Development of orthotopic tumour models using ultrasound-guided intrahepatic injection.

Mouse models of human diseases are an essential part of the translational pipeline. Orthotopic tumour mouse models are increasingly being used in cancer research due to their increased clinical relevance over subcutaneous xenograft models, particularly in relation to metastatic disease. In this study, we have developed orthotopic colorectal cancer liver metastases (CRCLM) and primary cholangiocarcinoma (CCA) models in BALB/c nude mice using minimally invasive ultrasound-guided intrahepatic injection. Due to its minimally invasive nature, the method reduced risk from surgical complications whilst being fast and easy to perform and resulted in measurable tumour volumes 1 to 3 weeks post-injection. Tumour volumes were monitored in vivo by weekly high-frequency ultrasound (HF-US) and/or twice weekly bioluminescence imaging (BLI) and confirmed with end-point histology. Take rates were high for human CRC cells (>73%) and for CCA cells (90%). We have demonstrated that this method reliably induces CRCLM and CCAs, in which tumour volume can be monitored throughout using HF-US and/or BLI. This provides a promising experimental tool for future testing of cancer therapeutics in an orthotopic model.

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid inhibits mouse MC-26 colorectal cancer cell liver metastasis via inhibition of PGE2-dependent cell motility.

BACKGROUND AND PURPOSE: The omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti-CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E-type PG synthesis. EXPERIMENTAL APPROACH: A BALB/c mouse model, in which intrasplenic injection of syngeneic MC-26 mouse CRC cells leads to development of liver metastases, was used. Dietary EPA was administered in the free fatty acid (FFA) form for 2 weeks before and after ultrasound-guided intrasplenic injection of 1 × 10(6) MC-26 cells (n= 16 each group). KEY RESULTS: Treatment with 5% (w w(-1)) EPA-FFA was associated with a reduced MC-26 mouse CRC cell liver tumour burden compared with control animals (median liver weight 1.03 g vs. 1.62 g; P < 0.034). Administration of 5% EPA-FFA was also linked to a significant increase in tumour EPA incorporation and lower intratumoural PGE(2) levels (with concomitant increased production of PGE(3)). Liver tumours from 5% EPA-FFA- treated mice demonstrated decreased 5-bromo-2-deoxyuridine-positive CRC cell proliferation and reduced phosphorylated ERK 1/2 expression at the invasive edge of tumours. A concentration-dependent reduction in MC-26 CRC cell Transwell® migration following EPA-FFA treatment (50-200 µM) in vitro was rescued by exogenous PGE(2) (10 µM) and PGE(1)-alcohol (1 µM). CONCLUSIONS AND IMPLICATIONS: EPA-FFA inhibits MC-26 CRC cell liver metastasis. EPA incorporation is associated with a 'PGE(2) to PGE(3) switch' in liver tumours. Inhibition of PGE(2)-EP(4) receptor-dependent CRC cell motility probably contributes to the antineoplastic activity of EPA.

Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo.

Hypoxia is an important factor in tumor growth. It is associated with resistance to conventional anticancer treatments. Gene therapy targeting hypoxic tumor cells therefore has the potential to enhance the efficacy of treatment of solid tumors. Transfection of a panel of tumor cell lines with plasmid constructs containing hypoxia-responsive promoter elements from the genes, vascular endothelial growth factor (VEGF) and erythropoietin, linked to the minimal cytomegalovirus (mCMV) or minimal interleukin-2 (mIL-2) promoters showed optimum hypoxia-inducible luciferase reporter gene expression with five repeats of VEGF hypoxic-response element linked to the mCMV promoter. Adenoviral vectors using this hypoxia-inducible promoter to drive therapeutic transgenes produced hypoxia-specific cell kill of HT1080 and HCT116 cells in the presence of prodrug with both herpes simplex virus thymidine kinase/ganciclovir and nitroreductase (NTR)/CB1954 prodrug-activating systems. Significant cytotoxic effects were also observed in patient-derived human ovarian cancer cells. The NTR/CB1954 system provided more readily controllable transgene expression and so was used for in vivo experiments of human HCT116 xenografts in nude mice. Subjects treated intratumorally with Ad-VEGFmCMV-NTR and intraperitoneal injection of CB1954 demonstrated a statistically significant reduction in tumor growth. Immunohistochemistry of treated xenografts showed a good correlation between transgene expression and hypoxic areas. Further investigation of these hypoxia-inducible adenoviral vectors, alone or in combination with existing modalities of cancer therapy, may aid in the future development of successful Gene-Directed Enzyme Prodrug Therapy systems, which are much needed for targeting solid tumors.

Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition.

Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable, whereas active phosphoinositide-3 kinase (PI3K)-Akt and mitogen-activated protein kinase (MAPK) were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFKs) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.

Role of cell surface molecules and autologous ascitic fluid in determining efficiency of adenoviral transduction of ovarian cancer cells.

Adenovirus is the most frequently used virus in gene therapy clinical trials. There have been conflicting reports on the ability of adenovirus to transduce primary ovarian cancer samples and the expression of relevant cell surface molecules. These factors were examined using primary ovarian cancer cells cultured from ascites and solid tumor to gain insights into the clinical use of adenovirus in ovarian cancer. The level of transduction of primary cultures was much higher than uncultured cells and established cell lines, and correlated with higher levels of coxsackie-adenovirus receptor (CAR) and integrin expression. Growth of primary cultures in autologous ascitic fluid prevented an increase in CAR expression and inhibited transduction compared with cells treated in supplemented RPMI. Cells at the periphery of solid tumor samples were transduced using a replication-incompetent virus and correlated with CAR expression. However, transduction was abolished by autologous ascitic fluid, despite the expression of CAR. We conclude that the use of adenoviruses for ovarian cancer gene therapy will require testing in the presence of inhibitory factors in ascitic fluid. The clinical use of adenoviral vectors may require circumvention of such inhibitory factors and the use of replication competent adenovirus to enable efficient viral penetration of the cancer.

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor.

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in beta-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The 'cassette' design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

Transcriptional targeting of acute hypoxia in the tumour stroma is a novel and viable strategy for cancer gene therapy.

Deregulated tumour growth and neovascularization result in an inadequate tumour blood supply, leading to areas of chronic hypoxia and necrosis. Irregular vascular structure and abnormal tumour physiology also cause erratic blood flow in tumour vessels. We reasoned that tumour stroma, including vascular endothelial cells, would consequently experience transient hypoxia that may allow transcriptional targeting as part of an antivascular gene therapy approach to cancer. To exploit hypoxia for transcriptional regulation, retroviral vectors were generated with modified LTRs: a 6-mer of hypoxia response elements in place of the viral enhancer produced near wild-type levels of expression in hypoxia but was functionally inert in normoxia. In a tumour xenograft model, expression was mainly around areas of necrosis, which were shown to be hypoxic; no expression was detected in tumour stroma. Time-course experiments in vitro demonstrated that expression was transient in response to a hypoxic episode, such that a reporter gene would be insensitive to acute hypoxia in vivo. In contrast, a significant therapeutic effect was seen upon ganciclovir administration with a vector expressing thymidine kinase (TK) in the tumour stroma. Expression of TK was more effective when targeted to acute hypoxia in the stroma compared to chronic hypoxia in the poorly vascularized regions of the tumour cell compartment. The data presented here are evidence that hypoxia in the stromal compartment does occur and that transient hypoxia constitutes a valid therapeutic target.

The research basis of health care decision making.

Regarding health care services, the decision-making process occurs at three primary levels: macro (national), mid-range (hospital) and micro (individual practitioner). The research basis for this process at each of these levels is briefly discussed, with an emphasis concerning which type of data is and/or should be utilized. The paradigm assumptions behind data generation are also explored with reference to methodologies which seek to combine different types of data. The nursing profession, within the changing structure of the NHS, needs to take account of data generation if it is to play an active research role, and therefore be able to influence the health care decision-making process.

Knowledge and level of consciousness: application to nursing practice.

Assessing a patient's level of consciousness is a skilled part of nursing practice. The epistemology of this activity is discussed using the four patterns of knowing identified by Carper. It is suggested that all four patterns and their interaction are necessary for a practitioner to be able to carry out this activity with the necessary reliability and accuracy that good safe practice dictates. A possible enhancement to how a practitioner gains this knowledge can be through the work of joint appointments, between education and clinical areas.

The young child's representation of depth in drawing: process and product.

Two experiments are reported from a series of studies in which plain blocks of various sizes were presented in various spatial orientations to children aged between 3 and 8 years in an attempt to establish how they represent three-dimensional spatial relations pictorially. The major results were that young children represent depth in the array vertically in the picture plane. Two important findings were that even from an early age drawings contain "view-centered" information and that children differentiate between the relative positions of objects in the array by the temporal order of their drawing. These results show the importance of studying the drawing process as well as its product.

A microsurgical technique for simultaneous pancreas and renal transplantation in the rat.

A successful technique of simultaneous pancreatico-renal transplantation into the streptozotocin-induced diabetic rat is described employing a sutureless cuff technique for three of four vascular anastomoses. Reversal of the diabetic state was uniformly observed within 24 hours postoperatively and near-normal renal function was evidenced by 50 days postoperatively. The technique is reliable and reproducible and represents the first report of successful combined transplantation into the diabetic rat.

The use of tacrine for tardive dyskinesia.

The authors administered 45-90 mg/day of tacrine to eight patients with tardive dyskinesia. After 2 weeks of treatment, there was a mean reduction in tardive dyskinesia scores of 43%. No improvement occurred when a placebo was used instead of tacrine.

Cold storage of the pancreas with a view to preservation of islet cell function following transplantation.

Three solutions, hyperosmolar citrate, modified Collins' C2, and Sacks' II solutions were compared as media for cold storage preservation (arterial infusion and subsequent cold storage in the same medium at 0-4 C) of the rat pancreas with a view to preservation of endocrine function. Pancreatic isotransplantation was performed following cold ischemic intervals of 0, 24, 30, and 36 hr, into streptozotocin-induced diabetic recipients. Results were assessed by normoglycemic survival and insulin response, together with K values following i.v. glucose tolerance tests at 3 months postoperatively; 24-hr preservation was achieved with equal success using modified Collins' C2 solution or hyperosmolar citrate-but not with Sacks' II solution. Preservation for 30 hr was consistently successful using modified Collins C2 solution only, but the period could not be extended with success to 36 hr. Hypoglycemia and hyperinsulinemia occurred 24 hr postoperatively in the majority of animals receiving grafts stored in Sacks' II solution, but to a much lesser extent using modified Collins' C2 and hyperosmolar citrate. This was also temporarily seen in grafts stored for 36 hr in modified Collins C2 solution. At 3 months postoperatively after 30 hr cold ischemia, i.v. glucose tolerance tests showed the hyperosmolar citrate cold-stored grafts had lower K values and significantly reduced insulin responses compared with grafts stored in modified Collins' C2 solution. The modified Collins' C2 solution proved to be the most effective of the three solutions tested.