Single-cell Survival Research Articles

Neurotransmitter receptor-related gene signature as potential prognostic and therapeutic biomarkers in colorectal cancer

Background: Colorectal cancer is one of the most common malignant tumors worldwide. A various of neurotransmitter receptors have been found to be expressed in tumor cells, and the activation of these receptors may promote tumor growth and metastasis. This study aimed to construct a novel neurotransmitter receptor-related genes signature to predict the survival, immune microenvironment, and treatment response of colorectal cancer patients.Methods: RNA-seq and clinical data of colorectal cancer from The Cancer Genome Atlas database and Gene Expression Omnibus were downloaded. Neurotransmitter receptor-related gene were collected from publicly available data sources. The Weighted Gene Coexpression Network Analysis (WGCNA), Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression, Support Vector Machine-Recursive Feature Elimination (SVM-RFE), and Random Forest (RF) algorithms were employed to construct the Neurotransmitter receptor-related gene prognostic signature. Further analyses, functional enrichment, CIBERSORTx, The Tumor Immune Single Cell Center (TISCH), survival analysis, and CellMiner, were performed to analyze immune status and treatment responses. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were carried out to confirm the expression levels of prognostic genes.Results: By combining machine learning algorithm and WGCNA, we identified CHRNA3, GABRD, GRIK3, and GRIK5 as Neurotransmitter receptor-related prognostic genes signature. Functional enrichment analyses showed that these genes were enriched with cellular metabolic-related pathways, such as organic acid, inorganic acid, and lipid metabolism. CIBERSORTx and Single cell analysis showed that the high expression of genes were positively correlated with immunosuppressive cells infiltration, and the genes were mainly expressed in cancer-associated fibroblasts and endothelial cells. A nomogram was further built to predict overall survival (OS). The expression of CHRNA3, GABRD, GRIK3, and GRIK5 in cancer cells significantly impacted their response to chemotherapy.Conclusion: A neurotransmitter receptor-related prognostic gene signature was developed and validated in the current study, giving novel sights of neurotransmitter in predicting the prognostic and improving the treatment of CRC.

Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells.

IntroductionHigh-efficacy single-cell cloning of human-induced pluripotent cells (IPSCs) remains a major challenge. The development of a culture method that supports single-cell passaging while maintaining reproducibility, homogeneity, scalability, and cell expansion to clinically relevant numbers is necessary for clinical application.MethodsTo address this issue, we combined the use of the rho-associated protein kinase (ROCK) inhibitor Y-27632 and hypoxic conditions in culture to produce a novel, efficient single-cell culture method for human IPSCs and embryonic stem cells.ResultsThrough immunocytochemistry, alkaline phosphatase assays, and flow cytometry, we demonstrated that our method enabled high single-cell proliferation while maintaining self-renewal and pluripotency abilities.DiscussionWe showed the beneficial effect of the interaction between hypoxia and ROCK inhibition in regulating cell proliferation, pluripotency, and single-cell survival of pluripotent cells.

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells.

Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2weeks. This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs

Selenium (Se) plays a vital role in reactive oxygen species (ROS) homeostasis and redox regulation in intracellular signaling via selenocysteine (Sec), known as the 21st proteinogenic amino acid, but its specific biological functions in development and disease remain undiscovered. In this study, we explored the role of selenophosphate synthetase 1 (SEPHS1) in the pluripotency maintenance and reprogramming. We found that high level of SEPHS1 is retained in undifferentiated embryonic stem cells (ESCs), which is decreased during their differentiation. SEPHS1 knockdown significantly reduced reprogramming efficiency, proving that SEPHS1 is required for acquisition of pluripotency. However, SEPHS1 knockdown did not affect the expression of significant pluripotency genes, suggesting that SEPHS1 may be involved in the survival of pluripotent stem cells rather than in the regulation of pluripotency genes. Transcriptome analysis revealed altered expression of the gene set related to the ROS pathway and apoptosis in SEPHS1-knockdown cells. We also demonstrated the role of SEPHS1 in human ESC clonogenicity, and we found improved single-cell survival of hESCs by selenium treatment in a concentration-dependent manner. Our study implies that hSEPHS1 is a regulator of selenium-mediated redox-signaling in human pluripotent stem cells and plays a role in their survival.

Reentrant Efficiency of Phototaxis in Chlamydomonas reinhardtii Cells

Phototaxis is one of the most fundamental stimulus-response behaviors in biology wherein motile microorganisms sense light gradients to swim toward the light source. Apart from single-cell survival and growth, it plays a major role at the global scale of aquatic ecosystems and bioreactors. We study phototaxis of single-celled algae Chlamydomonas reinhardtii as a function of cell number density and light stimulus using high spatiotemporal video microscopy. Surprisingly, the phototactic efficiency has a minimum at a well-defined number density, for a given light gradient, above which the phototaxis behavior of a collection of cells can even exceed the performance obtainable from single isolated cells. We show that the origin of enhancement of performance above the critical concentration lies in the slowing down of the cells, which enables them to sense light more effectively. We also show that this steady-state phenomenology is well captured by modeling the phototactic response as a density-dependent torque acting on an active Brownian particle.

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy.

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

3127 - Retention of Single Long Term Hematopoietic Stem Cells in Minimal Culture Medium Reveals Essential Components of HSC Function

Recent descriptions of single cell gene expression datasets from highly purified long-term hematopoietic stem cells (LT-HSCs) have significantly advanced our understanding of the molecular state of HSCs. Emerging concomitantly, however, is the knowledge that LT-HSCs share a significant amount of their transcriptome with cells that do not possess the functional properties of durable self-renewal and multi-lineage cell output. To discern which genes and pathways drive individual HSC properties and which are bystanders is a major outstanding challenge for the field. We have recently demonstrated that single HSCs (EPCR++CD150+CD48-CD45+Sca-1++, >50% LT-HSCs by single cell transplantation) can be cultured without undergoing division for a period of 710 days with minimal cytokine stimulation (21% single cell survival, 99.2% undivided). Limiting dilution assays of HSCs cultured for 7 days (7dHSCs) estimated the durable multi-lineage HSC frequency at 22%. Single cell transplantation experiments had a similar frequency with both primary (3/10) and secondary (3/3) transplantations demonstrating that single 7dHSCs retained HSC function. Since 7dHSCs share the functional properties of freshly isolated HSCs, they represent a robust comparator population to identify which genes are indispensable for the maintenance of HSC self-renewal and quiescence. We generated RNA-sequencing datasets from freshly isolated HSCs and 7dHSCs and identified 960 genes and a number of biological processes that were significantly down-regulated in 7dHSCs, suggesting that these are dispensable for HSC function. Major down-regulated processes included the response to oxidative stress, ligand-independent apoptosis, and cellular senescence. Interestingly, in all 3 mice successfully transplanted with single 7dHSCs, a lymphoid-deficient (or -HSC) program was observed, suggesting that -HSCs are more resilient to the stress induced by cytokine depletion and represent a potential mechanism of obtaining the molecular program of -HSC. Together, these data highlight the importance of studying HSCs in different contexts to identify common molecular features of their functional properties.

Abstract LB-043: Generation of clonal CRISPR/Cas9-edited human iPSC derived cellular models and its applications in physiologically relevant assays

Abstract Induced pluripotent stem cell (iPSC)-derived cells are widely used in disease models, safety testing and phenotypic and functional assays. The CRISPR/Cas9 system has revolutionized genome editing thus enabling rapid generation of disease models. Cutting edge technologies like genome editing paired with iPSC-derived cell models have reshaped our approach to disease modeling in vitro. Although, CRISPR/Cas9 has had huge impact on generation of complex cellular models, there are challenges in replicating disease-in-a-dish. One of the challenges is the implementation of genome editing tools in iPSC, particularly issues associated with editing tools delivery, cell recovery and clonal isolation of genome-edited cells. Here, we describe a workflow that facilitates the generation and isolation of clonal CRISPR-edited iPSCs through the use of a novel toolset. We observed high genome editing efficiency using an iPSC line that stably expresses the Cas9 protein and obtained improved survival of the iPSCs after delivery of the editing tools through culture in StemFlex™ medium, a novel PSC culture system. Furthermore, we have demonstrated that single edited iPSCs can be isolated through the combination of an optimized cell sorting method and expanded as single cell clones with reagents that support single cell survival. Through this workflow we have generated several iPSC lines carrying disease relevant SNP mutations. These tools and workflows not only contribute to improved success in the derivation of homogenous genome edited human iPSC clones but also provide novel alternatives to study disease-causing mutations in vitro. Additionally, we have also tested these cell models in commonly used cell health assays. These cell models can be further used to generate organoids to mimic conditions found in tissues. iPSC- derived cell models not only permit the study of disease mechanism in the cell types specifically affected in the disease but also deepen our understanding of the molecular mechanisms and in turn help development of novel therapeutics. Citation Format: Chetana M. Revankar, Erik Willems, Rex Lacambacal, Tufan gokirmak, Jordan Dizon, Jacquelyn Webb, Raquel Vega, David Piper. Generation of clonal CRISPR/Cas9-edited human iPSC derived cellular models and its applications in physiologically relevant assays [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 LB-043.

The development of induced pluripotent stem cell-derived mesenchymal stem/stromal cells from normal human and RDEB epidermal keratinocytes

BackgroundEpidermolysis bullosa (EB) is a group of hereditary disorders caused by mutations in the genes encoding structural molecules of the dermal-epidermal junction (DEJ). Cell-based therapies such as allogeneic mesenchymal stem/stromal cell (MSC) transplantation have recently been explored for severe EB types, such as recessive dystrophic EB (RDEB). However, hurdles exist in current MSC-based therapies, such as limited proliferation from a single cell source and limited cell survival due to potential allogenic rejection. ObjectivesWe aimed to develop MSCs from keratinocyte-derived induced pluripotent stem cells (iPSCs). MethodsKeratinocyte-derived iPSCs (KC-iPSCs) of a healthy human and an RDEB patient were cultured with activin A, 6-bromoindirubin-3′-oxime and bone morphogenetic protein 4 to induce mesodermal lineage formation. These induced cells were subjected to immunohistochemical analysis, flow cytometric analysis and RNA microarray analysis in vitro, and were injected subcutaneously and intravenously to wounded immunodeficient mice to assess their wound-healing efficacy. ResultsAfter their induction, KC-iPSC-induced cells were found to be compatible with MSCs. Furthermore, with the subcutaneous and intravenous injection of the KC-iPSC-induced cells into wounded immunodeficient mice, human type VII collagen was detected at the DEJ of epithelized areas. ConclusionsWe successfully established iPSC-derived MSCs from keratinocytes (KC-iPSC-MSCs) of a normal human and an RDEB patient. KC-iPSC-MSCs may have potential in therapies for RDEB.

Identifying drug resistant cancer cells using microbubble well arrays.

Drug resistance is a characteristic of tumor initiating cells that can give rise to metastatic disease. In this work we demonstrate the use of microbubble well arrays as a cell culture platform to enumerate and characterize drug resistant cells in a human derived tumorigenic squamous cell carcinoma cell line. The spherical architecture and compliant hydrophobic composition of the microbubble well favors single cell survival, clonal proliferation and formation of spheres that do not grow on standard tissue culture plastic and are resistant to cisplatin. Spheres form in isolation and in microbubble wells containing proliferating cells and to some degree they stain positive for common stem cell markers CD44 and CD133. Spheres are also observed in cellularized primary human tumors cultured in microbubble arrays. This proof-of-concept study illustrates the potential for microbubble array technology to enumerate cancer cells resistant to standard care drugs with the ability to test alternative drug combinations. This capability can be developed for designing patient specific treatment strategies. Recovery of drug-resistant cells will allow amore full characterization of their gene expression profile thereby expanding our fundamental knowledge and abilityto develop new targets to fight metastatic disease.

EGFR Family Members' Regulation of Autophagy Is at a Crossroads of Cell Survival and Death in Cancer.

The epidermal growth factor receptor (EGFR) signaling pathways are altered in many cancers contributing to increased cell survival. These alterations are caused mainly through increased expression or mutation of EGFR family members EGFR, ErbB2, ErbB3, and ErbB4. These receptors have been successfully targeted for cancer therapy. Specifically, a monoclonal antibody against ErbB2, trastuzumab, and a tyrosine kinase inhibitor against EGFR, gefitinib, have improved the survival of breast and lung cancer patients. Unfortunately, cancer patients frequently become resistant to these inhibitors. This has led to investigating how EGFR can contribute to cell survival and how cancer cells can overcome inhibition of its signaling. Indeed, it is coming into focus that EGFR signaling goes beyond a single signal triggering cell proliferation and survival and is a sensor that regulates the cell’s response to microenvironmental stresses such as hypoxia. It acts as a switch that modulates the ability of cancer cells to survive. Autophagy is a process of self-digestion that is inhibited by EGFR allowing cancer cells to survive under stresses that would normally cause death and become resistant to chemotherapy. Inhibiting EGFR signaling allows autophagy to contribute to cell death. This gives new opportunities to develop novel therapeutic strategies to treat cancers that rely on EGFR signaling networks and autophagy. In this review, we summarize the current understanding of EGFR family member regulation of autophagy in cancer cells and how new therapeutic strategies could be developed to overcome drug resistance.

Noisy Response to Antibiotic Stress Predicts Subsequent Single-Cell Survival in an Acidic Environment

Antibiotics elicit drastic changes in microbial gene expression, including the induction of stress response genes. While certain stress responses are known to "cross-protect" bacteria from other stressors, it is unclear whether cellular responses to antibiotics have a similar protective role. By measuring the genome-wide transcriptional response dynamics of Escherichia coli to four antibiotics, we found that trimethoprim induces a rapid acid stress response that protects bacteria from subsequent exposure to acid. Combining microfluidics with time-lapse imaging to monitor survival and acid stress response in single cells revealed that the noisy expression of the acid resistance operon gadBC correlates with single-cell survival. Cells with higher gadBC expression following trimethoprim maintain higher intracellular pH and survive the acid stress longer. The seemingly random single-cell survival under acid stress can therefore be predicted from gadBC expression and rationalized in terms of GadB/C molecular function. Overall, we provide a roadmap for identifying the molecular mechanisms of single-cell cross-protection between antibiotics and other stressors.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

In the linear quadratic model, the Poisson approximation and the Zaider-Minerbo formula agree on the ranking of tumor control probabilities, up to a critical cell birth rate

Purpose: To provide a rule for the agreement or disagreement of the Poisson approximation (PA) and the Zaider-Minerbo formula (ZM) on the ranking of treatment alternatives in terms of tumor control probability (TCP) in the linear quadratic model.Materials and methods: A general criterion involving a critical cell birth rate was formally derived. For demonstration, the criterion was applied to a distinct radiobiological model of fast growing head and neck tumors and a respective range of 22 conventional and nonconventional head and neck schedules.Results: There is a critical cell birth rate bcrit below which PA and ZM agree on which one out of two alternative treatment schemes with single-cell survival curves S′(t) and S′′(t) offers better TCP:For cell birth rates b above this critical cell birth rate, PA and ZM disagree if and only if b >bcrit > 0. In case of the exemplary head and neck schedules, out of 231 possible combinations, only 16 or 7% were found where PA and ZM disagreed. In all 231 cases the prediction of the criterion was numerically confirmed, and cell birth rates at crossovers between schedules matched the calculated critical cell birth rates.Conclusions: TCP estimated by PA and ZM almost never numerically coincide. Still, in many cases both formulas at least agree about which one out of two alternative fractionation schemes offers better TCP. In case of fast growing tumors featuring a high cell birth rate, however, ZM may suggest a re-evaluation of treatment options.

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture.

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.

CAF-specific markers: role of the TGFβ pathway.

CAF-specific markers: role of the TGFβ pathway.

Abstract IA30: How cells survive: Single cell analysis reveals properties of non-genetic apoptosis resistance.

Abstract The mechanism of non-genetic resistance to TRAIL-induced apoptosis remains unresolved as the regulatory pathways involved are interconnected and complex. Single-cell mass cytometry was used to profile functional changes in regulatory factors while simultaneously monitoring apoptotic progression in TRAIL-treated cells. Cells resistant to TRAIL possessed diverse signaling states, however only cells within a subset of these states, termed “essential persisters” (EPs), were able to survive long-term and replicate. Constriction of the EP signaling states with kinase perturbagens decreased cell survival, however, the EPs occupied unique signaling states for each cell line tested. In order to overcome cell-type specific resistance, single peturbogen data was used to predict the effects of perturbagen combinations on survival. The level of EP signaling state constriction by a single perturbagen was a more robust predictor of combinatorial effects than extrapolating from single perturbogen cell survival data directly. These findings provide a population-level understanding of non-genetic resistance and explain the shortcoming of previous ‘bulk’ methods for identifying mechanisms of TRAIL-resistance. The use of signaling space constriction in persisters may guide design of therapies against non-genetic resistance in the future. Citation Format: Douglas R. Green. How cells survive: Single cell analysis reveals properties of non-genetic apoptosis resistance. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA30.

Abstract 3305: The role of mTOR in mediating nicotine induced alpha-7 nicotinic acetylcholine receptor activation and the regulation of radiotherapeutic response

Abstract Tobacco use is the single largest preventable risk factor for developing cancer. Furthermore, tobacco is associated with poor therapeutic outcomes in part because its continued use has been shown to decrease the efficacy of conventional chemotherapy and/or radiotherapy (RT). Preclinical data suggests that nicotine administration decreases RT efficacy by activating the alpha-7-nicotinic acetylcholine receptor (a7-nAChR). Our study evaluated the effects of nicotine and selective a7-nAChR agonists (LY 2087101, PHA 543613, PNU 282987, and TC 1698) administration on radiotherapeutic efficacy. H460 and A549 human lung cancer cell lines were dose escalated with nicotine and the a7-nAChR agonists alone or in combination with single fraction X-ray radiation (2, 6 Gy) and cell survival was assessed by clonogenic assay. Nicotine administration and activation of a7-nAChR by LY 2087101 or PHA 543613 was found to have a radioprotective effect in both cell lines by increasing cell survival following RT. Conversely, activation of a7-nAChR by PNU 282987 or TC 1698 did not modulate radiotherapeutic response. Western blot analysis demonstrated that although all four tested a7-nAChR agonists increased phosphorylation of AKT (S473), only PHA 543613 and LY 2087101 were found to also activate mTOR (S2448). Additional clonogenic studies were performed to further examine the role of mTORC1 in governing response to RT. Inhibition of mTORC1 by rapamycin alone sensitized both A549 and H460 cells to the effect of RT. Interestingly, rapamycin treatment was sufficient to block the radioprotective effects of nicotine, PHA 543613, and LY 2087101 in A549 cells, however, in H460 cells rapamycin did not restore radiotherapeutic efficacy. Moreover, examination of Deptor, a key component of mTOR complexes shown to be associated with RT response, demonstrated that a7-nAChR activation by nicotine, PHA 543613, or LY 2087101 increased Deptor degradation in A549 cells, while a7-nAChR activation decreased Deptor degradation in H460 cells. These observations suggest that differences in the regulation of Deptor level may contribute to the variation in both rapamycin sensitivity and restoration of therapeutic efficacy to RT between H460 and A549 cell lines. Additional studies will be necessary to further elucidate the role of the mTOR complexes in mediating a7-nAChR induced modulation of response to RT. Citation Format: Samantha L. Sobus, Sundaravadivel Balasubramanian, Michelle A. Romano, Graham W. Warren. The role of mTOR in mediating nicotine induced alpha-7 nicotinic acetylcholine receptor activation and the regulation of radiotherapeutic response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3305. doi:10.1158/1538-7445.AM2015-3305

The broad-spectrum anti-DNA virus agent cidofovir inhibits lung metastasis of virus-independent, FGF2-driven tumors.

The FDA-approved anti-DNA virus agent cidofovir (CDV) is being evaluated in phase II/III clinical trials for the treatment of human papillomavirus (HPV)-associated tumors. However, previous observations had shown that CDV also inhibits the growth of vascular tumors induced by fibroblast growth factor-2 (FGF2)-transformed FGF2-T-MAE cells. Here, we demonstrate that CDV inhibits metastasis induced by FGF2-driven, virus-independent tumor cells. Pre-treatment of luciferase-expressing FGF2-T-MAE cells with CDV reduced single cell survival and anchorage-independent growth in vitro and lung metastasis formation upon intravenous inoculation into SCID mice. This occurred in the absence of any effect on homing of FGF2-T-MAE cells to the lungs and on the growth of subconfluent cell cultures or subcutaneous tumors in mice. Accordingly, CDV protected against lung metastasis when given systemically after tumor cell injection. Lung metastases in CDV-treated mice showed reduced Ki67 expression and increased nuclear accumulation of p53, indicating that CDV inhibits metastasis by affecting single cell survival properties. The anti-metastatic potential of CDV was confirmed on B16-F10 melanoma cells, both in zebrafish embryos and mice. These findings suggest that CDV may have therapeutic potential as an anti-metastatic agent and warrants further study to select those tumor types that are most likely to benefit from CDV therapy.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.