Inhibitor of DNA-binding 4 contributes to the maintenance and expansion of cancer stem cells in 4T1 mouse mammary cancer cell line

The cancer stem cell (CSC) hypothesis proposes that CSCs are the root of cancer. CSC-targeted therapies may prevent cancer relapse and provide more effective treatment. The expression of aldehyde dehydrogenase 1, as assessed by the Aldefluor assay, has been recognized as a marker of CSCs in breast cancer. Inhibitors of DNA-binding proteins (IDs) have an important role in stem cell differentiation. In this study, we examined IDs necessary for the regulation of stem properties in Aldefluorpos 4T1 cells. When the expression profile of IDs in Aldefluorneg and Aldefluorpos 4T1 cells was compared, qRT-PCR analysis showed that ID4 expression was highly upregulated in Aldefluorpos 4T1 cells. In addition, knockdown of ID4 expression suppressed the properties of CSCs, including their sphere-forming ability and side population phenotype. The findings suggest that ID4 may be a therapeutic target for the treatment of advanced breast cancer

Generation and characterisation of two D2A1 mammary cancer sublines to model spontaneous and experimental metastasis in a syngeneic BALB/c host

Studying the complex mechanisms underlying breast cancer metastasis and therapy response necessitates relevant in vivo models, particularly syngeneic models with an intact immune system. Two syngeneic spontaneously metastatic sublines, D2A1-m1 and D2A1-m2, were generated from the poorly metastasising BALB/c-derived D2A1 cell line by serial in vivo passaging. In vivo and in vitro analyses revealed distinct and shared characteristics of the metastatic D2A1-m1 and D2A1-m2 sublines. In particular, D2A1-m1 cells are more aggressive in experimental metastasis assays, while D2A1-m2 cells are more efficient at disseminating from the primary tumour in spontaneous metastasis assays. Surprisingly, classical metastasis-associated in vitro phenotypes such as enhanced proliferation, migration and invasion are reduced in the sublines compared to the parental cell line. Further, evasion of immune control cannot fully explain their enhanced metastatic properties. By contrast, both sublines show increased resistance to apoptosis when cultured in non-adherent conditions and, for the D2A1-m2 subline, increased 3D tumour spheroid growth. Moreover, the enhanced spontaneous metastatic phenotype of the D2A1-m2 subline is associated with an increased ability to recruit an activated tumour stroma. The metastatic D2A1-m1 and D2A1-m2 cell lines provide additional syngeneic models for investigating the different steps of the metastatic cascade and thereby represent valuable tools for breast cancer researchers. Finally, this study highlights that morphology and cell behaviour in 2D cell-based assays cannot be used as a reliable predictor of metastatic behaviour in vivo

Post-mortem estimation of temperature distribution on a power transformer: Physicochemical and mechanical approaches

Power transformers are electrical machines that allow us to transport electric energy, with reduced losses, from generation stations to consumption points. This definition gives us an idea of the number of transformers that are used in power distribution systems worldwide. The main problem that may affect power transformers is the operation at high temperature. This paper summarizes the results of a postmortem temperature estimation carried out on an 800 kVA distribution transformer. Two methodologies are considered for estimating the temperature distribution in the windings of the machine. The first is based on the calculation of the degree of polymerization and the second is obtained from the tensile strength index. By knowing the value of these two magnitudes for a new and for an aged paper, and the period of operation of a transformer, the temperature distribution along the height of the windings can be estimated. None other previous works have used the tensile strength of winding paper for temperature distribution estimation. With these results and the loading regime records, similar transformers still in operation can be operated in an alternative manner and future designs can be improved

Thermal degradation assessment of Kraft paper in power transformers insulated with natural esters

Kraft paper in combination with dielectric oil is the most common as insulation system used in power transformers. The most used oil in power transformers is mineral oil. However, dielectric oils based on natural esters possess some advantages in comparison with mineral oil such as higher biodegradability, fire safety and availability. Therefore, they might be the most ideal substitute for mineral oil. The introduction of a new material requires the evaluation of its degradation rate because this determines transformers’ life span. In order to assess the performance of new insulating systems based on vegetable oils, simulations to obtain the hot-spot temperatures in power transformers using vegetable oils and controlled laboratory experiments of ageing were carried out. The reason is that hot spot temperature inside windings of the oil-immersed power transformers is one of the main manifestations of the thermal stress which leads to aging of insulation systems.The research leading to these results has received funding from multiple sources during years but we would specifically like to acknowledge the support received in the later stages from the Spanish Plan Estatal de I+D under the grant agreement DPI2013- 43897-P

Nontransgenic models of breast cancer

Numerous models have been developed to address key elements in the biology of breast cancer development and progression. No model is ideal, but the most useful are those that reflect the natural history and histopathology of human disease, and allow for basic investigations into underlying cellular and molecular mechanisms. We describe two types of models: those that are directed toward early events in breast cancer development (hyperplastic alveolar nodules [HAN] murine model, MCF10AT human xenograft model); and those that seek to reflect the spectrum of metastatic disease (murine sister cell lines 67, 168, 4T07, 4T1). Collectively, these models provide cell lines that represent all of the sequential stages of progression in breast disease, which can be modified to test the effect of genetic changes

Imagable 4T1 model for the study of late stage breast cancer

<p>Abstract</p> <p>Background</p> <p>The 4T1 mouse mammary tumor cell line is one of only a few breast cancer models with the capacity to metastasize efficiently to sites affected in human breast cancer. Here we describe two 4T1 cell lines modified to facilitate analysis of tumor growth and metastasis and evaluation of gene function <it>in vivo</it>. New information regarding the involvement of innate and acquired immunity in metastasis and other characteristics of the model relevant to its use in the study of late stage breast cancer are reported.</p> <p>Methods</p> <p>The lines were engineered for stable expression of firefly luciferase to allow tracking and quantitation of the cells <it>in vivo</it>. Biophotonic imaging was used to characterize growth and metastasis of the lines <it>in vivo </it>and an improved gene expression approach was used to characterize the basis for the metastatic phenotype that was observed.</p> <p>Results</p> <p>Growth of cells at the primary site was biphasic with metastasis detected during the second growth phase 5–6 weeks after introduction of the cells. Regression of growth, which occurred in weeks 3–4, was associated with extensive necrosis and infiltration of leukocytes. Biphasic tumor growth did not occur in BALB/c SCID mice indicating involvement of an acquired immune response in the effect. Hematopoiesis in spleen and liver and elevated levels of circulating leukocytes were observed at week 2 and increased progressively until death at week 6–8. Gene expression analysis revealed an association of several secreted factors including colony stimulatory factors, cytokines and chemokines, acute phase proteins, angiogenesis factors and ECM modifying proteins with the 4T1 metastatic phenotype. Signaling pathways likely to be responsible for production of these factors were also identified.</p> <p>Conclusion</p> <p>The production of factors that stimulate angiogenesis and ECM modification and induce hematopoiesis, recruitment and activation of leukocytes suggest that 4T1 tumor cells play a more direct role than previously appreciated in orchestrating changes in the tumor environment conducive to tumor cell dissemination and metastasis. The new cell lines will greatly facilitate the study of late stage breast and preclinical assessment of cancer drugs and other therapeutics particularly those targeting immune system effects on tumor metastasis.</p

Maspin is a tumour suppressor that inhibits breast cancer tumour metastasis in vivo

Maspin is a member of the serpin family of serine proteases and functions as a tumour suppressor. A study using a new syngeneic mouse model for breast cancer suggests that maspin can inhibit metastasis in vivo

Functional and molecular characterisation of EO771.LMB tumours, a new C57BL/6-mouse-derived model of spontaneously metastatic mammary cancer.

The translation of basic research into improved therapies for breast cancer patients requires relevant preclinical models that incorporate spontaneous metastasis. We have completed a functional and molecular characterisation of a new isogenic C57BL/6 mouse model of breast cancer metastasis, comparing and contrasting it with the established BALB/c 4T1 model. Metastatic EO771.LMB tumours were derived from poorly metastatic parental EO771 mammary tumours. Functional differences were evaluated using both in vitro assays and spontaneous metastasis assays in mice. Results were compared to non-metastatic 67NR and metastatic 4T1.2 tumours of the 4T1 model. Protein and transcript levels of markers of human breast cancer molecular subtypes were measured in the four tumour lines, as well as p53 (Tp53) tumour-suppressor gene status and responses to tamoxifen in vivo and in vitro. Array-based expression profiling of whole tumours identified genes and pathways that were deregulated in metastatic tumours. EO771.LMB cells metastasised spontaneously to lung in C57BL/6 mice and displayed increased invasive capacity compared with parental EO771. By immunohistochemical assessment, EO771 and EO771.LMB were basal-like, as was the 4T1.2 tumour, whereas 67NR had a luminal phenotype. Primary tumours from all lines were negative for progesterone receptor, Erb-b2/Neu and cytokeratin 5/6, but positive for epidermal growth factor receptor (EGFR). Only 67NR displayed nuclear estrogen receptor alpha (ERα) positivity. EO771 and EO771.LMB expressed mutant p53, whereas 67NR and 4T1.2 were p53-null. Integrated molecular analysis of both the EO771/EO771.LMB and 67NR/4T1.2 pairs indicated that upregulation of matrix metalloproteinase-3 (MMP-3), parathyroid hormone-like hormone (Pthlh) and S100 calcium binding protein A8 (S100a8) and downregulation of the thrombospondin receptor (Cd36) might be causally involved in metastatic dissemination of breast cancer

Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer

BACKGROUND: Insulin-like growth factor 1 (IGF1) promotes breast cancer and disease progression. Bioavailability of IGF1 is modulated by IGF-binding proteins (IGFBPs). IGFBP4 inhibits IGF1 activity but cleavage by pregnancy-associated plasma protein-A (PAPP-A) protease releases active IGF1. METHODS: Expression of IGF pathway components and PAPP-A was assessed by western blot or RT-PCR. IGFBP4 (dBP4) resistant to PAPP-A cleavage, but retaining IGF-binding capacity, was used to block IGF activity in vivo. 4T1.2 mouse mammary adenocarcinoma cells transfected with empty vector, vector expressing wild-type IGFBP4 or vector expressing dBP4 were implanted in the mammary fat pad of BALB/c mice and tumour growth was assessed. Tumour angiogenesis and endothelial cell apoptosis were assessed by immunohistochemistry. RESULTS: 4T1.2 cells expressed the IGF1R receptor and IGFBP4. PAPP-A was expressed within mammary tumours but not by 4T1.2 cells. Proliferation and vascular endothelial growth factor (VEGF) production by 4T1.2 cells was increased by IGF1(E3R) (recombinant IGF1 resistant to binding by IGFBPs) but not by wild-type IGF1. IGF1-stimulated microvascular endothelial cell proliferation was blocked by recombinant IGFBP4. 4T1.2 tumours expressing dBP4 grew significantly more slowly than controls or tumours expressing wild-type IGFBP4. Inhibition of tumour growth by dBP4 was accompanied by the increased endothelial cell apoptosis. CONCLUSION: Protease-resistant IGFBP4 blocks IGF activity, tumour growth and angiogenesis