Xenograft models can be established from the histological spectra of NSCLC encountered in the clinical setting and mimic closely the features of their primary tumors. The capacity of these tumors to engraft may be predictive of a more aggressive phenotype and poorer clinical outcome. No significant financial relationships to disclose.

Inhibitory effects of chitooligosaccharides on tumor growth and metastasis

Chitooligosaccharides (COS) are hydrolyzed products of chitosan and have been proven to exhibit various biological functions. The objectives of this study were to evaluate the anti-tumor growth, anti-metastatic potency and related pathways of COS extracted from fungi. In in vitro studies, we found that COS significantly inhibited human hepatocellular carcinoma (HepG2) cell proliferation, reduced the percentage of S-phase and decreased DNA synthesis rate in COS-treated HepG2 cells. Expressions of cell cycle-related genes were analyzed and the results indicated that p21 was up-regulated, while PCNA, cyclin A and cdk-2 were down-regulated. Moreover, we also found that the activity of metastatic related protein (MMP-9) could be inhibited by COS in Lewis lung carcinoma (LLC) cells. In in vivo studies, we found that COS inhibited the tumor growth of HepG2 xenografts in severe combined immune deficient (SCID) mice. In a LLC-bearing mouse tumor growth and lung metastasis model, COS inhibited tumor growth and the number of lung colonies in LLC-bearing mice as well as the lung metastasis, and it prolonged the survival time of the LLC-mice. These results suggest a potential anti-tumor growth and anti-metastatic potency of COS in cancer chemoprevention.

Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex

Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rgamma(null)) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rgamma(null) mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 x 10(6) PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-alpha signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.

Human Embryonic Stem Cells with Maintenance under a Feeder-Free and Recombinant Cytokine-Free Condition

We previously reported that cynomolgus monkey embryonic stem (ES) cells could be maintained under a feeder-free condition without using recombinant cytokines if sizes and numbers of ES colonies were kept within an appropriate range. Here we show that this finding is also true with human ES cells (hESCs). The two lines of hESCs, khES-1 and khES-3, were appropriately maintained in the absence of feeder layers or exogenous cytokines such as fibroblast growth factors, Noggin, transforming growth factor beta, and Activin by closely controlling the size and number of hESC colonies. High-level expressions of immature markers including SSEA-4, Oct-4, and Nanog were detected in feeder-free and cytokine-free hESCs, and they formed teratomas when implanted into severe combined immunedeficiency (SCID) mice. No chromosomal abnormalities were observed over 20 passages, ruling out the possibility that special clones with growth advantages had been selected. Global protein expression profiles were quite similar among the hESCs maintained by our feeder- and cytokine-free method, by coculture with mouse embryonic fibroblasts (MEFs) and by a feeder-free method using conditioned media of MEFs. However, the activation level of Akt, an important player for the maintenance of ES cells, was highest and the activation level of extracellular signal-regulated kinase, a critical player for differentiation of ES cells, was lowest in the hESCs maintained by our cytokine-free method. Our results not only show a technical improvement for the maintenance of hESCs but also open a new avenue for the understanding of autocrine signaling networks of hESCs.

In vitro and in vivo study of phloretin‐induced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter

Phloretin (Ph), a natural product found in apples and pears with glucose transporter (GLUT) inhibitory activity, exerts antitumor effects. However, little is known about its effects on human liver cancer. The purpose of this study is to test the cytotoxic effects of Ph on HepG2 cells and to identify the underlying molecular pathways. Human hepatocellular carcinoma specimens and HepG2 show a high level of GLUT2 transporter activity in the cell membrane. Real-time PCR and MTT assays demonstrate that Ph-induced cytotoxicity correlates with the expression of GLUT2. Flow cytometry and DNA fragmentation studies show that 200 microM Ph induces apoptosis in HepG2, which was reversed by glucose pretreatment. GLUT2 siRNA knockdown induced HepG2 apoptosis, which was not reversed by glucose. Western blot analysis demonstrates that both intrinsic and extrinsic apoptotic pathways in addition to Akt and Bcl-2 family signaling pathways are involved in Ph-induced cell death in HepG2 cells. Furthermore, using flow cytometry analysis, a mitochondrial membrane potential assay and Western blot analysis, we show that cytochalasin B, a glucose transport inhibitor, enhances the Ph-induced apoptotic effect on HepG2 cells, which was reversed by pretreatment with glucose. Furthermore, we found significant antitumor effects in vivo by administering Ph at 10 mg/kg intraperitoneally to severe combined immune deficiency mice carrying a HepG2 xenograft. A microPET study in the HepG2 tumor-bearing mice showed a 10-fold decrease in (18)F-FDG uptake in Ph-treated tumors compared to controls. Taken together, these results suggest that Ph-induced apoptosis in HepG2 cells involves inhibition of GLUT2 glucose transport mechanisms.