Passage In Mice Research Articles

Propagation of ovine prions from “poor” transmitter scrapie isolates in ovine PrP transgenic mice

Ovine prion strains have typically been identified by their transmission properties, which include incubation time and lesion profile, in wild type mice. The existence of scrapie isolates that do not propagate in wild type mice, defined here as “poor” transmitters, are problematic for conventional prion strain typing studies as no incubation time or neuropathology can be recorded. This may arise because of the presence of an ovine prion strain within the original inoculum that does not normally cross the species barrier into wild type mice or the presence of a low dose of an infectious ovine prion strain that does. Here we have used tg59 and tg338 mouse lines, which are transgenic for ovine ARQ or VRQ PrP, respectively, to strain type “poor” transmitter ovine scrapie isolates. ARQ and VRQ homozygous “poor” transmitter scrapie isolates were successfully propagated in both ovine PrP transgenic mouse lines. We have used secondary passage incubation time, PrPSc immunohistochemistry and molecular profile, to show that different prion strains can be isolated from different “poor” transmitter samples during serial passage in ovine PrP transgenic mice. Our observations show that poor or inadequate transmissibility of some classical scrapie isolates in wild type mice is associated with unique ovine prion strains in these particular sheep scrapie samples. In addition, the analysis of the scrapie isolates used here revealed that the tg338 mouse line was more versatile and more robust at strain typing ovine prions than tg59 mice. These novel observations in ovine PrP transgenic mice highlight a new approach to ovine prion strain typing.

Evolution of Sub-Clones with KRAS Mutations In Pediatric Patients with MLL-AF4 Rearrangements

Abstract 2454Mixed Lineage Leukemia (MLL) gene rearrangements are common genetic translocations occurring in human leukemia. Numerous MLL-partner genes have been identified so far; of these MLL-AF4 was found to characterize the largest subgroup of fusion proteins in pediatric acute lymphoblastic leukemia (ALL). A recent study reported that mutations in kras accelerate leukemo-lymphogenesis in a MLL-AF4 transgenic mouse model. Activating mutations in RAS genes, frequently occuring in codons 12, 13 and 61, prevent the hydrolysis of RAS-GTP and result in the constitutive activation of the RAS proteins enhancing cell survival and proliferation. Mutations in RAS genes were found in several types of human cancers, including leukemia and ALL with MLL rearrangements.We analyzed the presence of KRAS mutations in pediatric MLL-AF4 rearranged ALL samples at diagnosis and relapse with the aim to establish the frequency of KRAS mutations in MLL-AF4 leukemia and to tentatively associate RAS mutations to the leukemogenic process.In this study 40 ALL patients were included (23 infant and 17 non infant); of these, 14 pairs at diagnosis and relapse, 22 at diagnosis only and 4 samples at relapse only. The study was approved by the institutional ethical committee and informed consent was obtained in accordance with the Declaration of Helsinki.Mutation screening was performed for exon 2 and exon 3 of KRAS by 454 technology (Roche Applied Science) with GS Junior Sequencing Instruments; to recognize each specific patient, primer pairs included a 10-base molecular identifier barcode sequence (MID). We generated amplicon ultra-deep sequencing of the 2 amplicons with a median coverage per amplicon of 1800 reads per sample.Twenty patients (55,6%) out of 36 with t(4;11) at diagnosis harbored KRAS mutations (range of penetrance of 0,4–29,7% for mutated sequences). Interestingly, in some patients more than one clone with different mutations at difference penetrance in KRAS codons were present. Of the 18 relapse samples 4 (22%) harbor KRAS mutations (penetrance of 1.3–38% of reads).Sanger sequencing was used to validate the presence of mutations in patients with more of 20% of penetrance identified with 454 technology.To evaluate the role of KRAS mutations in the leukemogenic process we analyzed matched paired diagnostic and relapse samples; in patients with mutations at diagnosis, 3 distinct situations were identified in the relapsed samples, pointing different changes between diagnosis and relapse: (1) presence of KRAS mutations in a specific codon at diagnosis and disappearance of this clone at relapse (6 of 9 cases, 67%); (2) decrease of the penetrance of a mutation between diagnosis and relapse and appearance of a new different mutation at relapse (2 of 9 cases, 22%); (3) clonal evolution of the same mutation and increase of the percentage of the mutated sequence from diagnosis to relapse (1 of 9 cases, 11%).Apparently, patients with MLL-AF4 rearrangements at diagnosis and relapse presented with subclones and during the relapse phase previous clones disappeared while new clones appeared and only in one case we can assume a selective advantage of the clone with the KRAS mutation.To further explore the propagation of KRAS mutated subclones in MLL-AF4, we transplanted 2 patients at diagnosis into a NOD/SCID xenograft mouse model. Xenografted samples obtained from leukemia bearing mice transplanted further onto subsequent recipients in serial passages. KRAS mutations were analyzed for each of the passages as mentioned above. One of the diagnostic samples used for transplantation harbored KRAS G12S mutation. The human cells of this patient isolated after the first passage in the mouse carried the same mutation of the diagnosis but with a very low penetration, suggesting a minority clone that decreased further in the second passage. No trace of mutations was found in the third passage. The other diagnostic sample for which serial transplanted samples were analyzed had no KRAS mutation at diagnosis and no new mutations appeared during further passages in the mice.The disappearance of the mutated KRAS subclone during passages in the recipient mice seems to reflect the negative selection for mutated clones observed in patients between diagnosis and relapse.Overall, these data showed that KRAS mutations are frequently occurring in MLL-AF4 leukemias at diagnosis however these mutations do not seem to add a selective advantage to the blast cells. Disclosures:No relevant conflicts of interest to declare.

Use of Murine Bioassay to Resolve Ovine Transmissible Spongiform Encephalopathy Cases Showing a Bovine Spongiform Encephalopathy Molecular Profile

Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK‐1 and UK‐2, were investigated further by transmissions to wild‐type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrPSc immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild‐type mice; PrPSc deposition pattern in wild‐type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrPSc deposition patterns in both wild‐type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK‐1 and UK‐2 cases were not compatible with BSE.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Reversion of PB2-627E to -627K during Replication of an H5N1 Clade 2.2 Virus in Mammalian Hosts Depends on the Origin of the Nucleoprotein

H5N1 highly pathogenic avian influenza viruses (HPAIV) of clade 2.2 spread from Southeast Asia to Europe. Intriguingly, in contrast to all common avian strains specifying glutamic acid at position 627 of the PB2 protein (PB2-627E), they carry a lysine at this position (PB2-627K), which is normally found only in human strains. To analyze the impact of this mutation on the host range of HPAIV H5N1, we altered PB2-627K to PB2-627E in the European isolate A/Swan/Germany/R65/2006 (R65). In contrast to the parental R65, multicycle growth and polymerase activity of the resulting mutant R65-PB2(K627E) were considerably impaired in mammalian but not in avian cells. Correspondingly, the 50% lethal dose (LD₅₀) in mice was increased by three orders of magnitude, whereas virulence in chicken remained unchanged, resulting in 100% lethality, as was found for the parental R65. Strikingly, R65-PB2(K627E) reverted to PB2-627K after only one passage in mice but did not revert in chickens. To investigate whether additional R65 genes influence reversion, we passaged R65-PB2(K627E) reassortants containing genes from A/Hong Kong/156/97 (H5N1) (carrying PB2-627E), in avian and mammalian cells. Reversion to PB2-627K in mammalian cells required the presence of the R65 nucleoprotein (NP). This finding corresponds to results of others that during replication of avian strains in mammalian cells, PB2-627K restores an impaired PB2-NP association. Since this mutation is apparently not detrimental for virus prevalence in birds, it has not been eliminated. However, the prompt reversion to PB2-627K in MDCK cells and mice suggests that the clade 2.2 H5N1 HPAIV may have had a history of intermediate mammalian hosts.

Conformational Transformation and Selection of Synthetic Prion Strains

Prion protein is capable of folding into multiple self-replicating prion strains that produce phenotypically distinct neurological disorders. Although prion strains often breed true upon passage, they can also transform or “mutate” despite being devoid of nucleic acids. To dissect the mechanism of prion strain transformation, we studied the physicochemical evolution of a mouse synthetic prion (MoSP) strain, MoSP1, after repeated passage in mice and cultured cells. We show that MoSP1 gradually adopted shorter incubation times and lower conformational stabilities. These changes were accompanied by structural transformation, as indicated by a shift in the molecular mass of the protease-resistant core of MoSP1 from approximately 19 kDa [MoSP1(2)] to 21 kDa [MoSP1(1)]. We show that MoSP1(1) and MoSP1(2) can breed with fidelity when cloned in cells; however, when present as a mixture, MoSP1(1) preferentially proliferated, leading to the disappearance of MoSP1(2). In culture, the rate of this transformation process can be influenced by the composition of the culture media and the presence of polyamidoamines. Our findings demonstrate that prions can exist as a conformationally diverse population of strains, each capable of replicating with high fidelity. Rare conformational conversion, followed by competitive selection among the resulting pool of conformers, provides a mechanism for the adaptation of the prion population to its host environment.

Preclinical xenograft models of human sarcoma show nonrandom loss of aberrations

Human tumors transplanted into immunodeficient mice (xenografts) are good preclinical models, and it is important to identify possible systematic changes during establishment and passaging in mice. High-resolution microarray-based comparative genomic hybridization (array CGH) was used to investigate how well a series of sarcoma xenografts, including 9 patient/xenograft pairs and 8 early versus late xenograft passage pairs, represented the patient tumor from which they originated. In all analyses, the xenografts were more similar to their tumor of origin than other xenografts of the same type. Most changes in aberration patterns were toward a more normal genome complement, and the increased aberrations observed were mostly toward more loss. In general, the changes were scattered over the genome, but some changes were significant in osteosarcomas. These were rather focused and consistent with amplifications frequent in patient samples, involving the genes platelet-derived growth factor receptor A (PDGFRA), cysteine-rich hydrophobic domain 2 (CHIC2), FIP-like 1 (FIP1L1), ligand of numb-protein X1 (LNX1), RAS-like family 11 member B (RASL11B), and sec1 family domain containing 2 (SCFD2), probably a sign of continued tumor progression. Some changes that disappeared may have been involved in host-stroma interactions or chemotherapy resistance, possibly because of the absence of selection in the mouse. Direct xenografts reflected well the genomic patterns of their tumors of origin. The few significant aberrations that were lost during passaging in immune-defective mice may have been caused by the lack of selection in the new host, whereas aberrations that were gained appeared to be the result of general tumor progression rather than model-specific artifacts.

Squirrel adenovirus type 1 in red squirrels (Sciurus vulgaris) in Germany

ENTERIC adenovirus (AdV) infection of red squirrels ( Sciurus vulgaris ) suffering from diarrhoea was first seen in Great Britain, in the county of Norfolk (Sainsbury and others 2001). The...

Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines

A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral “contamination”, much like routine mycoplasma testing.

Abstract 1598: Extensive use of fresh patient-derived tumor samples for drug discovery and drug positioning in oncology

Abstract Most preclinical investigations in Oncology research are performed using permanent cancer cell lines that have been kept in continuous in vitro passages since decades. These plastic-cultured cells became very different from their original tumors since accumulated genetic abnormalities and multiple selections of subclones occurred over time. The use of fresh patient-derived tumor samples for ex vivo assays and for establishment of new relevant in vivo tumor models allows investigating the anti-tumor activity of new therapies directly linked with clinical reality. In this respect, Oncodesign has developed a large and international network of clinical centers for the collection of a large number of fresh patient-derived tumor biopsies from all cancer pathologies and from healthy tissues. The collection of these samples is done under ethically approved master agreements and with the signed consent of each patient. The patient's clinical history, the serology results (HIV, HBV and HCV) and tissue banking are centralized in our internal approved biological resource center. Examples of ex vivo assays will be presented based on patient-derived acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia and other hemopathologies using chromium release and Annexin V FACS assays. These studies aimed to demonstrate the CDC, ADCC and apoptosis induction of new therapeutics antibodies whereby approved antibodies such as rituximab and alemtuzumab were used as positive controls. Similarly, these fresh patient-derived models were used to study the direct dose-response effect of new chemotherapeutic agents through apoptosis induction (bortezomib used as positive control). The freshly collected tumors were also used to establish new in vivo tumor models in different strains of mice and rats. The full characterization of the genetic patterns of the xenograft derived tumors was compared with the original collected human tumors and with the clinical data of the patient. A panel of “standard of care” compounds was tested in these new tumor models for pharmacological characterization. The expansion of such tumors at early passages in mice was used to implement a reproducible ex vivo 3D assay. The assay was developed to investigate the potential anti-tumor activity of conventional and targeted therapies, and allows sufficient throughput for use during drug discovery lead optimisation. Ex vivo drug effects were then correlated with in vivo results using the same panel of tumorgrafts. The use of fresh patient-derived tumors in drug discovery and early preclinical development of new therapies aimed at corroborating results with clinical reality. Altogether, these processes from the clinical tumor collection to the in vivo drug efficacy study through ex vivo assays should help the preclinical drug selection, development and clinical positioning as well as companion biomarker identification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2011-1598

Abstract 2828: Antitumor activity of the investigational proteasome inhibitor MLN9708 in preclinical models of NSCLC

Abstract The successful development of VELCADE® (bortezomib) for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for hematological malignancies. The investigational drug MLN9708 is a modified dipeptidyl boronic acid and a potent, reversible and specific inhibitor of the proteasome. MLN9708 is currently being evaluated in Phase I trials for hematologic malignancies as well as solid tumors. In preclinical models, MLN9708 showed better antitumor activity and PD response compared to bortezomib.Upon exposure to aqueous solutions or plasma, MLN9708 immediately hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all preclinical studies described in this report. We evaluated the antitumor activity of MLN2238 in several non small cell lung cancer (NSCLC) xenografts. More than 70% tumor growth inhibition was observed in the HCC-827 xenograft model with 13 mg/kg, IV, twice weekly (BIW) dosing. In the H1650 NSCLC xenograft model, 40-50% tumor growth inhibition was found at 8 and 11 mg/kg, IV, BIW dosing. However, a synergistic effect on H1650 tumor growth inhibition was found when MLN2238 was combined with chemotherapeutic agents including doxorubicin 8 mg/kg IV, weekly (QW) dosing (90% tumor growth inhibition) and taxotere 5 and 10 mg/kg, IV, QW (80-100% tumor growth inhibition). Furthermore, the combination of MLN2238 (8 and 11 mg/kg, IV, BIW) and taxotere (10 mg/kg, IV, QW) resulted in tumor regression in the H1650 xenograft model, whereas no regressions were observed with either drug as a single agent. Similar single agent activity and combination effect with taxotere was observed We also evaluated single agent activity and combination with taxotere in an early-passage primary human tumor xenograft model, PHTX-132Lu. In an effort to understand the factors that may determinecontributing sensitivity vs resistance to proteasome inhibitors, we tested the ability of MLN2238 to inhibit anchorage independent growth and colony formation of NSCLC tumor cells in semi-solid medium. For these studies, tumor cells are derived from primary human tumor explants which have been maintained by serial passage in immunocompromised mice. Initial experiments show a range of sensitivity to MLN2238 among the primary tumors, Comparisons of genomic data, including gene expression profiles, copy number variation, and sequencing of commonly mutated genes, may illuminate factors contributing to sensitivity and resistance. In addition, in vivo studies will be performed on selected models to examine the value of this in vitro colony formation assay in predicting in vivo sensitivity. Data suggest that MLN2238 has antitumor activity alone and in combination with cytotoxic agents in preclinical models of NSCLC, and these results support the ongoing Phase I clinical investigation of MLN9708 in solid tumor types including NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2828. doi:10.1158/1538-7445.AM2011-2828

Abstract 1585: Novel human renal medullary carcinoma mouse model to evaluate chemotherapy efficacy and tumor biology

Abstract Renal medullary carcinoma (RMC) is a rare disease with only ∼100 cases having been reported to date with the majority of these patients also having the sickle cell disease or trait. Multiple therapies, including chemotherapy, radiation, and resection, have been tried; however, this disease is almost uniformly fatal. We developed new mouse models of RMC that were created using patient derived tumor xenografts in the NOD SCID IL2 receptor γ chain null (NSG) mouse. RMC was diagnosed in a 9 year old male with sickle cell trait and multiple metastases in liver and lungs. The patient was treated by resection of the renal tumor, followed by chemotherapy, including bortezomib, paclitaxel/carboplatin/ bevacizumab, doxil/bevacizumab, and sunitinib. The patient showed partial or no response to each regimen and developed malignant pleural effusion and died 9 months p=0.0043 from diagnosis. NSG xenograft models were formed from both the patient primary renal tumor specimen and malignant pleural effusion. Comparative microarray gene expression data collection, genomic SNP data collection for copy number variance (CNV), and histology analyses were performed with the original patient renal tumor specimen and the two xenograft models for determining predictive chemotherapy responses for therapeutic efficacy. Additionally, the xenograft tumors were used for chemotherapy efficacy studies for using these models for future predictive efficacy studies. Both primary renal tumor and the metastatic pleural effusion developed large mouse xenograft tumors (P0) that were passaged into secondary mouse passages (P1). H & E histology showed that both the patient renal tumor and the renal tumor-derived xenograft tumors were moderately differentiated and morphologically similar. The malignant pleural effusion xenograft tumors were highly differentiated and not morphologically similar to the primary patient renal tumor or the renal tumor-derived xenograft. Sunitinib and temsirolimus were evaluated in comparison to vehicle control for tumor growth delay in P1 malignant pleural effusion xenografted mice and sunitinib efficacy was established vs. vehicle controls. Sunitinib demonstrated was highly efficacious compared to vehicle control with 110% tumor growth delay (p=0.0043) and temsirolimus tumor growth delay was not efficacious (p>0.05). Since RMC is a rare disease for which there is currently no effective therapy, these new mouse models will be a useful for developing new treatments for this lethal disease. Furthermore, with a series of samples (normal, sickle cell trait kidney, RMC at diagnosis, and tumor cells from pleural effusion), we expect to understand molecular mechanisms underlying the disease progression pathway (pathogenesis). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1585. doi:10.1158/1538-7445.AM2011-1585

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

399 DEVELOPMENT AND CHARACTERIZATION OF CLINICALLY RELEVANT TUMOR MODELS FROM PATIENTS WITH RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011399 DEVELOPMENT AND CHARACTERIZATION OF CLINICALLY RELEVANT TUMOR MODELS FROM PATIENTS WITH RENAL CELL CARCINOMA Jose Karam, Xiu-Ying Zhang, Pheroze Tamboli, Vitaly Margulis, Hua Wang, E. Jason Abel, Stephen Culp, and Christopher Wood Jose KaramJose Karam Houston, TX More articles by this author , Xiu-Ying ZhangXiu-Ying Zhang Houston, TX More articles by this author , Pheroze TamboliPheroze Tamboli Houston, TX More articles by this author , Vitaly MargulisVitaly Margulis Dallas, TX More articles by this author , Hua WangHua Wang Houston, TX More articles by this author , E. Jason AbelE. Jason Abel Houston, TX More articles by this author , Stephen CulpStephen Culp Houston, TX More articles by this author , and Christopher WoodChristopher Wood Houston, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.487AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Animal models are instrumental in understanding disease pathophysiology and mechanisms of therapy action and resistance in vivo. We sought to establish and characterize a panel of mouse models of renal cell carcinoma (RCC) derived from patients undergoing radical nephrectomy. METHODS Renal cell carcinoma mouse models were established from four patients with distinct histologies of renal cell carcinoma. Tumor tissues obtained during surgery were implanted into subcutaneous space of female BALB/c nude mice and serially passaged into new mice. Tumors obtained directly from patients and those derived from serial passages in nude mice were characterized by histology, Short Tandem Repeat (STR) fingerprinting, VHL gene sequencing, and Single Nucleotide Polymorphism (SNP) analysis for each of the four histologies. Tumor bearing mice were treated with sunitinib or everolimus and growth was compared between different histologies and different tumor passages. Primary cell cultures were also derived from patient tumors and were transfected with a lentiviral vector carrying the luciferase gene. Bioluminescence imaging (BLI) of mice was performed after intraperitoneal injection of luciferin in 2 of the models. RESULTS Four subcutaneous xenograft mouse models were developed: MDA−RCC−48, MDA−RCC−55, MDA−RCC−62, and MDA−RCC−80, representing papillary type 1, papillary type 2, clear cell, and clear cell with sarcomatoid features renal cell carcinoma, respectively. Tumor growth was dependent on the histologic type, size of implanted tumor chip, and possibly the passage number. We confirmed that the mouse tumors accurately and faithfully represent their respective original patient tumors as STR fingerprints were matching, histology on H&E staining was comparable, and SNP profiles and VHL mutation status were conserved with multiple passages. BLI results were commensurate with the subcutaneous xenograft growth patterns and enabled sequential and non−invasive monitoring of growth in an orthotopic implantation setting. Mice treated with sunitinib and everolimus exhibited an initial response to these agents, followed by a later stage of resistance to these agents, which mimics the clinical observations in patients with RCC. CONCLUSIONS We developed four mouse xenograft models of RCC with clear cell and papillary histologies, with stable histologic and molecular characteristics. We are currently using these models to identify mechanisms of action, molecular correlates of response, and resistance to novel targeted therapies. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e161 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jose Karam Houston, TX More articles by this author Xiu-Ying Zhang Houston, TX More articles by this author Pheroze Tamboli Houston, TX More articles by this author Vitaly Margulis Dallas, TX More articles by this author Hua Wang Houston, TX More articles by this author E. Jason Abel Houston, TX More articles by this author Stephen Culp Houston, TX More articles by this author Christopher Wood Houston, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Establishment of a novel chinese human lung adenocarcinoma cell line CPA-Yang3 and its real bone metastasis clone CPA-Yang3BM in immunodeficient mice

背景与目的肺癌复发和转移仍是世界性医学难题。本研究旨在建立国人肺腺癌转移细胞株，为研究肺癌转移的分子机制提供新颖的实验材料。方法本细胞取自首诊双侧淋巴结转移，65岁女性肺腺癌(Ⅲb期)患者的胸水细胞原代培养即获成功。致瘤率实验，细胞生长曲线，染色体核型分析，荧光定量PCR检测，小鼠心室造模，核素骨显像和X片活体成像检测小鼠骨转移，取骨转移灶细胞原代培养，in vivo-in vitro重复多个cycles筛选骨转移细胞。结果传自第4代细胞小鼠致瘤率达100%，经裸鼠左心室种植(1×106细胞/只)后第3周经核素骨显像、X片和病理检测确定骨转移率为：下颌骨100%，肱骨50%，股骨66.7%，脊柱50%，肩胛骨33%。同时伴有多脏器转移，肺(33%)、肝(50%)、肾上腺(17%)、颌下腺(33%)。荧光定量PCR检测ESM1、VEGF-C、IL-6、IL-8、AR、SVIL、FN1等基因过表达。将股骨转移细胞经体内外3个cycles筛选获得骨转移细胞CPA-Yang3BM。结论新建的CPAYang3是个取自首诊双侧淋巴结转移肺腺癌病人的胸水原代细胞培养成功并经裸鼠心室造模、体内外4个cycles筛选后获得的多发骨转移的国人肺腺癌细胞株。

The typing of Trypanosoma evansi isolates using mobile genetic element (MGE) PCR

The mobile genetic element PCR (MGE-PCR) is a simple and sensitive technique that can be used to detect genetic variability in Trypanosoma brucei ssp. To investigate the reliability of MGE-PCR in genotyping Trypanosoma evansi, stocks that were isolated directly from camels and after their respective passage in mice were analyzed. Construction of a dendrogram using the MGE-PCR banding profiles revealed a clear distinction between T. evansi and T. brucei, as well as discriminating the T. evansi strains (T. evansi with minicircle types B and A). A minor host-dependent clustering shows a genetic difference of <15%. Changes in the banding profiles were observed after serial passage of T. evansi type B in mice, while those of T. evansi type A were identical. It is apparent that significant random insertion mobile element positional variation occurs when T. evansi isolates are introduced into a new host, a factor that needs to be considered when MGE-PCR is used to determine genetic variation in T. evansi isolates that have different host origins.

Emergence of multiple prion strains from single isolates of ovine scrapie

The infectious agent associated with prion diseases such as ovine scrapie shows strain diversity. Ovine prion strains have typically been identified by their transmission properties in wild-type mice. However, strain typing of ovine scrapie isolates in wild-type mice may not reveal properties of the infectious prion agent as they exist in the original host. This could be circumvented if ovine scrapie isolates are passaged in ovine prion protein (PrP)-transgenic mice. This study used incubation time, lesion profile, immunohistochemistry of the disease-associated PrP (PrP(Sc)) and molecular profile to compare the range of ovine prion strains that emerged from sheep scrapie isolates following serial passage in wild-type and ovine PrP transgenic mice. It was found that a diverse range of ovine prion strains emerged from homozygous ARQ and VRQ scrapie isolates passaged in wild-type and ovine PrP transgenic mice. However, strain-specific PrP(Sc) deposition and PrP27-30 molecular profile patterns were identified in ovine PrP transgenic mice that were not detected in wild-type mice. Significantly, it was established that the individual mouse brain selected for transmission during prion strain typing had a significant influence on strain definition. Serial passage of short- and long-incubation-time animals from the same group of scrapie-inoculated mice revealed different prion strain phenotypes. These observations are consistent with the possibility that some scrapie isolates contain more than one prion strain.

Treatment of patient tumor-derived colon cancer xenografts by a TRAIL gene-armed oncolytic adenovirus

Oncolytic virus-armed gene therapy may offer new treatment options and improve the prognosis for patients with colon cancer. In this study, we sought to further confirm the antitumor activity of oncolytic virus-armed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy in xenografts, which derived from the tumors of patients with colon cancer. To this end, we established xenotransplantable tumors from fresh surgical specimens. The histology of these xenografts maintained the features of the original tumors during passaging in nude mice. We next treated these xenografts with adenoviruses carrying TRAIL and the adenovirus E1A gene (Ad/TRAIL-E1) driven by the human telomerase reverse transcriptase promoter. The vector expressing the TRAIL gene (Ad/gTRAIL) or the E1A gene (Ad/GFP-E1) alone was used as control vector. The results demonstrated that Ad/TRAIL-E1 had more significant inhibitory effects on tumor growth than Ad/gTRAIL or Ad/GFP-E1 alone. Furthermore, we did not find any obvious treatment-related toxicity in the mice. Our results indicate that the use of an oncolytic adenoviral vector, in combination with TRAIL gene therapy, is a promising novel approach for cancer therapy.

Systemically Injected Mesenchymal Stem Cells Traffic to Myelomatous Bone and Inhibit Bone Disease, and Intralesionally Promotes Bone Formation and Delays Myeloma Progression During the Disease Active Stage or Remission

AbstractAbstract 980Mesenchymal stem cells (MSCs) cytotherapy has been clinically tested in various applications including bone regeneration, autoimmune diseases, and cancer. The aims of the study were to test the effect of MSCs cytotherapy on myeloma (MM) bone disease and tumor growth, and determine their ability to traffic into myelomatous bone during active disease stage and induction of remission by melphalan. We exploited the SCID-rab model for MM and a novel human myeloma cell line, Hg, established through our previously reported procedure (Xin et al., BJH 2007). Hg myeloma cells do not grow in culture but are capable of sequential passaging in SCID-rab mice. Microarray analysis revealed similar gene expression profiling between the Hg cell line and the original patient's myeloma plasma cells, and that these cells are classified in the MMSET subgroup and express DKK1, indicating their authenticity and clinical relevancy. The human fetal MSCs were transduced with a luciferase/GFP reporter in a lentiviral vector for in vivo tracking. In Hg-bearing hosts (5 mice/group), intra-bone injection of MSCs (1×106 cells/mouse) increased bone mineral density (BMD) by 21±4% while in control, diluent injected bones it was reduced by 14±5% (p<0.001). Increased bone formation by MSCs was associated with reduced tumor growth by 50% (p<0.01). Bioluminescence analysis revealed disappearance of the majority of the intralesionally injected MSCs within 4 weeks, suggesting that MSCs exert their effects on bone remodeling as a bystander cells (trophic effect). To test effect on relapse, remission was induced by treating Hg-bearing hosts with a total of 4 subcutaneous injections of melphalan (10 mg/kg/4 days), followed by intra-bone injection of diluent or MSCs (10 mice/group). Three weeks post-cytotherapy BMD was increased by 23±5% in bones injected with MSCs and reduced by 23±3% in bones injected with diluent (p<0.001). Eleven weeks post-cytotherapy, BMD was reduced by 33±6% and 9±7% in bones injected with diluent and MSCs, respectively (p<0.03). Following melphalan treatment circulating immunoglobulin (Ig) level (MM burden) was undetected while at 2 weeks after cytotherapy it was detected in 80% and 30% of hosts injected with diluent and MSCs, respectively. At experiment's end, Ig levels were significantly lower by 6 folds in hosts treated with MSCs than diluent (p<0.01). To further validate clinical relevancy, MSCs or diluent were intravenously injected into Hg-bearing hosts. In contrast to a single injection, 4 weekly, intravenous injections of MSCs (10 mice/group) prevented reduction of the BMD of the myelomatous bone while in control hosts the BMD was reduced by 14±3% (p<0.006 vs. pretreatment). MM growth was not affected by single or multiple intravenous injections of MSCs. Ex vivo imaging of tissues from hosts with active MM or treated with melphalan detected MSCs in implanted bones and murine lungs indicating that myeloma cells or conditions induced by MM or melphalan attract MSCs to myelomatous bones. We conclude that intra-bone injection of MSCs effectively promotes bone formation and delays MM progression during the disease active stage or remission. We also conclude that exogenous MSCs are capable of trafficking to myelomatous bone and that systemic, weekly injections of MSCs inhibit MM bone disease. Disclosures:No relevant conflicts of interest to declare.

A Tissue Microarray Study of Osteosarcoma: Histopathologic and Immunohistochemical Validation of Xenotransplanted Tumors as Preclinical Models

Osteosarcomas (OS) are aggressive neoplasms with a wide range of morphologic patterns. OS cases (primary and xenotransplanted) with paraffin blocks available were collected and included in tissue microarrays (TMAs). A morphologic evaluation including the different passages in mice was carried out according to the new WHO criteria. In addition, TMAs were analyzed with a wide panel of immunohistochemical (IHC) markers (osteonectin, osteocalcin,cytokeratin, S100, Sox-9, Ki-67, Bcl-2, p53, p16, survivin, CD99, and caveolin-1). A total of 61 cases were collected. The distribution of the cases according to the histopathologic pattern was: 38 osteogenic OS, 8 primary chondrogenic OS, 2 primary telangiectatic OS, 6 parosteal OS, 2 primary small cell OS, 2 primary poorly differentiated OS, 1 primary dedifferentiated OS, and 3 primary pleomorphic MFH-like OS. The tumor morphology in xenotransplants was similar to the primary or metastatic tumor of origin and was generally maintained over the passages. The IHC results were heterogeneous and osteonectin and osteocalcin were the most expressed in original tumor and xenografts. S100 and Sox-9 were expressed in chondrogenic areas. Caveolin and survivin showed significant IHC variation between the subsequent passages. p16 displayed heterogenic expression. p53 expression increased over the passages, and Ki-67 expression was not associated with a more undifferentiated pattern, but increased over the passages. An accurate morphologic evaluation using TMAs in original tumor is essential for the OS diagnosis; hence there is no IHC marker that alone distinguishes the OS subtypes. Xenografts in OS allow the study of tumor progression in this type of aggressive neoplasm.