Tool For Preclinical Studies Research Articles

Abstract 1845: Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. The vast majority of the human HNSCCs contain p53 mutations, some of which acquire oncogenic gain-of-function (GOF) activities. Previous mouse models in which either the p53R172H GOF mutation or deletion of the p53 gene were co-activated with oncogenic K-ras showed that p53R172H can predispose to oral tumor initiation, accelerate tumor growth and promote progression to SCC. However, the impact of p53 gain- and loss-of-function (LOF) mutations in metastasis could not be assessed because of the low rates of progression to SCC in that model. To overcome this limitation, we used the tobacco-surrogate 4NQO to induce oral lesions that may progress to advance carcinomas following stepwise changes that resemble the gradual accumulation of histological and molecular abnormalities observed during human oral cancer progression. To examine the role of p53 mutations during SCC malignant progression we applied 4NQO to mice in which the p53R172H mutation or homozygous deletion of p53 were activated in oral epithelial cells. We observed that oral tumors appeared faster in the presence of p53 mutations than in mice with wtp53. No significant difference was observed between the p53R172H mutation and p53 deletion. However, survival was shorter in mice expressing p53R172H than in mice with loss of p53. Notably, regional and distant metastasis were only observed in mice with oral tumors expressing p53R172H. Therefore, these mice are excellent tools for pre-clinical studies designed to target early stages of tumor development or latest stages of progression and metastasis. As immunotherapy is becoming adopted as part of the standard treatment for head and neck cancer patients, we conducted a detailed characterization of the immune infiltrates of the lesions that developed in these mice in order to assess their immunogenicity. We observed a progressive increase in T-cell infiltration from normal oral mucosa to oral premalignant lesions (OPL) to SCC, in the presence or absence of p53 mutations. Similarly, the expression of immune checkpoint proteins, including PD1, PDL1 and CTLA4 increased during oral tumor progression, regardless of the p53 status. Interestingly, we observed a sharp increase in the presence of immune infiltrates expressing the co-stimulatory OX40 in SCCs relative to premalignant lesions, with different extent depending on the nature of the p53 mutation. The significance of these observations is currently being analyzed. In summary, p53R172H promotes metastasis in oral tumors induced by 4NQO. In addition, T-cell infiltration and activation of immune checkpoints during oral tumor progression suggest that this is an excellent model to test immunotherapy-based strategies. Furthermore, our findings suggest that OX40-based therapies may be tailored to patients according to their p53 status. Citation Format: Jin Wang, Yuanyuan Zhang, Bingbing Wang, Adel K. El-Naggar, Jeffrey N. Myers, Carlos Caulin. Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1845. doi:10.1158/1538-7445.AM2017-1845

Geroscience approaches to increase healthspan and slow aging.

For decades, researchers in the biology of aging have focused on defining mechanisms that modulate aging by primarily studying a single metric, sometimes described as the “gold standard” lifespan. Increasingly, geroscience research is turning towards defining functional domains of aging such as the cardiovascular system, skeletal integrity, and metabolic health as being a more direct route to understand why tissues decline in function with age. Each model used in aging research has strengths and weaknesses, yet we know surprisingly little about how critical tissues decline in health with increasing age. Here I discuss popular model systems used in geroscience research and their utility as possible tools in preclinical studies in aging.

Positron Emission Tomography: Current Challenges and Opportunities for Technological Advances in Clinical and Preclinical Imaging Systems.

Positron emission tomography (PET) imaging is based on detecting two time-coincident high-energy photons from the emission of a positron-emitting radioisotope. The physics of the emission, and the detection of the coincident photons, give PET imaging unique capabilities for both very high sensitivity and accurate estimation of the in vivo concentration of the radiotracer. PET imaging has been widely adopted as an important clinical modality for oncological, cardiovascular, and neurological applications. PET imaging has also become an important tool in preclinical studies, particularly for investigating murine models of disease and other small-animal models. However, there are several challenges to using PET imaging systems. These include the fundamental trade-offs between resolution and noise, the quantitative accuracy of the measurements, and integration with X-ray computed tomography and magnetic resonance imaging. In this article, we review how researchers and industry are addressing these challenges.

The future of patient-derived tumor xenografts in cancer treatment.

Over the last decades, major technological advancements have led to a better understanding of the molecular drivers of human malignancies. Nonetheless, this progress only marginally impacted the cancer therapeutic approach, probably due to the limited ability of experimental models to predict efficacy in clinical trials. In an effort to offset this limitation, there has been an increasing interest in the development of patient-derived xenograft (PDX) models where human tumors are xenotransplanted into immunocompromised mice. Considering their high resemblance to human tumors and their stability, PDX models are becoming the preferred translational tools in preclinical studies. Nonetheless, several limitations hamper a wider use of PDX models and tarnish the concept that they might represent the missing piece in the personalized medicine puzzle.

Genetically engineered mouse and orthotopic human tumor xenograft models of retinoblastoma.

Retinoblastoma is a rare pediatric cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Murine models of retinoblastoma provide excellent tools for preclinical studies as well as for the study of the biological processes that drive tumorigenesis following Rb loss. In this chapter, we describe several genetically engineered mouse and orthotopic human xenograft models of retinoblastoma and discuss the advantages and disadvantages of these models.

Abstract 1200: Characterization of a novel radiation-induced sarcoma cell line

Abstract Background: Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models currently exist to facilitate studies of RIS. Methods: We derived a primary human cell line, UACC-SARC1, from a RIS. Short tandem repeat (STR) genotyping was used to confirm that this cell line was propagated from the tumor. Further characterization of this cell line involved comparing 24 markers using immunocytochemistry (ICC) to immunohistochemistry (IHC) of the tumor, a Matrigel invasion assay, karyotyping of the cell line, comparative genomic hybridization (CGH), DNA sequencing using the Ion AmpliSeq Cancer panel and in vivo mouse xenografts after subcutaneous injection of UACC-SARC1 in immunodeficient mice. Results: STR profiling of UACC-SARC1 was virtually identical to its parental tumor. IHC analysis of the tumor and ICC analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. CGH provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. SCID mice xenografts exhibited tumor formation but resulting tumors failed to metastasize. Further xenografts with NOD SCID gamma (NSG) mice are planned. The doubling time of UACC-SARC1 was 28.3 hours. Conclusion: Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. Citation Format: Julie E. Lang, Brandon Nokes, Grishma Sheth, Petr Novak, Laura Fuchs, George S. Watts, Bernard W. Futscher, Neal Mineyev, Weizhu Zhu, Lauren LeBeau, Ray Nagle, Lee Cranmer. Characterization of a novel radiation-induced sarcoma cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1200. doi:10.1158/1538-7445.AM2014-1200

An In Vivo Mouse Model of Metastatic Human Thyroid Cancer

Mouse models of metastatic human cancers are important tools in preclinical studies for testing new systematic therapies and studying effectors of cancer metastasis. The major drawbacks of current mouse models for metastatic thyroid cancer are that they have low metastasis rates and do not allow in vivo tumor detection. Here, we report and characterize an in vivo detectable metastasis mouse model of human thyroid cancer using multiple thyroid cancer cell lines. Human anaplastic thyroid cancer cell lines 8505C, C-643, SW-1736, and THJ-16T; follicular thyroid cancer cell lines FTC-133, FTC-236, and FTC-238; and Hürthle cell carcinoma cell line XTC-1 were transfected with a linearized pGL4.51[luc2/CMV/Neo] vector or transduced with lentivirus containing Luc2-eGFP reporter genes. The stably transfected cells were injected intravenously into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Tumors were detected with an in vivo imaging system-Xenogen IVIS. Vemurafenib, a BRAF inhibitor, was used to treat lung metastases generated from 8505C-Luc2 cells with a BRAF(V600E) mutation to test the accuracy of the model to evaluate response to therapy. Intravenous injection of as few as 30,000 8505C-Luc2 cells produced lung metastases in 100% of the injected mice, and many of these mice also developed bone metastases at a later stage of the disease. Similarly, metastatic tumors also developed in all mice injected with C-643-Luc2, THJ-16T-Luc2, FTC-133-Luc2, FTC-236-Luc2, FTC-238-Luc2, and XTC-1-Luc2 cells. The metastases were easily detectable in vivo, and tumor progression could be dynamically and accurately followed and correlated with the actual tumor burden. Furthermore, disease progression could be easily controlled by adjusting the number of injected cells. The in vivo treatment of 8505C xenograft lung metastases with vemurafenib dramatically reduced the growth and signal intensity with good correlation with actual tumor burden. Herein we report an in vivo detectable mouse model of metastatic human thyroid cancer that is reliable and reproducible. It will serve as a useful tool in the preclinical testing of alternative systematic therapies for metastatic thyroid cancer, and for functional studies of thyroid cancer tumor biology in vivo.

Transgenic Pig Expressing the Red Fluorescent Protein Kusabira-Orange as a Novel Tool for Preclinical Studies on Hepatocyte Transplantation

IntroductionResearch on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as pigs. We used transgenic pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells. Materials and MethodsDonor hepatocytes were isolated from transgenic Kusabira-Orange (KO)-expressing pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a transgenic-cloned KO pig by Matsunari et al. and ova from a domestic pig. The hepatocyte transplant recipients were the nontransgenic, KO-negative littermates. In these recipient pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the transgenic male pig were isolated using collagenase perfusion. Hepatocytes (1 × 109 cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys. ResultsStrong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the transgenic male donor pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7. ConclusionTransgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.

Genomic biomarkers for cardiotoxicity in rats as a sensitive tool in preclinical studies

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver-operating characteristic (ROC) analysis using more cardiotoxic and non-cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi-gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi-gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi-gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs.

Synthesis and Evaluation of 99mTc Chelate-conjugated Bevacizumab

Vascular endothelial growth factor (VEGF) is one of the classic factors involved in tumor-induced angiognesis in several solid tumors. Bevacizumab, a monoclonal antibody against VEGF, can be used as an imaging tool in preclinical studies. The aim of this study was to radiolabel Bevacizumab with (99m)Tc and to evaluate in vivo its imaging properties in an adenocarcinoma animal model. For this purpose, Bevacizumab was derivatized with Suc-HYNIC as a bifunctional coupling agent. A mixture of Tricine/SnCl(2).2H(2)O was added to Bevacizumab-HYNIC and radiolabeled with (99m)TcO(4)(-). The radiochemical stability of the radiolabeled antibody was assessed. Biodistribution and scintigraphy imaging were performed in normal CD1 female mice and in spontaneous adenocarcinoma tumor bearing CD1 mice (n = 5). We demonstrated that 99mTc-HYNIC-Bevacizumab was stable. In vivo biodistribution studies revealed that tumor uptake of (99m)Tc-HYNIC-Bevacizumab was 1.37 ± 0.51% and 5.33 ± 2.13% at 4 and 24 h postinjection, respectively. Scintigraphy image studies showed tumor selective uptake of (99m)Tc-HYNIC-Bevacizumab in the tumor-bearing mice. We conclude that (99m)Tc-HYNIC-Bevacizumb has the potential to be used as a tracer for tumor imaging in preclinical studies.

In Vivo Imaging Enables High Resolution Preclinical Trials on Patients’ Leukemia Cells Growing in Mice

BackgroundXenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients’ acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter.Design and MethodsPatients’ acute lymphoblastic leukemia cells were lentivirally transduced to express the membrane-bound form of Gaussia luciferase. In vivo imaging was established in individual patients’ leukemias and extensively validated.ResultsBioluminescence in vivo imaging enabled reliable and continuous follow-up of individual mice. Light emission strictly correlated to post mortem quantification of leukemic burden and revealed a logarithmic, time and cell number dependent growth pattern. Imaging conveniently quantified frequencies of leukemia initiating cells in limiting dilution transplantation assays. Upon detecting a single leukemia cell within more than 10,000 bone marrow cells, imaging enabled monitoring minimal residual disease, time to tumor re-growth and relapse. Imaging quantified therapy effects precisely and with low variances, discriminating treatment failure from partial and complete responses.ConclusionsFor the first time, we characterized in detail how in vivo imaging reforms preclinical studies on patient-derived tumors upon increasing monitoring resolution. In the future, in vivo imaging will enable performing precise preclinical studies on a broad range of highly demanding clinical challenges, such as treatment failure, resistance in leukemia initiating cells, minimal residual disease and relapse.

Fluorescent Nanoprobes Dedicated to in Vivo Imaging: From Preclinical Validations to Clinical Translation

With the fast development, in the last ten years, of a large choice of set-ups dedicated to routine in vivo measurements in rodents, fluorescence imaging techniques are becoming essential tools in preclinical studies. Human clinical uses for diagnostic and image-guided surgery are also emerging. In comparison to low-molecular weight organic dyes, the use of fluorescent nanoprobes can improve both the signal sensitivity (better in vivo optical properties) and the fluorescence biodistribution (passive “nano” uptake in tumours for instance). A wide range of fluorescent nanoprobes have been designed and tested in preclinical studies for the last few years. They will be reviewed and discussed considering the obstacles that need to be overcome for their potential everyday use in clinics. The conjugation of fluorescence imaging with the benefits of nanotechnology should open the way to new medical applications in the near future.

Oxygen Breathing Affects 3′-Deoxy-3′-18F-Fluorothymidine Uptake in Mouse Models of Arthritis and Cancer

Noninvasive in vivo imaging of biologic processes using PET is an important tool in preclinical studies. We observed significant differences in 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) uptake in arthritic ankles and carcinomas between dynamic and static PET measurements when mice breathed oxygen. Thus, we suspected that air or oxygen breathing and the anesthesia protocol might influence (18)F-FLT tracer uptake. We injected arthritic, healthy, and CT26 colon carcinoma-bearing mice with (18)F-FLT before static or dynamic small-animal PET measurements. The spontaneously oxygen- or air-breathing mice were kept conscious or anesthetized with ketamine and xylazine during (18)F-FLT uptake before the 10-min static PET measurements. For dynamic PET scans, mice were anesthetized during the entire measurement. (18)F-FLT uptake was reported in percentage injected dose per cubed centimeter by drawing regions of interest around ankles, carcinomas, and muscle tissue. Additionally, venous blood samples were collected before (18)F-FLT injection and after PET measurement to analyze pH, carbon dioxide partial pressure (pCO(2)), and lactate values. A significantly reduced (18)F-FLT uptake was measured in arthritic ankles and in CT26 colon carcinomas when the mice breathed oxygen and were conscious during tracer uptake, compared with mice that were anesthetized during (18)F-FLT uptake. Breathing air completely abolished this phenomenon. Analysis of blood samples that were obtained from the mice before (18)F-FLT injection and after the PET scan implicated respiratory acidosis that was induced by oxygen breathing and consciousness during tracer uptake. Acidosis was found to be the primary factor responsible for the reduced (18)F-FLT uptake, as reflected by increased pCO(2) and reduced pH and lactate values. Oxygen-breathing conscious mice sustained respiratory acidosis and, consequently, reduced cell proliferation and (18)F-FLT uptake in arthritic ankles and CT26 colon carcinomas. Thus, we suggest the use of air instead of oxygen breathing for (18)F-FLT PET measurements.

The role of metabotropic glutamate receptor 7 (mGluR7) in drug dependence

The successful pharmacotherapy for drug addiction is a challenge for pharmacology in XXI century. We still do not have any registered medication for cocaine by FDA, alcohol therapies are not enough successful. High hopes concern metabotropic glutamate 7 receptor as a target for pharmacotherapy development of drug dependence. It is well documented the role of the mGluR7 in rewarding and motivational effects of cocaine and alcohol. The mGluR7 allosteric agonist AMN082 and antagonist MMPIP have become new tools in preclinical studies. AMN082 attenuated cocaine-enhanced electrical Brain Stimulation Reward (eBSR) in rats decreased cocaine self-administration, too, probably by counteracting cocaine-induced inhibition of the ventral pallidal GABA release. The mGluR7 activation reduced alcohol intake and preference in rats. The mGluR7 receptors take part in reinstatement of drug-seeking behavior after abstinence. The investigations showed that AMN082 suppressed cocaine-induced reinstatement of self-administrated rats via mGluR2/3 mechanism. It was mentioned also that mGluR7 allosteric agonist inhibited reinstatement of ethanol-induced CPP. Moreover, activation of the mGluR7 receptor exhibits less adverse reaction in comparison to other mGluRs and may give a promise for further development of drug dependence therapies in humans. However, some nonspecific effects of the mGluR7 in ethanol addiction have been recognized, so there are further investigations needed to explain the mechanisms of the mGluR7 action in rewarding and motivational effects of drugs.

Biochemical characterization of fluorescent‐labeled recombinant human alpha‐l‐iduronidase in vitro

In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low-molecular-weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the protein's characteristics. We tested the in vitro properties of fluorescent-labeled recombinant human alpha-l-iduronidase (rhIDU, the enzyme deficient in Hurler syndrome) and compared labeled to unlabeled proteins. Labeled rhIDU retained full enzymatic activity and showed similar kinetics to nonlabeled rhIDU. Uptake of labeled rhIDU into human Hurler fibroblasts, measured by activity assay, was equivalent to unlabeled rhIDU enzyme and showed an uptake constant of 0.72 nM. Labeled rhIDU was also able to enter cells via the mannose 6-phospate receptor pathway and reduce glycosaminoglycan storage in Hurler fibroblasts. Subcellular localization was verified within lysosomes by confocal microscopy. These findings suggest that fluorescent labeling does not significantly interfere with enzymatic activity, stability, or uptake, and validates this method as a way to track exogenously administered enzyme.

The Multiple Faces of Valosin-Containing Protein-Associated Diseases: Inclusion Body Myopathy with Paget’s Disease of Bone, Frontotemporal Dementia, and Amyotrophic Lateral Sclerosis

Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a progressive, fatal genetic disorder with variable penetrance, predominantly affecting three main tissue types: muscle (IBM), bone (PDB), and brain (FTD). IBMPFD is caused by mutations in the ubiquitously expressed valosin-containing protein (VCP) gene, a member of the AAA-ATPase superfamily. The majority of individuals who develop IBM have progressive proximal muscle weakness. Muscle biopsies reveal rimmed vacuoles and inclusions that are ubiquitin- and TAR DNA binding protein-43 (TDP-43)-positive using immunohistochemistry. PDB, seen in half the individuals, is caused by overactive osteoclasts and is associated clinically with pain, elevated serum alkaline phosphatase, and X-ray findings of coarse trabeculation and sclerotic lesions. FTD diagnosed at a mean age of 55 years in a third of individuals is characterized clinically by comprehension deficits, dysnomia, dyscalculia, and social unawareness. Ubiquitin- and TDP-43-positive neuronal inclusions are also found in the brain. Genotype-phenotype correlations are difficult with marked intra-familial and inter-familial variations being seen. Varied phenotypes within families include frontotemporal dementia, amyotrophic lateral sclerosis, Parkinsonism, myotonia, cataracts, and anal incompetence, among others. Cellular and animal models indicate pathogenetic disturbances in IBMPFD tissues including altered protein degradation, autophagy pathway alterations, apoptosis, and mitochondrial dysfunction. Currently, mouse and drosophila models carrying VCP mutations provide insights into the human IBMPFD pathology and are useful as tools for preclinical studies and testing of therapeutic strategies. In this review, we will explore the pathogenesis and clinical phenotype of IBMPFD caused by VCP mutations.

Highlights of This Issue

Despite satisfactory remission rates, resistance to imatinib is an important issue for therapy of chronic myeloid leukemia. Deville and colleagues showed that the emergence of resistance occurred faster in cells overexpressing the catalytic subunit of the telomerase, showing that this enzyme represents an additional factor in the development of imatinib resistance. Furthermore, strategies targeting either telomerase expression or activity restored imatinib sensitivity in the resistant cells. Therefore, combining antitelomerase strategies to imatinib treatment represents an attractive approach to prevent the emergence of imatinib-resistant clones and increase the probability to eradicate the disease.PP2A is a major serine/threonine phosphatase and a potent tumor suppressor; however, its role in prostate cancer has remained underexplored. Bhardwaj and colleagues have now shown that PP2A activity is inversely associated with androgen-independent growth of prostate cancer cells. Their data reveal a novel mechanism, whereby loss of PP2A-mediated checkpoints leads to the activation of Akt and ERK and partially sustains androgenreceptor signaling under steroiddeprived condition. Their findings offer potential therapeutic implications for targeting PP2A in castration-resistant prostate cancer.Exposure of cells to microtubuletargeting cancer drugs such as paclitaxel causes mitotic arrest by activation of the mitotic checkpoint. Some cells can escape mitotic arrest by entering interphase without dividing, a process termed mitotic slippage. Riffell and colleagues examine mechanisms underlying mitotic slippage using two chemicals found to induce slippage. SU6656 and geraldol induced mitotic slippage through caspase-3-dependent degradation of the checkpoint kinase BubR1, thus permitting proteasomedependent degradation of cyclin B1 and escape from drug-induced mitotic arrest. The identification of this pathway linking apoptosis with mitotic control may have implications for cancer therapy.The epithelial to mesenchymal transition (EMT) of malignant hepatocytes is a crucial event in hepatocellular carcinoma (HCC) progression and recurrence. In this study, van Zijl and colleagues established a novel and unique cellular EMT model of human HCC to identify molecular mechanisms and to assess therapeutic drug efficacy during liver carcinoma progression. Most remarkably, they found that the combined treatment with doxorubicin and sorafenib caused increased susceptibility of HCC cell types before and after EMT, resulting in enhanced drug efficacy. This model of EMT that reliably reflects human HCC progression is an invaluable tool in preclinical studies for the identification of molecular mechanisms underlying HCC progression, the pharmacological determination of dose-effect relationships and thus the efficacy of single and combined treatments with novel and currently used anticancer drugs, and the (re)-evaluation of drug target specificity and pleiotropic effects.

Hybrid ferritin nanoparticles as activatable probes for tumor imaging.

Near-infrared fluorescence (NIRF) imaging is emerging as an important tool in preclinical studies.[1–5] As the technology is being established and implemented worldwide, a wave of effort has been spurred to develop novel imaging probes that can accurately recognize and report diseases, such as cancer.[3,6] One common drawback of NIRF imaging, however, is its high background and as a consequence of that, low specificity.[7,8] To address this issue, we and others have developed activatable probes with various approaches. Such probes, constructed by bringing into proximity an energy donor and receptor, are designed to be fluorescently quenched at a quiescent stage but activatable when encountering a specific trigger, which leads to a structural or conformational change and restores fluorescence activities.[7–9] This technique allows the signals to be only amplified at diseased areas upon a designated environment change, a feature which can greatly improve the signal-to-background ratio.

Differentiation and enrichment of expandable chondrogenic cells from human embryonic stem cells in vitro

Human embryonic stem cells (hESCs) are considered as useful tools for pre-clinical studies in regenerative medicine. Although previous reports have shown direct chondrogenic differentiation of mouse and hESCs, low yield and cellular heterogenicity of the resulting cell population impairs the generation of sufficient numbers of differentiated cells for further testing and applications. Based on our previously established high-density micromass model system to study hESC chondrogenesis, we evaluated the effects of transforming growth factor (TGF)-β1 and bone morphogenetic protein-2 on early stages of chondrogenic differentiation and commitment by hESCs. Significant chondrogenic induction of hESCs, as determined by quantitative measurements of cartilage-related gene expression and matrix protein synthesis, was achieved in the presence of TGF-β1. By means of selective growth factor combination (TGF-β1, FGF-2 and platelet-derived growth factor-bb) and plating on extracellular matrix substratum, we report here the reproducible isolation of a highly expandable, homogenous and unipotent chondrogenic cell population, TC1, from chondrogenically committed hESCs. Like primary chondrocytes, TC1 rapidly dedifferentiates upon isolation and monolayer expansion but retains the chondrogenic differentiation potential and responds to TGF-β1 for cartilaginous tissue formation both in vitro and in vivo. In addition, TC1 displays a somatic cell cycle kinetics, a normal karyotype and does not produce teratoma in vivo. Thus, TC1 may provide a potential source of chondrogenic cells for drug testing, gene therapy and cell-based therapy.

Immunohistopathological and neuroimaging characterization of murine orthotopic xenograft models of glioblastoma multiforme recapitulating the most salient features of human disease.

Tumorigenesis in human glioblastoma multiforme (GBM) is driven by several genetic abnormalities with disruption of important molecular pathways, such as p53/MDM2/p14ARF and EGFR/PTEN/Akt/mTOR. The malignant progression of human GBM is also primarily associated with a peculiar multistep pathophysiological process characterized by intratumoral ischemic necrosis (i.e. pseudopalisading necrosis) and activation of the hypoxia-inducible factor (HIF)-1alpha pathway with consequent peritumoral microvascular proliferation and infiltrative behaviour. Predictable preclinical animal models of GBM should recapitulate the main pathobiological hallmarks of the human disease. In this study we describe two murine orthotopic xenograft models using U87MG and U251 human cell lines. Ten Balb/c nude male mice were orthotopically implanted with either U87MG (5 mice) or U251 (5 mice) cell lines. Intracranial tumor growth was monitored through Magnetic Resonance Imaging (MRI). Immunohistopathological examination of the whole cranium was performed 30 days after implantation. U251 orthotopic xenografts recapitulated the salient pathobiological features described for human GBM, including invasive behaviour, wide areas of pseudopalisading necrosis, florid peripheral angiogenesis, GFAP and vimentin expression, nonfunctional p53 expression, striking active-caspase-3 and HIF-1alpha expression along pseudopalisades. U87MG orthotopic xenografts proved to be very dissimilar from human GBM, showing expansile growth, occasional necrotic foci without pseudopalisades, intratumoral lacunar pattern of angiogenesis, lack of GFAP expression, functional p53 expression and inconsistent HIF-1alpha expression. Expression of pAkt was upregulated in both models. The results obtained suggest that the U251 orthotopic model may be proposed as a predictive and reliable tool in preclinical studies since it recapitulates the most salient pathobiological features reported for human GBM.