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Karyotype analysis for Spermatogonia at passage 16, before transfection with XTNs demonstrating a normal male 40, XY karyotype.

ANOVA with multiple comparisons and Tukey's post-hoc test

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC.Significance:A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

Supplementary Figure from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

Supplementary Data from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

RAS family proteins are important drug targets in multiple cancers due to their central role in hypertrophic and pro-mitotic signaling. KRAS is a small GTPase that is activated in response to growth factors including HGF, TGFα, and EGF. KRAS then activates the MAP kinase (MAPK; RAF/MEK/ERK) signaling pathway, driving ERK translocation to the nucleus, and resulting in transcription of pro-mitotic and pro-growth genes. Additionally, KRAS contributes to PI3K/AKT/mTOR signaling, leading to transcriptional activation as well as hypertrophic protein synthesis via mTOR. We sought to validate several RAS mutant cell-derived tumor xenograft models in the SRG OncoRat. Cells were inoculated subcutaneously in the flank of SRG rats. To date, we have validated tumor growth using the HT-1080 fibrosarcoma NRAS mutant (Gln61Lys (c.181C>A)) cell line. We have also validated a suite of KRAS mutant lines in the SRG OncoRat, including: A-549 lung adenocarcinoma (Gly12Ser (c.34G>A)); NCI-H2122 NSCLC adenocarcinoma (Gly12Cys (c.34G>T)); MIA PaCa-2 pancreatic ductal adenocarcinoma and H-358 NSCLC adenocarcinoma (Gly12Cys (c.34G>T)); Capan-2 pancreatic ductal adenocarcinoma (Gly12Val (c.35G>T)); NCI-H441 NSCLC papillary adenocarcinoma (Gly12Val (c.35G>T)); NCI-H1734 NSCLC adenocarcinoma (Gly13Cys (c.37G>T)); and MDA-MB-231 breast adenocarcinoma (Gly13Asp (c.38G>A)). All cell lines showed robust and consistent tumor growth when injected subcutaneously into the SRG OncoRat. These models provide valuable tools for pre-clinical testing of RAS inhibition. Citation Format: R. Grace Walton, Diane Begemann, Cynthia Dunn, Valeria Steffey, Abigail Ross, Razoanul Haque, Fallon K. Noto. The SRG OncoRat supports growth of numerous RAS mutant cell lines, expanding pre-clinical RAS-inhibitor testing [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A040.

Human cancer xenografts are a vital tool for understanding tumor biology, growth kinetics, and therapeutic efficacy using animal models. Historically, immunodeficient mice have been the standard rodent species for cancer xenograft modeling. However, an immunodeficient rat that supports a wide variety of human cancer cell types would provide a larger rodent strain for easier surgical manipulation, serial blood sampling, and provide a single model in which efficacy, pharmacokinetics, and toxicology can be performed. We have created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRGTM OncoRat®) that provides a highly supportive environment for growing tumors of human origin. The SRG rat lacks B, T, and NK cells and readily supports the growth of multiple human cancer cell lines. The SRG rat is more immunodeficient than the Nude rat, suggesting it may be permissive to a wider variety of human cancer types. Here we demonstrate the utility of the SRG rat for both subcutaneous and orthotopic xenograft modeling. The SRG rat supports the growth of both lung and liver orthotopic cancers. In addition, the SRG rat supports the growth of orthoptic human glioblastoma multiforme in the brain. We use in vivo imaging to visualize tumor establishment and growth in subcutaneous, orthotopic, and metastatic models. Furthermore, our data show the ability of the SRG rat to support the growth of multiple different human cancer cell types subcutaneously in two different matrices, Matrigel® and VitroGel®. These data confirm that the SRG rat is an excellent host for studying human cancer. Our data demonstrate that the SRG rat has a high utility for studies using in vivo imaging, orthotopic tumor implantation, and standard subcutaneous tumor modeling. As the most immunodeficient rat commercially available, the SRG rat supports the growth of multiple human cancer types in a larger rodent strain relative to commercially available mouse models. Citation Format: Diane Begemann, Aida Javidan, Cynthia Dunn, Nicolas Johnston, R. Grace Walton, Valeriya Steffey, Ian Corbin, Niveen Fulcher, Cleusa De Oliveira, Hu Xu, Mila Uzelac, Andrew Deweyert, John A. Ronald, Susanne Schmid, Matthew O. Hebb, Fallon K. Noto. In vivo subcutaneous and orthotopic cancer xenograft modeling in the SRG immunodeficient rat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 42.

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. Significance: A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, encoding the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30-40% of cases, the clinical actionability of these mutations has not been studied. Here, we show that mutation to Aα results in synthetic lethality to treatment with inhibitors of ribonucleotide reductase (RNR), and multiple models of Aα mutant uterine serous tumors were sensitive to Clofarabine, an RNR inhibitor in vivo. Aα mutant cells displayed impaired checkpoint signaling upon RNRi treatment, and subsequently accumulated more DNA damage than wild type cells. This was PP2A dependent as complete inhibition of PP2A activity using LB-100, a catalytic site inhibitor, sensitized wild type USC cells to RNRi. Analysis of TCGA data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of USC patients harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNR inhibitors are not routinely used for uterine cancers, we identified a cohort of patients with recurrent disease treated with gemcitabine at MSKCC as a second or later line therapy. In a retrospective analysis of this cohort there was a trend for improved outcomes in USC patients treated with RNRi gemcitabine compared to patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. Citation Format: Caitlin M. O'Connor, Sarah E. Taylor, Kathryn M. Miller, Lauren Hurst, Terrance J. Haanen, Tahra K. Suhan, Kaitlin P. Zawacki, Fallon K. Noto, Jonida Trako, Arathi Mohan, Jaya Sangodkar, Dmitriy Zamarin, Analisa DiFeo, Goutham Narla. Synthetic lethality by targeting ribonucleotide reductase in PP2A deficient uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3341.

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non–small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481–9. ©2018 AACR.

Human tumor xenografts are a staple tool for understanding tumor biology, growth kinetics, and therapeutic efficacy. While these studies are most commonly done in immunocompromised mice, we have created a Sprague Dawley Rag2 null, Il2rgamma null SRGTM rat that is an excellent host for human xenografts (OncoRat®). Lacking B, T, and NK cells, the SRG rat readily supports the growth of multiple human cancer cell lines, including lines that do not engraft well or grow consistently in existing mouse models. The tumor microenvironment (TME) is a critical factor for supporting xenograft tumors, and the microenvironment of a human tumor grown in the rat has yet to be fully characterized. In this study, a collaborative effort between research institutions discovered that the tumor microenvironment in the SRG rat is more robust, involved, and more supportive of human tumor growth than in NSG mice. To characterize the aforementioned differences in rat and mouse TME, human prostate cancer cell lines LNCaP and VCaP were grown in NSG mice and SRG rats. Formalin fixed paraffin embedded sections were stained via immunohistochemistry (IHC) for both rat and mouse tumor microenvironment markers. Collagen marker CD29, endothelial cell marker CD31, macrophage marker CD45, smooth muscle actin, and stromal markers CD54 and vimentin were analyzed in both animal hosts. When applicable, staining was quantified via counting positive cells per high powered field of view. When VCaP and LNCaP xenograft tumors are hosted by SRG rats, the host TME is significantly more involved within the human tumor, and readily supports tumor growth. Comparing the same markers in SRG rat and NSG mouse hosts revealed a stark difference - the SRG rat TME is more prevalent than the mouse. Results show significantly increased stromal cells per high powered field in the SRG rat when compared to tumors of the same cell line grown in the NSG mouse. There is heavily increased endothelial and stromal cell infiltration from the host into the human tumor, and higher heterogeneity within the cell population in tumors hosted by SRG rats. These data show that there are more human tumor epithelial cell interactions within the TME of the SRG rat than in NSG mouse. Increased stromal involvement more accurately recapitulates a human TME and may help explain the better take rates and faster growth rates of xenografts in SRG rats versus NSG mice. It is well known that recapitulating the tumor cell population heterogeneity is a study limitation when using animal models. Utilization of the SRG rat TME has great value in nonclinical research by more accurately translating into human disease, while remaining in a readily available immunodeficient animal model (i.e., OncoRat). Citation Format: Diane Begemann, Bisoye Towobola Adedeji, Valeriya Steffey, Sam Moody, Goutham Narla, Fallon Noto. Sprague-Dawley Rag2 null Il2rgamma null SRG rat (OncoRat®) has enhanced tumor microenvironment in human prostate cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2949.

The development of Novel cancer immunotherapies has relied on the use of immune humanized mice. The field of study can be enhanced using an immune humanized rat, which will provide several advantages over the currently available humanized mouse models. The SRG OncoRat®; A Rag2/Il2rg double-knockout rat supports the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. Using this rat, we developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). We demonstrated the development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Furthermore, we demonstrated the presence of Human CD45+, CD3+, and CD20+ cells in peripheral blood, spleen, and bone marrow of the SRG OncoRat® engrafted with human PBMCs, hence humanizing the rat's immune system. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Bisoye Towobola Adedeji, Fallon K. Noto, Tseten Yeshi Jamling, Yash Argawal, Cole Jamison Beatty, Sara Grace Ho, Antu Das, Rajeev Kishore Salunke, Moses Turkle Bility. Humanized rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5621.

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a pre-clinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicological testing and PK/PD studies in the same model animal. Here we created an immunodeficient rat with a functional deletion of the Rag2 gene, using genetically modified spermatogonial stem cells (SSCs). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2 deficient SSCs into sterile recipients. Offspring were genotyped and a founder with a 27bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2(tm1Hera) (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. Additionally a human KRAS mutant non-small cell lung cancer cell line (H358), a patient derived high grade serous ovarian cancer cell line (OV81), and a patient derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies.

Current cell line-derived cancer and PDX mouse models are hindered by low engraftment rates and slow tumor growth kinetics. Furthermore, serial passaging of patient derived tissue results in changes to the genomic landscape such that the transplanted tumor no longer reflects the primary tumor. To address these limitations, we produced an immunodeficient rat, a Sprague-Dawley Rag2/Il2rg double knockout (SRG) rat that lacked mature B cells, T cells, and circulating NK cells. We demonstrated that the SRG rat has improved tumor take rates and growth kinetics using a variety of human cell lines and PDXs. Cell lines tested include VCaP and LNCaP, which have poor or highly variable growth kinetics in commercially available immunodeficient mouse models. We have demonstrated that both of these cell lines have superior take rate and growth kinetics in the SRG rat, providing a more efficient model for drug efficacy studies. In addition, the rat can accommodate a larger tumor volume, allowing for serial fine needle aspirate biopsy for PK/PD analysis in the same animal throughout the course of the study without significantly affecting normal tumor growth. The PDXs in the SRG developed larger tumor volume, over 20,000 mm3 in the first passage, which provided an ample source of tissue for characterization and/or subsequent passage into SRG for drug efficacy studies. Larger tumor volumes enabled fewer animals to be needed for a study, allowed for faster timelines to drug efficacy data, and reduced the need for serial passaging to generate enough tissue, which limited genomic divergence from the parental tumor tissue. As proof of principle, we used next generation sequencing based genomic analysis to direct a precision medicine strategy to guide in vivo efficacy studies. Specifically, using this approach we found a novel mutation in the MET pathway in a primary patient derived sample and tested therapies that were predicted to target this mutation and compared efficacy to standard of care agents in this unique SRG rat derived PDX model. Collectively, this data suggests that this novel rat model could serve as a patient avatar to better predict outcomes to genomically-directed therapies. In addition, we tested the ability of the SRG rat to support the growth of patient-derived tissue that was cryopreserved directly from patient harvested and had not previously been xenografted into an animal model. This concept may provide a means for establishing PDX models from cryopreserved samples when animals are not immediately available for xenografting. Citation Format: Fallon K Noto, Bisoye Towobola Adedeji, Sam Moody, Chris Brenzel, Jack Crawford, Goutham Narla, B. Mark Evers, Tseten Yeshi Jamling. A Rag2/Il2rg double-knockout rat (SRG OncoRat) enables precision-medicine based cancer studies with cell line- and patient-derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B067. doi:10.1158/1535-7163.TARG-19-B067

Immune humanized mice have been valuable in the development of novel cancer immunotherapies and have demonstrated stronger efficacy when combined with standard of care chemotherapy. An immune humanized rat could provide several advantages over the currently available humanized mouse models, including supporting the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. We developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). hSIS-humanized rat support development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Finally, we have demonstrated humanization of the rat immune system using human PBMCs. Human CD45+, CD3+, and CD20+ cells can be found in the peripheral blood, spleen, and bone marrow of engrafted rats. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Fallon K Noto, Bisoye Towobola Adedeji, Yash Agarwal, Cole Beatty, Sara Ho, Antu Das, Rajeev Salunke, Moses Bility, Tseten Yeshi Jamling. A Rag2/Il2rg double-knockout rat supports engraftment of human immune system for immunotherapy-based cancer efficacy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A007. doi:10.1158/1535-7163.TARG-19-A007

Current treatment options for advanced castration-resistant prostate cancers (CRPC) are effective for a brief period before becoming refractory. It is important to use relevant in vivo models for candidate selection in the development of next-generation mechanistically-distinct drugs to treat castration- and drug- resistant prostate cancers. Here, we describe a series of AR pan-antagonists (selective AR degraders (SARDs)) that degrade the AR and AR splice variants. SARDs inhibit the wild-type and LBD mutant ARs comparably and inhibit the in vitro proliferation and in vivo growth of enzalutamide-sensitive and resistant prostate cancer xenografts. Xenograft studies conducted in immune-compromised SRG rats (Sprague Dawley Rag2-/- Il2rg -/-; Hera Biolabs) with three lead SARDs demonstrated regression of enzalutamide-sensitive and -resistant VCaP tumors both in castrated and in intact rats. SRG rats provide the benefit of abundant blood samples for weekly evaluation of rising PSA, which also indicated that the SARDs inhibit the rising PSA, while enzalutamide was inactive in enzalutamide-resistant model. Drug metabolism and pharmacokinetic (DMPK) studies, also conducted with SRG and wild-type rats and in combination with efficacy, indicate that the molecules possess all the necessary drug-like properties. The molecules exhibit a broad safety margin with no cross-reactivity with G-Protein Coupled Receptor, kinase, and nuclear receptor family members. Collectively, the SARDs exhibit the properties necessary for a next-generation prostate cancer drug and that use of SRG rats facilitated thorough characterization of their in vivo properties. Citation Format: Suriyan Udhayasuriyan Ponnusamy, Fallon K Noto, Bisoye Towobola Adedeji, Yali He, Dong-Jin Hwang, Sam Moody, Thirumagal Thiyagarajan, Tseten Yeshi Jamling, Duane D Miller, Ramesh Narayanan. Potential next-generation androgen receptor-targeted therapeutic for enzalutamide-resistant prostate cancer; In vivo characterization in immune-compromised SRG rats [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B037. doi:10.1158/1535-7163.TARG-19-B037

Human cancer xenografts in rodents can provide predictive data on the success of candidate drugs in clinical trials and have been a pivotal tool in moving new drugs from the bench to the clinic. However, currently available immunodeficient mouse models have shown some limitation and variability in tumor take rates and growth kinetics in cancer cell lines. In addition, commercially available human cancer cell lines aren’t representative of the genomic and molecular diversity of cancers found in patients. Patient Derived Xenograft (PDX), in which tumor tissue is transplanted directly into rodents after biopsy from the patient, better represents that molecular signature, heterogeneity, and pathology of the original tumor. Therefore, in vivo efficacy studies with PDX models could be highly predictive for treatment sensitivity. Despite the many advantages of PDXs for preclinical research, PDX mouse models are hindered by low engraftment rates and slow tumor growth kinetics. The loss of patient tumor heterogeneity and stromal cells as the PDX is passaged multiple times to generate sufficient tumor tissue to inoculate a cohort of animals for efficacy studies is also a disadvantage in the immunodeficient mouse models. To address these limitations, we have introduced the OncoRat®; built on the SRGTM Platform, a Sprague-Dawley Rag2/Il2rg double knockout rat that lacks mature B cells, T cells, and circulating NK cells. We have demonstrated that the OncoRat has improved tumor take rate and growth kinetics for non-small cell lung cancer (NSCLC) PDXs. The NSLSC PDXs in the OncoRat have a much larger tumor volume, over 20,000 mm3 in the first passage (P0) in the rat, which provides an ample source of tissue for characterization and/or subsequent passage (P1) into OncoRat for drug efficacy studies. This leads to fewer animals used for study and faster timelines to drug efficacy data, resulting in a reduction in cost. In addition, we have used genomic analysis for guidance in planning in vivo efficacy studies. One of our NSCLC PDX models harbors a novel mutation in the MET pathway, suggesting this tumor would not be responsive to standard of care treatment. An efficacy study we performed in the OncoRat suggests that this particular tumor would respond well to Type II MET inhibitors, such as Cabonzantinib. This proof of concept study demonstrates that genomic and molecular analysis can provide insight into treatment outcomes and that PDX models in the OncoRat could serve as patient avatars for predicting treatment outcomes. Citation Format: Fallon K. Noto, Bisoye Towobola Adedeji, Sam Moody, Chris Brenzel, Jack Crawford, Goutham Narla, Tseten Yeshi Jamling. A case study: OncoRat is a viable patient avatar for a NSCLC patient with a Y1248H Met activating mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1059.

Oncology drug and therapeutic developments are hindered by lack of appropriate preclinical models that faithfully recapitulate tumor pathology, tumor growth and metastasis, genetics, and the tumor microenvironment. In particular, in vitro modeling of tumors often results in a cell line that differs from the patient’s tumor in key genetic aspects directly relating to the cancer phenotype and which thus differs in response to various treatments. Immunodeficient mice have paved the way for in vivo modeling of tumors and disseminated cancers. Growth kinetics and response to therapeutics can be more appropriately modeled in these systems. In addition, the advent of patient-derived xenografts (PDXs), whereby a piece of tumor is taken directly from the patient and grown in the mouse, demonstrates that personalized cancer treatments could be the standard of care. Recent advances in genetically modified rats provide another platform for modeling human cancer and PDX tissues. The rat offers a number of advantages over the mouse, including easier surgical manipulation, larger tumor size and greater blood volume for downstream analyses, as well as being the preferred model for drug efficacy and toxicology studies. This chapter reviews advances in human PDX modeling in the mouse and rat.

Elevated levels of low-density lipoprotein cholesterol (LDL-C) in plasma are a major contributor to cardiovascular disease, which is the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL-C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently, we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSCs). This finding raises the possibility of using patient-specific iPSC-derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach, we produced iPSCs from JD a patient with mutations in the low-density lipoprotein receptor (LDLR) gene that result in familial hypercholesterolemia (FH). We demonstrate that (1) hepatocytes can be efficiently generated from FH iPSCs; (2) in contrast to control cells, FH iPSC-derived hepatocytes are deficient in LDL-C uptake; (3) control but not FH iPSC-derived hepatocytes increase LDL uptake in response to lovastatin; and (4) FH iPSC-derived hepatocytes display a marked elevation in secretion of lipidated apolipoprotein B-100. Conclusion: Cumulatively, these findings demonstrate that FH iPSC-derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient-specific iPSC-derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of cardiovascular disease. (HEPATOLOGY 2012)

In vivo modeling of human cancer in genetically engineered rodents can provide insights into tumor kinetics, genetics and molecular biology, and allow for the testing of drug efficacy. Over the recent years, studies have demonstrated that immunodeficient mice, such as the NSG, reconstituted with functional human immune cells, such as peripheral blood mononuclear cells (PBMCs), are promising models for immuno-oncology efficacy studies. Immune humanized mice engrafted with human cancer cells show human-specific immune responses when treated with drugs that target immune pathways, such as CTLA-4 and PD-1, leading to the inhibition of tumor growth. These models provide a critical platform to study how the immune system can be engaged to drive anti-cancer efficacy. Although the NSG and similar immunodeficient mouse strains have been beneficial for human immuno-oncology studies, there are many caveats to performing these studies in mice, including inconsistent tumor kinetics, small tumor size for downstream analyses, limited blood for PK/PD studies due to the small size of the mouse, and graft vs. host disease (GvHD) onset around 4-6 weeks post-engraftment. Humanized rat models would allow for the development of larger tumors and the ability to perform serial blood sampling on a routine basis throughout the course of treatment. We have created a Rag2 null, Il2rg null rat on the Sprague Dawley background (SRGTM) that lacks B, T, and NK cells and supports the growth of multiple human cancer cell lines, including lines that do not engraft or grow well in existing mouse models: HCT-116, H358, and VCaP. The SRGTM rat is also permissive to immune humanization with PBMCs. PBMC-engrafted SRGTM rats have a significant amount of human CD45+ lymphocytes in peripheral blood, of which the majority are T cells, comparable to immune-humanized NSG and NOG mice. Some animals also show significant levels of circulating human B cells. Interestingly, the incidence of GvHD is delayed to 12-14 weeks post-engraftment in the immune humanized SRG™ rat. We are currently assessing functionality of the PBMC-immune humanized SRG™ rat in immuno-oncology studies as well as characterizing the ability of the SRG™ rat to support the growth of human patient derived xenografts (PDX). The generation of this novel humanized SRG™ rat model could allow for a more permissive host system to test existing and novel immunomodulatory strategies for the treatment of human disease. Citation Format: Fallon K. Noto, Bisoye Towobola, Angela Arey, Goutham Narla, Christopher Chengelis, Tseten Yeshi. The SRGTM rat: A novel SCID rat for humanization studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1155.

Mouse models of human cancer have paved the way for studying cancer biology and genomics and their effects on cancer growth kinetics, propensity for metastasis, and treatment response. In addition, human cancer xenografts provide the opportunity to study cancer cell interactions with host stroma and tumor morphology. A plethora of genetically immunodeficient mouse models exist with different immune phenotypes, resulting in significant variability in tumor take rates and growth kinetics for a wide range of human cancer cell lines and patient-derived xenografts (PDX). Inconsistent or poor growth in these immunodeficient models have made downstream analysis and drug efficacy testing difficult. As a result, a significant number of mice are needed for drug efficacy screening to achieve a cohort of animals with tumors of similar size with similar tumor growth kinetics for treatment. It is possible that these cell lines might grow more consistently in a different immunodeficient model, such as an immunodeficient rat. Until recently, the only immunodeficient rat that existed was the nude (NIH-Foxn1rnu; RNU) rat. This rat lacks T cells, but maintains a normal repertoire of all other immune cells, including B and NK cells. As such, there are a limited number of human cancer cell lines that can survive in the nude rat. We have created a genetically modified rat with a functional mutation of the Rag2 gene (Sprague Dawley – Rag2 null; SDR), resulting in a loss of mature B and T cells. In addition, we have created a Rag2/Il2rg double knockout rat (Sprague Dawley – Rag2; Il2rg null; SRG) that lacks mature B cells, T cells, and has fewer NK cells than wild-type Sprague Dawley rats. We have shown that the SDR rat is permissive for solid tumor growth of the human acute lymphocytic leukemia REH cell line, human glioblastoma U87MG cell line, human non-small cell lung cancer H358 cell line, and cell lines derived from ovarian and endometrial PDX samples. In some cases, tumor growth kinetics are superior in the SDR rat compared with immunodeficient mouse models. We have demonstrated that the human prostate cancer cell line, VCaP, which has poor engraftment efficiency and growth kinetics in the mouse, grows well in the SRG rat. The SRG rat is currently being validated for growth kinetics of other human cancer cell lines and PDX tissues. In addition, we are developing several disseminated human leukemia models in the SRG rat and creating immune-humanized mice and rats to be used in conjunction with human tumor xenografts for immunotherapy efficacy studies. Citation Format: Fallon K. Noto, Kamesh Ravi, Angela Arey, Christopher McClain, Wei Zhang, Goutham Narla, Jack Crawford, Tseten Yeshi. Novel immunodeficient rat models capable of supporting the growth of human tumor xenografts [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B36.

The JT-60 Super Advanced (JT-60SA) tokamak aims to perform the ITER support and to demonstrate steady-state high-beta plasma project with the collaboration between Japan and EU. To attain these objectives, the negative-ion-based NBI (N-NBI) system is required to inject 10 MW for 100 s at the beam energy of 500 keV. On JT-60U, the present N-NBI ion source has injected 3.2 MW for 21 s at 320 keV; however, three key issues should be solved for the JT-60SA N-NBI ion source. One is to improve the voltage holding capability of the large negative ion source, where the available acceleration voltage has been limited to less than ~400 kV due to breakdowns. The accelerator of the JT-60U ion source is composed of large three-stage grids and three fiberglass reinforced plastic (FRP) insulators. Recent R&D tests suggested that the FRP insulators were not the main factor to trigger the breakdowns at the early conditioning stage. The accelerator with a large area of grids and their supporting structure may need a high margin in the design of electric field and a long time for conditioning. The second issue is to reduce the power loading of the acceleration grids. It was found that some beamlets were strongly deflected due to beamlet-beamlet interaction and strike on the grounded grid in the accelerator. Moreover, the electrons generated in the accelerator caused the grid loading and the overheating of the beamline components. The acceleration grids for JT-60SA are to be designed by taking account of the beamlet-beamlet interaction and the applied magnetic field in 3-D simulation. Third is to maintain the D production for 100 s. Although a constant D- beam power was confirmed on JT-60U for 21 s, an active cooling system is required to keep the temperature of the plasma grid (PG) under optimum condition during 100-s operation. A simple cooling structure is proposed for the active cooled PG, where a key is the temperature gradient on the PG for uniform D- production. In the present schedule, design work, reflecting the latest R&D progress, will continue until ~2011. The modified N-NBI ion source will start on JT-60SA in 2015.

Modification of JT-60U to a superconducting device (so called JT-60SA) has been planned to contribute to ITER and DEMO. The NBI system is required to inject D 0 beams of 34 MW for 100 s. The upgraded NBI system for JT-60SA consists of 12 positive-ion-based NBI (P-NBI) units and one negative-ion-based NBI (N-NBI) unit. The injection powers of each P-NBI and N-NBI units are 2 MW at 85 keV and 10 MW at 500 keV, respectively. On JT-60U, the long pulse operations of 30 s at 2 MW and 20 s at 3.2 MW have been achieved on the P-NBI and N-NBI units, respectively. Both units have demonstrated no injection power degradation for long pulse operation. It has been also found that the thermal time constants of the main key components with active cooling, such as the ion source of the present NBI system, are less than ∼20 s. Therefore, the pulse extension up to 100 s is expected to need some modifications mainly the power supply system. In addition, the voltage-holding capability of the negative ion source is required to be improved. The detailed technical design of the NBI system for JT-60SA is presented.

The 500 keV negative-ion based neutral beam injector for JT-60U started operations in 1996. The availability of the negative ion based neutral beam injection system has been improved gradually by modifying the ion source and optimizing its operation parameters. Recently, the extension of the pulse duration up to 30 s has been intended to study quasi-steady state plasma on JT-60U. The most serious issue is to reduce the heat load on the grids for long pulse operation. Two modifications have been proposed to reduce the heat load. One is to suppress the spread of beamlet-bundle which may be caused by beamlet–beamlet interaction in the multi-aperture grid due to the space charge force. Indeed, the investigation of the beam deflection, which was measured by the infrared camera on the target plate set 3.5 m away from the grid, indicates that the spread of beamlet-bundle is in proportion to the current density. Field-shaping plates were attached on the extraction grid to modify the local electric field. The plate thickness was optimized to steer the beamlet deflection. The other is to reduce the stripping loss, where the electron of the negative ion beam is stripped and accelerated in the accelerator and then collides with the grids. The ion source was modified to reduce the pressure in the accelerator column to suppress the beam-ion stripping loss. To date, long pulse injection of 19 s of 1.5–1.6 MW at a high energy beam of 360 keV, 9–10 A for D− has been obtained by one ion source with these modifications.

We have developed a 64-channel magnetocardiograph (MCG) system for the diagnosis of heart disease. The MCG is characterized by its display of a unique three-dimensional image of bio-currents flowing inside the human body. The outline of the heart can be displayed without use of MRI. A mobile SQUID-based non-distractive evaluation apparatus was realized by the active shielding technique. The system can offer information from beneath the surface of the specimen by using a saw-wave excitation method. This mobile technology enables us to inspect ferromagnetic materials, whose high magnetic field rules out the use of a conventional SQUID apparatus near them.

One of the advantages of MBR is its excellent effluent quality, which is suitable for a wide range of reuse purposes. We investigated the characteristics of MBR effluent and evaluated them based on the Japanese guideline for the reuse of treated wastewater. As the result, MBR effluent showed qualitative characteristics that satisfy the requirement except chromaticity for recreational purpose. Further treatment, such as by ozone or activated carbon, will be required to remove the remaining color. MBR shows high removal efficiency of bacteria and other hazardous microorganisms such as Cryptosporidium. We investigated the removal efficiency of virus by MBR using coliphage as an alternative index. The results showed that high removal efficiency for coliphage could be obtained by MBR. The removal mechanism appears to be that coliphage are attached to the activated sludge and thus rejected by the membrane together with activated sludge particles. With regard to the endocrine disrupters, no significant differences were observed between MBR and CAS in the removal of main endocrine disrupters.

After a brief review on advanced applications of magnetic fields generated by high-T/sub c/ bulk superconductors in Japan, the outline and present status of the CREATE (Collaboration of Regional Entities for the Advancement of Technological Excellence) Iwate Project "Advanced Application of Magnetic Fields- The creation of industries based on magnetic fields application -" will be described.

The authors have succeeded in constructing a superconducting permanent magnet system capable of generating the highest magnetic field of 3.15 T in the open space between the face-to-face magnetic poles containing the melt-processed RE-Ba-Cu-O bulk superconductors. A pair of bulk samples was cooled to 38 K by using small GM-refrigerators (15 W at 77 K), and magnetized by the pulsed field magnetization (PFM) process called "IMRA" method. The values and distributions of trapped magnetic fields were measured. The authors will refer to a novel face-to-face field generator using seven bulk superconductors arranged on a couple of the cold stages. The size of the magnetic pole is 150 mm in diameter. The highest magnetic field obtained by the field cooling method operated at 5 T has reached 1.4 T on the right-hand side magnetic pole surface and 0.9 T at the center of the open space of 34 mm between the magnetic poles, respectively. The intense magnetic fields are investigated to be applied to various industries such as the magnetic separation technique and so on.

Fracture toughness of Sm123 bulk superconductors with 16.7 and 28.6 mol% of Sm211 particles have been investigated by Vickers hardness test at cryogenic temperatures. The hardness test was carried out on [001] plane for two types of SmBCO bulk materials during 40-293 K. However, the hardness of Sm29 increased to 7,380 MPa with decreasing temperatures. In addition, fracture toughness of Sm17 and Sm29 are discussed calculating from Vickers hardness, median cracks length, Young's modulus and applied load. The fracture toughness of Sm17 became constant to 0.8 MPa/spl radic/m following the hardness property. The fracture toughness of Sm29 decreased from 0.73 Mpa/spl radic/m at 293 K to 0.67 Mpa/spl radic/m at 40 K.

The negative-ion based neutral beam injection system developed by JAERI for JT-60U has been intended to generate a 500 keV and 10 MW beam for 10 seconds with two ion sources. Technical efforts to d...

Immunodeficient mouse models seek to recapitulate the clinical features of advanced human cancers. However, drug efficacy testing and downstream analysis such as pharmacokinetic (PK) /pharmacodynamic (PD)-based studies are limited because of variability in tumor growth and kinetics, limited tumor growth potential, requiring the enrollment of larger numbers of mice in each treatment group to achieve required cohort sizes. Considering the distinctive advantages of rats in terms of their larger size, and ease of sampling at different time points for drug efficacy and clinical testing, we have developed unique immunodeficient rat models: the Rag2 knockout SDRTM rat (absence of mature B and T cells) and the Rag2/Il2rg double knockout SRGTM rat (absence of mature B and T cells, and lower NK cells) on a Sprague- Dawley background. Our group has previously demonstrated higher engraftment, better tumor kinetics, and increased tumor volume of xenografts in these rat models compared to available immunodeficient mouse strains for various cancer xenograft models. The purpose of the present study is to demonstrate the feasibility of drug efficacy studies using targeted molecular agents directed against cancer-associated drivers in our newly developed SDR and SRG rat models. We tested the efficacies of a combination treatment of an AKT inhibitor (MK2206) and MEK inhibitor (AZD6244), and a small-molecule activator of PP2A (SMAP) (DT-061) in a SDR rat xenograft model of human KRAS-mutant non-small cell lung cancer. 1 x 106 H358 cells were transplanted subcutaneously on the left hind flank in SDR rats. When tumor size reached 100-150 mm3, the rats were randomized to either control or treatment groups. The treatment groups received either a combination of AZD6244 with MK2206, or DT-061, and the vehicle-only control received n, n-dimethylacetamide + Solutol®/Kolliphor® HS 15, twice daily for 30 days by oral gavage. Both the kinase-inhibitors combination and SMAP treatment inhibited the growth of lung tumor xenograft in the SDR rats as shown by decreased mean tumor volume and fold changes in tumor volume compared to the control group. The combination treatment dose was just as effective at half the dose reported in previous mouse studies, demonstrating that lower dosing is sufficient for efficacy testing in rats. Importantly, no mortality, behavioral abnormalities, or changes in body weight were observed during the study, indicating a favorable tolerability profile in this model. We have also demonstrated that a human prostate cancer cell line VCaP, which has very poor engraftment and growth profiles in existing mouse models, grows well in our SRG rats. The SRG rat is now being used to study the chemotherapeutic efficacy of the benchmark androgen receptor inhibitor enzalutamide, in this VCaP tumor xenograft model. In addition, weekly blood sampling to measure PSA levels in the serum is being performed in the VCaP tumor xenograft efficacy study to demonstrate the flexibility of frequent sampling in the rat model to evaluate correlations between tumor growth and serum PSA levels. Citation Format: Kamesh Ravi, Fallon Noto, Christopher McClain, Angela Arey, Goutham Narla, Jack Crawford, Tseten Yeshi. Novel immunodeficient rat models offer a unique platform for drug efficacy studies in human tumor xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B170.

Animal models of human cancer offer the potential to study human tumor growth kinetics, genetic variance among human cancers, and provide in vivo platforms for drug efficacy testing. In particular, immunodeficient mouse models have been invaluable in modeling a wide range of human cancers. However, some cancer lines don’t grow well in the available mouse models or show variability in growth kinetics from mouse to mouse, making drug efficacy studies difficult due to differences in tumor size at the onset of treatment. These challenges are also seen in patient derived xenograft (PDX) models, in addition to long timeframes to obtain sufficient mice with PDX tissue growth for drug efficacy studies. Mice are also limited in tumor growth potential with regard to humane endpoints and small size also limits the volume of blood that can be collected for analysis. An immunodeficient rat model could provide a solution to some of these issues. A rat model would allow for larger tumor size, easier surgical manipulation, and greater volume of tissue and blood sampling for downstream analysis. In addition, large tumors from rats could be serially transplanted into mice for drug efficacy testing and could provide a large number of transplanted mice in a shorter period of time compared with serially transplanting from mouse to mouse. We have created an immunodeficient rat model with a functional deletion of the Rag2 gene. This knockout, created using spermatogonial stem cells, lacks mature B and T cells. To assess the capability of the Rag2 knockout rat to accept human xenografts, we transplanted 2 commercially available human cancer cell lines into our animals. The human REH acute lymphocytic leukemia cell line was transplanted via intravenous injection and the human glioblastoma cell line U87MG was transplanted subcutaneously. Both cell lines survived in the Rag2 knockout rat and resulted in the growth of tumors comprised of human cells. Studies are underway to characterize the Rag2 knockout rat’s ability to grow other human cell lines, including those that do not grow well in mice, and PDX tissues. Citation Format: Fallon K. Noto, Angela Arey, Christopher McClain, Wei Zhang, Tseten Yeshi. A novel immunodeficient rat for modeling human cancer [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 807. doi:10.1158/1538-7445.AM2017-807

Efforts to identify pharmaceuticals to treat heritable metabolic liver diseases have been hampered by the lack of models. However, cells with hepatocyte characteristics can be produced from induced pluripotent stem cells (iPSCs). Here, we have used hepatocyte-like cells generated from homozygous familial hypercholesterolemia (hoFH) iPSCs to identify drugs that can potentially be repurposed to lower serum LDL-C. We found that cardiac glycosides reduce the production of apolipoprotein B (apoB) from human hepatocytes in culture and the serum of avatar mice harboring humanized livers. The drugs act by increasing the turnover of apoB protein. Analyses of patient medical records revealed that the treatment of patients with cardiac glycosides reduced serum LDL-C levels. These studies highlight the effectiveness of using iPSCs to screen for potential treatments for inborn errors of hepatic metabolism and suggest that cardiac glycosides could provide an approach for reducing hepatocyte production of apoB and treating hypercholesterolemia.

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.

Multifilamentary (Nb,Ti)/sub 3/Sn superconducting wires with reinforcing stabilizer of CuNb composite, CuNb/(Nb,Ti)/sub 3/Sn, were developed. The outstanding point is that the 0.2% proof stress at 4.2 K in CuNb/(Nb,Ti)/sub 3/Sn was extremely improved to 320 MPa even after the heat treatment at 670/spl deg/C for 192 hours, which is 1.8 times higher than that in ordinary Cu/(Nb,Ti)/sub 3/Sn. The overall J/sub c/ value in CuNb/(Nb,Ti)/sub 3/Sn was obtained to be 110 A/mm/sup 2/ at 16 T and 4.2 K. These results show that the bronze processed multifilamentary (Nb,Ti)/sub 3/Sn superconducting wire with CuNb reinforcing stabilizer is actually employable for a high field superconducting magnet. >

This study eluidates the onset of wake oscillation and the generation process of the oscillating bubble behind a circular cylinder by visualizing computed results obtained by solving numerically the two-dimensional, time-dependent Navi-er-Stokes equations at the low Reynolds numbers. The zero-streamline begins to oscillate near the rear stagnant point at Re=1-4. A bubble composed of two standing vortices adhered to the cylinder certainly begins to oscillate at Re=5.60-5.6 2. Instability generated by motion of the zero-streamline and the bubble is not transported into the downstream and is the absolute instability, since the flow outside the neighborhood of the rear stagnnant point is completely steady and symmetric at Re≤16.

This study elucidates stability in a cylinder wake at the low Reynolds number by numerical calculation solving the two-dimensional Navier-Stokes equations and the continuity equation. The wake has a bubble and begins to oscillate at Re=6.0-6.5, where Re is the Reynolds number. The bubble is not stable but unstable; the bubble is composed of two asymmetric standing eddies. Since the flow state outside the bubble is steady and symmetric, instability occurring near the rear stagnant point at 6.5≤Re≤6.0 is not transported in the downstream. Since centipedes in the unite streaklines occur in the downstream at 16.0≤Re, instability is transported in the downstream. A change of the centipedes with an increase of the Reynolds number can give a clear insight on a process of the shift of the wake patterns, viz. the centipede flow, the wavy wake, and the Karman vortex street.

It is desired to develop a high-strength high-conductivity material for reinforcing composite superconductors which will be employed in large-scale and/or high-field superconducting magnets in emerging large-scale projects such as fusion magnets, SMES magnets, hybrid magnets, and so on. A review of our decade long study of reinforcing stabilizers, and recent results on CuNb composite wires and high-purity Ta wires are reported. Mechanical and electrical properties are reported for CaO crucible melted and drawn CuNb composite wires and for heavily cold worked high-purity Ta wires. Tensile tests at room temperature show a yield stress of about 556 MPa even after annealing at 750°C for 1 h for CuNb composite wires and 0.2% proof stress of about 666 MPa for the Ta wires. Resistivity at 4.2 K is about 0.25 μ ω cm for CuNb wires and about 0.40 μω cm for the Ta wires, respectively at a field of 15 T. It is concluded that the CuNb composite wires, the high-purity Ta wires and the Al2O3 dispersion-strengthened copper are suitable as a reinforcing stabilizer for conductors of large-scale and/or high-field superconducting magnets.

There exists a worldwide shortage of donor livers available for orthotropic liver transplantation and hepatocyte transplantation therapies. In addition to their therapeutic potential, primary human hepatocytes facilitate the study of molecular and genetic aspects of human hepatic disease and development and provide a platform for drug toxicity screens and identification of novel pharmaceuticals with potential to treat a wide array of metabolic diseases. The demand for human hepatocytes, therefore, heavily outweighs their availability. As an alternative to using donor livers as a source of primary hepatocytes, we explored the possibility of generating patient-specific human hepatocytes from induced pluripotent stem (iPS) cells. Conclusion: We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe a procedure that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells that display key liver functions and can integrate into the hepatic parenchyma in vivo. (HEPATOLOGY 2010.)

Mechanical and electrical properties have been studied for a heavily cold-worked high-purity Ta wire. Tensile test at room temperature shows a large 0.2% proof stress of about 70 kg/mm/sup 2/, which is probably caused by work-hardening in the wire drawing process. Observations of fracture surfaces using a scanning electron microscope indicate that this material is very ductile. Moreover, this sample has a low resistivity of about 0.4 mu Omega -cm even at the high field of 15 T. The residual resistivity ratio is about 80, which is consistent with the high purity of the sample. Therefore, it has been found that the cold-worked Ta wire is applicable to a reinforcing stabilizer for conductors of large-scale and/or high-field superconducting magnets. >

The T/sub c/, B/sub c2/, and J/sub c/ were investigated for superconductors in a residual strain state for bronze processed commercial multifilamentary Nb/sub 3/Sn wires. It was found that the temperature dependence of B/sub c2/ under strain obeys (1-(2/3)( pi T/1.75T/sub c/)/sup 2/) near T=0 and (1-T/T/sub c/) near T/sub c/. The establishment of the temperature scaling law of a global pinning force F/sub p/=J/sub c/*B was confirmed in commercial Nb/sub 3/Sn wires.

The present numerical computations and flow visualizations showed the generation of the Karman vortex street and the change of its vortex shedding upon cooling a circular cylinder submerged in an upward free-stream of air at the low Reynolds number. The dominant causes of the generation of the vortex and the decrease of the Strouhal number in the opposing flow were made clear from points of view of changes of the kinematic viscosity, separation points, wake velocity, and vorticity upon cooling the cylinder.

The first long pulse production of high power D(-) ion beams has been demonstrated in the JT-60 U negative ion sources, each of which was designed to produce 22 A, 500 keV D(-) ion beams. Voltage holding capability and the grid power loading were examined for long pulse production of high power D(-) ion beams. From the correlation between voltage holding and the light intensity of cathodoluminescence from the Fiber Reinforced Plastic insulators, the acceleration voltage for stable voltage holding capability was found to be less than 320-340 kV where the light was sufficiently suppressed. By tuning the extraction voltage, the grid power loadings in the ion sources were decreased to the allowable levels for long pulse injection without a significant reduction of the beam power. After tuning the acceleration and extraction voltages, D(-) ion beams of 12.5 and 9.8 A were produced at 340 keV with cesium seeding at a rate of approximately 14 microg/s into the ion sources. The pulse duration of these D(-) ion beams was extended step by step, and then was successfully extended up to 18 s without degradation of the negative ion production. The D(-) ion beams were neutralized to yield 3.6 MW D(0) beams by a gas cell, and then injected into the JT-60 U plasma. Further, a slight reduction of D(-) ion beam power allowed the longer injection duration of 21 s at a D(0) beam power of 3.2 MW. The success in the long pulse production of a high power D(-) ion beam shows that negative ion beams can be produced during a few tens of seconds without degradations of negative ion production and the voltage holding in a large Cs-seeded negative ion source.

The Nb/Al composite wires with various average thicknesses of niobium layer, from 43 to 300 nm, were fabricated by the method using aluminum-clad niobium foils as a starting material and the dependency of the critical current density J c on the niobium layer thickness was studied. Even at a thickness of 43 nm, J c gave no tendency for saturation. In contrast, the transition temperature was saturated at about 17K. Although the obtained J c values exceeded the best ones for the Nb/Al composite wires fabricated by the powder metallurgy process and niobium-tube method, they were still far from the results for stoichiometric Nb 3 Al. The J c dependency on the niobium layer thickness was compared with the data in other methods and discussed in detail.

Recent improvements and developmental research on several high field superconducting wires are reviewed. High field characteristics of NbTiHf wires, wound in a 13.08 T superconducting magnet in a 1.8 K pressurized superfluid helium bath, have been studied in detail. Ti-alloyed Nb 3 Sn fm wires prepared by the Nb-tube method showed excellent high field J c -characteristics and were wound in a 16.7 T superconducting magnet. One of these wires was employed in the HOMER facility (a multipurpose superconducting magnet at KfK) and generated 20.15 T, recently. A new process called the clad chip extrusion (CCE) method has been developed for the preparation of superconducting Nb 3 Al wires. Short samples of the CCE processed Nb 3 Al wires showed J c higher than 10 4 A/cm 2 up to 18.8 T at 4.2 K. One of the in-situ processed V 3 Ga short sample wires prepared by an induction melting using a CaO crucible showed J c =10 4 A/cm 2 at 16.2 T and 4.2 K, which will be improved by further optimization of the process and the composition.

A thermal plume occurs above a heat island. This plume affects the real urban climate and the heat island phenomenon. Because of its complexity, a real heat island, from which the thermal plume occurs, is simplified. A simplified heat island is a line heat source. A thermal plume above this simplified heat island was investigated. In general, a thermal plume sways naturally from side to side, and, in most cases, a plume is occurring in a thermally stable stratified fluid. Therefore, a thermal plume with a swaying motion in a thermally stable stratified fluid inside a comparatively large enclosure was formed and experimentally investigated. The thermal plume was observed by flow visualization. The plume temperature was measured, and the statistical analysis was carried out in time series data of the plume temperature. As a result, the fumigation motion of the plume above the simplified heat island with extremely low heat rate is firstly visualized and then clarified by this laboratory experiment. This fumigation behavior clarified in this investigation is quite important in the heat island phenomenon and the urban climatology.

Investigation is made clear on an identification method of a vortex from a circular cylinder by CAFV. The streamlines in coordinates for a cylinder is at rest or moves, the velocity vector, the streakline, the pathline, the vorticity, the isotherm, the timeline, and the isobar for the circular cylinder wake with the Reynolds number 44 and 70 are obtained and discussed.

The pulse duration of the negative ion based NBI system has been extended from 10 s to 25 s to study long pulse plasmas on JT-60U. A feedback control technique has been demonstrated to keep the arc power constant by controlling the filament voltage for long pulse operation. Thus it was clearly observed that the negative ion beam current increased with the temperature of the plasma grid at constant arc power. A tapered filament is employed to improve its durability for the next operational campaign. Moreover, a high voltage holding test indicates that the reduction in the outgassing from the FRP (fiberglass-reinforced plastic) insulator may be a key to suppress the breakdowns in the ion source