Schreiber, Claire A.

Abstract Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. Significance Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature β cell phenotype would lead to safe islet replacement therapy for diabetes.

Altered myocardial structure and function, secondary to chronically elevated blood pressure, are leading causes of heart failure and death. B-type natriuretic peptide (BNP), a guanylyl cyclase A agonist, is a cardiac hormone integral to cardiovascular regulation. Studies have demonstrated a causal relationship between reduced production or impaired BNP release and the development of human hypertension. However, the consequences of BNP insufficiency on blood pressure and hypertension-associated complications remain poorly understood. Therefore, the goal of this study was to create and characterize a novel model of BNP deficiency to investigate the effects of BNP absence on cardiac and renal structure, function, and survival. Genetic BNP deletion was generated in Dahl salt-sensitive rats. Compared with age-matched controls, BNP knockout rats demonstrated adult-onset hypertension. Increased left ventricular mass with hypertrophy and substantially augmented hypertrophy signaling pathway genes, developed in young adult knockout rats, which preceded hypertension. Prolonged hypertension led to increased cardiac stiffness, cardiac fibrosis, and thrombi formation. Significant elongation of the QT interval was detected at 9 months in knockout rats. Progressive nephropathy was also noted with proteinuria, fibrosis, and glomerular alterations in BNP knockout rats. End-organ damage contributed to a significant decline in overall survival. Systemic BNP overexpression reversed the phenotype of genetic BNP deletion. Our results demonstrate the critical role of BNP defect in the development of systemic hypertension and associated end-organ damage in adulthood.

Abstract Tumor-associated hypoxia has been associated with malignant progression, metastasis and resistance to therapy. We studied the role of oxygen tension (pO2) in modulating the properties of cultured human prostate cancer (CaP) cells. In air, where oxygen tension (pO2) is approximately 20 kPa, cultured CaP cells expressed lower levels of transcripts associated with cancer and urologic disease; reducing pO2 to 2 kPa made these levels comparable to those in surgical CaP specimens suggesting that hypoxically cultured cells, rather than normoxically cultured cells, express genes at levels akin to those in tumor cells in situ. In human primary CaP tissue, transcripts of hypoxia-associated genes DLG7, CCNB1 and HMMR were overexpressed and associated with Gleason score and disease prognosis; this implies the potential prognostic value of these transcripts. While hypoxia affected the CaP cell proteome mostly quantitatively, it did affect expression of tumor–associated antigens and suggested the potential therapeutic relevance of the hypoxic antigenic landscape. Overall, our results suggest that hypoxia modifies cellular properties of cultured CaP cells towards a phenotype more similar to tumor cells in situ. Introduction of pO2 as a variable can constitute a tool for the identification of more effective prognostic biomarkers and development of better therapy for CaP and, possibly, other tumors. Citation Format: Christian R. Gomez, Stanimir Vuk-Pavlovic, Farhad Kosari, Jan Marie Munz, Claire Schreiber, Gaylord Knutson, Cristine Charlesworth, R. Jeffrey Karnes, John Cheville, George Vasmatzis. Prognostic value of hypoxia-associated genes in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C43.

Abstract Tumor-associated hypoxia facilitates malignant progression, metastasis and poor prognosis in prostate cancer (CaP); however, effects of oxygen tension (pO2) in CaP are just beginning to be investigated. To establish mechanisms whereby hypoxia enhances malignant properties and survival of CaP and to identify pO2-regulated tumor-associated macromolecules, we propagated CaP cell lines LnCaP, VCaP and DU145 in hypoxia (pO2=2 kPa) and compared them with normoxically grown cells (pO2=20 kPa); hypoxic cells proliferated faster and secreted more vascular endothelial growth factor (p<0.05). Transcriptome studies revealed different gene expression in cells grown in hypoxia relative to those in normoxia. Interestingly, in hypoxic cells transcripts associated with cancer and urologic disease were overexpressed in comparison to normoxic cells (p<0.001) suggesting an association of low pO2 and aggressive features of CaP. Further, we analyzed the transcriptome in primary CaP cells isolated by laser-capture microdissection (LCM) and whole CaP tissue. We compared the data to those from CaP cells cultured at pO2=20 kPa or pO2=2 kPa. Notably, hypoxia increased transcript levels for pyruvate dehydrogenase kinase isozyme 1, nuclear prelamin A recognition factor, glucose phosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase in all three cell lines (p<0.05) to levels comparable to those found in primary bulk tissue and LCM isolated cells (p<0.005). This finding suggests that gene expression in hypoxically cultured cells is more akin to that in tumor cells in situ than are cells grown normoxically. By 2D-gel electrophoresis, we found that change in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Our results suggest that hypoxia affects transcriptome and proteome in CaP cells. In addition, we screened 20 patient sera and 20 healthy control sera for spontaneous antibodies cross-reactive with VCaP cells and found that the sera reacted with numerous proteins, some previously reported to elicit an immune response in CaP patients (e.g., nucleoporin 62 and transitional endoplasmic reticulum ATPase). By this method we detected numerous novel autoantigens (under validation). Currently we are clarifying the pO2 effects on the relationship of transcriptome, proteome and tumor-associated antigens in CaP cells. Support: DOD PC094680 (CRG), Minnesota Partnership for Biotechnology and Medical Genomics, Mayo Clinic Prostate SPORE 5P50CA091956 (FK); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). 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 LB-308. doi:10.1158/1538-7445.AM2011-LB-308

Abstract In early clinical trials, vaccination with allogeneic cancer cells enhanced the overall survival in some patients with malignancies. Because the administered vaccine cells were cultured at pO2=20 kPa, it is unclear whether they provided an adequate antigen match to tumor cells in situ where pO2 is generally much lower. Thus, we postulate that hypoxically grown vaccine cells will provide a better antigen match to tumors in situ. We are testing this hypothesis by studying the effects of hypoxia on prostate cancer (CaP) cells. LnCaP and VCaP cells were cultured at pO2=2 kPa or 20 kPa and analyzed by 2-D gel electrophoresis. The difference in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Using a threshold of fivefold change, we found that hypoxia decreased the levels of thirteen proteins and increased of four in VCaP cells. These results are in line with the reports showing that hypoxia affects expression only of a small fraction of total cellular protein and that the content of total protein is not altered significantly. To determine if the hypoxia-dependent changes affected immune reactivity of the cells, we took advantage of the spontaneous autoantibodies against tumor-associated antigens (TAAs). Hence, we compared the binding to lysates of LnCaP and VCaP cells of antibodies pooled from CaP patients (n=25), healthy controls (n=25), rheumatoid arthritis (n=17), colorectal cancer (n=10) and lung cancer (n=10). CaP patient sera plasma reacted with numerous spots, some even in control groups and thus considered nonspecific. CaP sera plasma specifically bound to seven spots; four were hypoxia-specific. All selected spots were excised from the gel, trypsin-digested, and identified by MALDI-TOF mass spectrometry as heat shock 70 kDa protein 4; protein disulfide isomerase A3; heterogeneous nuclear ribonucleoprotein L and leucine-rich repeat-containing protein 47. With the exception of the latter, the proteins have been identified or validated as TAAs before, but apparently none in CaP. CaP patient antibodies bound to lysates of LnCaP cells grown at pO2=2 kPa, but less than to such VCaP cell lysates. Our results confirmed the findings that hypoxia affects the proteome mostly quantitatively. Lysates of hypoxic CaP cells revealed novel potential TAAs suggesting the potential immunologic relevance of antigens in the hypoxic proteome. Thus, pO2 control can provide a tool for the development of more effective immunotherapy for CaP and possibly other cancers. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C223.