Jill E. Shea

Phenotype and Genotype of Pancreatic Cancer Cell Lines

Abstract The dismal prognosis of pancreatic adenocarcinoma is due in part to a lack of molecular information regarding disease development. Established cell lines remain a useful tool for investigating these molecular events. Here we present a review of available information on commonly used pancreatic adenocarcinoma cell lines as a resource to help investigators select the cell lines most appropriate for their particular research needs. Information on clinical history; in vitro and in vivo growth characteristics; phenotypic characteristics, such as adhesion, invasion, migration, and tumorigenesis; and genotypic status of commonly altered genes (KRAS, p53, p16, and SMAD4) was evaluated. Identification of both consensus and discrepant information in the literature suggests careful evaluation before selection of cell lines and attention be given to cell line authentication.

Controlled and targeted tumor chemotherapy by ultrasound-activated nanoemulsions/microbubbles

The paper reports the results of nanotherapy of ovarian, breast, and pancreatic cancerous tumors by paclitaxel-loaded nanoemulsions that convert into microbubbles locally in tumor tissue under the action of tumor-directed therapeutic ultrasound. Tumor accumulation of nanoemulsions was confirmed by ultrasound imaging. Dramatic regression of ovarian, breast, and orthotopic pancreatic tumors was observed in tumor therapy through systemic injections of drug-loaded nanoemulsions combined with therapeutic ultrasound, signifying efficient ultrasound-triggered drug release from tumor-accumulated nanodroplets. The mechanism of drug release in the process of droplet-to-bubble conversion is discussed. No therapeutic effect from the nanodroplet/ultrasound combination was observed without the drug, indicating that therapeutic effect was caused by the ultrasound-enhanced chemotherapeutic action of the tumor-targeted drug, rather than the mechanical or thermal action of ultrasound itself. Tumor recurrence was observed after the completion of the first treatment round; a second treatment round with the same regimen proved less effective, suggesting that drug-resistant cells were either developed or selected during the first treatment round.

Ultrasound-Mediated Tumor Imaging and Nanotherapy using Drug Loaded, Block Copolymer Stabilized Perfluorocarbon Nanoemulsions

Perfluorocarbon nanoemulsions can deliver lipophilic therapeutic agents to solid tumors and simultaneously provide for monitoring nanocarrier biodistribution via ultrasonography and/or (19)F MRI. In the first generation of block copolymer stabilized perfluorocarbon nanoemulsions, perfluoropentane (PFP) was used as the droplet forming compound. Although manifesting excellent therapeutic and ultrasound imaging properties, PFP nanoemulsions were unstable at storage, difficult to handle, and underwent hard to control phenomenon of irreversible droplet-to-bubble transition upon injection. To solve the above problems, perfluoro-15-crown-5-ether (PFCE) was used as a core forming compound in the second generation of block copolymer stabilized perfluorocarbon nanoemulsions. PFCE nanodroplets manifest both ultrasound and fluorine ((19)F) MR contrast properties, which allows using multimodal imaging and (19)F MR spectroscopy for monitoring nanodroplet pharmacokinetics and biodistribution. In the present paper, acoustic, imaging, and therapeutic properties of unloaded and paclitaxel (PTX) loaded PFCE nanoemulsions are reported. As manifested by the (19)F MR spectroscopy, PFCE nanodroplets are long circulating, with about 50% of the injected dose remaining in circulation 2h after the systemic injection. Sonication with 1-MHz therapeutic ultrasound triggered reversible droplet-to-bubble transition in PFCE nanoemulsions. Microbubbles formed by acoustic vaporization of nanodroplets underwent stable cavitation. The nanodroplet size (200nm to 350nm depending on a type of the shell and conditions of emulsification) as well as long residence in circulation favored their passive accumulation in tumor tissue that was confirmed by ultrasonography. In the breast and pancreatic cancer animal models, ultrasound-mediated therapy with paclitaxel-loaded PFCE nanoemulsions showed excellent therapeutic properties characterized by tumor regression and suppression of metastasis. Anticipated mechanisms of the observed effects are discussed.

Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas.

Notch signaling regulates numerous developmental processes, often acting either to promote one cell fate over another or else to inhibit differentiation altogether. In the embryonic pancreas, Notch and its target gene Hes1 are thought to inhibit endocrine and exocrine specification. Although differentiated cells appear to downregulate Hes1, it is unknown whether Hes1 expression marks multipotent progenitors, or else lineage-restricted precursors. Moreover, although rare cells of the adult pancreas express Hes1, it is unknown whether these represent a specialized progenitor-like population. To address these issues, we developed a mouse Hes1CreERT2 knock-in allele to inducibly mark Hes1+ cells and their descendants. We find that Hes1 expression in the early embryonic pancreas identifies multipotent, Notch-responsive progenitors, differentiation of which is blocked by activated Notch. In later embryogenesis, Hes1 marks exocrine-restricted progenitors, in which activated Notch promotes ductal differentiation. In the adult pancreas, Hes1 expression persists in rare differentiated cells, particularly terminal duct or centroacinar cells. Although we find that Hes1+ cells in the resting or injured pancreas do not behave as adult stem cells for insulin-producing beta (β)-cells, Hes1 expression does identify stem cells throughout the small and large intestine. Together, these studies clarify the roles of Notch and Hes1 in the developing and adult pancreas, and open new avenues to study Notch signaling in this and other tissues.

Ultrasonic Nanotherapy of Pancreatic Cancer: Lessons from Ultrasound Imaging

Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer death in the United States, with a median survival time of only 3-6 months for forty percent of patients. Current treatments are ineffective, and new PDA therapies are urgently needed. In this context, ultrasound-mediated chemotherapy by polymeric micelles and/or nanoemulsion/microbubble encapsulated drugs may offer an innovative approach to PDA treatment. PDA xenografts were orthotopically grown in the pancreas tails of nu/nu mice by surgical insertion of red fluorescence protein (RFP)-transfected MiaPaCa-2 cells. Tumor growth was controlled by fluorescence imaging. Occasional sonographic measurements correlated well with the formal tumor tracking by red fluorescence. Tumor accumulation of paclitaxel-loaded nanoemulsion droplets and droplet-to-bubble transition under therapeutic ultrasound was monitored by diagnostic ultrasound imaging. MiaPaCa-2 tumors manifested resistance to treatment by gemcitabine (GEM). This drug is the gold standard for PDA therapy. The GEM-resistant tumors proved sensitive to paclitaxel. Among six experimental groups studied, the strongest therapeutic effect was exerted by the following drug formulation: GEM + nanodroplet-encapsulated paclitaxel (nbGEN) combined with tumor-directed 1-MHz ultrasound that was applied for 30 s four to five hours after the systemic drug injection. Ultrasound-mediated PDA therapy by either micellar or nanoemulsion encapsulated paclitaxel resulted in substantial suppression of metastases and ascites, suggesting ultrasound-enhanced killing of invasive cancerous cells. However, tumors relapsed after the completion of therapy, indicating survival of some tumor cells. The recurrent tumors manifested development of paclitaxel resistance. Ultrasound imaging suggested nonuniform distribution of nanodroplets in the tumor volume due to irregular vascularization, which may result in the development of zones with subtherapeutic drug concentration. This is implicated as a possible cause of the resistance development, which may be pertinent to various modes of tumor nanotherapy.

Polymeric micelles and nanoemulsions as drug carriers: Therapeutic efficacy, toxicity, and drug resistance

The manuscript reports the side-by-side comparison of therapeutic properties of polymeric micelles and nanoemulsions generated from micelles. The effect of the structure of a hydrophobic block of block copolymer on the therapeutic efficacy, tumor recurrence, and development of drug resistance was studied in pancreatic tumor bearing mice. Mice were treated with paclitaxel (PTX) loaded poly(ethylene oxide)-co-polylactide micelles or corresponding perfluorocarbon nanoemulsions. Two structures of the polylactide block differing in a physical state of micelle cores or corresponding nanodroplet shells were compared. Poly(ethylene oxide)-co-poly(d,l-lactide) (PEG-PDLA) formed micelles with elastic amorphous cores while poly(ethylene oxide)-co-poly(l-lactide) (PEG-PLLA) formed micelles with solid crystalline cores. Micelles and nanoemulsions stabilized with PEG-PDLA copolymer manifested higher therapeutic efficacy than those formed with PEG-PLLA copolymer studied earlier. Better performance of PEG-PDLA micelles and nanodroplets was attributed to the elastic physical state of micelle cores (or droplet shells) allowing adequate rate of drug release via drug diffusion and/or copolymer biodegradation. The biodegradation of PEG-PDLA stabilized nanoemulsions was monitored by the ultrasonography of nanodroplets injected directly into the tumor; the PEG-PDLA stabilized nanodroplets disappeared from the injection site within 48h. In contrast, nanodroplets stabilized with PEG-PLLA copolymer were preserved at the injection site for weeks and months indicating extremely slow biodegradation of solid PLLA blocks. Multiple injections of PTX-loaded PEG-PDLA micelles or nanoemulsions to pancreatic tumor bearing mice resulted in complete tumor resolution. Two of ten tumors treated with either PEG-PDLA micellar or nanoemulsion formulation recurred after the completion of treatment but proved sensitive to the second treatment cycle indicating that drug resistance has not been developed. This is in contrast to the treatment with PEG-PLLA micelles or nanoemulsions where all resolved tumors quickly recurred after the completion of treatment and proved resistant to the repeated treatment. The prevention of drug resistance in tumors treated with PEG-PDLA stabilized formulations was attributed to the presence and preventive effect of copolymer unimers that were in equilibrium with PEG-PDLA micelles. PEG-PDLA stabilized nanoemulsions manifested lower hematological toxicity than corresponding micelles suggesting higher drug retention in circulation. Summarizing, micelles with elastic cores appear preferable to those with solid cores as drug carriers. Micelles with elastic cores and corresponding nanoemulsions both manifest high therapeutic efficacy, with nanoemulsions exerting lower systemic toxicity than micelles. The presence of a small fraction of micelles with elastic cores in nanoemulsion formulations is desirable for prevention of the development of drug resistance.

Focused ultrasound-mediated drug delivery to pancreatic cancer in a mouse model.

BackgroundMany aspects of the mechanisms involved in ultrasound-mediated therapy remain obscure. In particular, the relative roles of drug and ultrasound, the effect of the time of ultrasound application, and the effect of tissue heating are not yet clear. The current study was undertaken with the goal to clarify these aspects of the ultrasound-mediated drug delivery mechanism.MethodsFocused ultrasound-mediated drug delivery was performed under magnetic resonance imaging guidance (MRgFUS) in a pancreatic ductal adenocarcinoma (PDA) model grown subcutaneously in nu/nu mice. Paclitaxel (PTX) was used as a chemotherapeutic agent because it manifests high potency in the treatment of gemcitabine-resistant PDA. Poly(ethylene oxide)-co-poly(d,l-lactide) block copolymer stabilized perfluoro-15-crown-5-ether nanoemulsions were used as drug carriers. MRgFUS was applied at sub-ablative pressure levels in both continuous wave and pulsed modes, and only a fraction of the tumor was treated.ResultsPositive treatment effects and even complete tumor resolution were achieved by treating the tumor with MRgFUS after injection of nanodroplet encapsulated drug. The MRgFUS treatment enhanced the action of the drug presumably through enhanced tumor perfusion and blood vessel and cell membrane permeability that increased the drug supply to tumor cells. The effect of the pulsed MRgFUS treatment with PTX-loaded nanodroplets was clearly smaller than that of continuous wave MRgFUS treatment, supposedly due to significantly lower temperature increase as measured with MR thermometry and decreased extravasation. The time of the MRgFUS application after drug injection also proved to be an important factor with the best results observed when ultrasound was applied at least 6 h after the injection of drug-loaded nanodroplets. Some collateral damage was observed with particular ultrasound protocols supposedly associated with enhanced inflammation.ConclusionThis presented data suggest that there exists an optimal range of ultrasound application parameters and drug injection time. Decreased tumor growth, or complete resolution, was achieved with continuous wave ultrasound pressures below or equal to 3.1 MPa and drug injection times of at least 6 h prior to treatment. Increased acoustic pressure or ultrasound application before or shortly after drug injection gave increased tumor growth when compared to other protocols.

Defective apical extrusion signaling contributes to aggressive tumor hallmarks

When epithelia become too crowded, some cells are extruded that later die. To extrude, a cell produces the lipid, Sphingosine 1-Phosphate (S1P), which activates S1P2 receptors in neighboring cells that seamlessly squeeze the cell out of the epithelium. Here, we find that extrusion defects can contribute to carcinogenesis and tumor progression. Tumors or epithelia lacking S1P2 cannot extrude cells apically and instead form apoptotic-resistant masses, possess poor barrier function, and shift extrusion basally beneath the epithelium, providing a potential mechanism for cell invasion. Exogenous S1P2 expression is sufficient to rescue apical extrusion, cell death, and reduce orthotopic pancreatic tumors and their metastases. Focal Adhesion Kinase (FAK) inhibitor can bypass extrusion defects and could, therefore, target pancreatic, lung, and colon tumors that lack S1P2 without affecting wild-type tissue. DOI: http://dx.doi.org/10.7554/eLife.04069.001

Dietary fish oil results in a greater bone mass and bone formation indices in aged ovariectomized rats

Postmenopausal bone loss and the possible progression to osteoporosis is a major health concern. Until recently, hormone replacement therapy (HRT) was the standard for preventing the development of osteoporosis and possible hip fractures following menopause. However, because of some adverse effects of HRT, new therapies, lifestyle habits, and nutritional interventions are being developed and better characterized in their ability to prevent bone loss after menopause. One such option is to increase the amount of fish oil consumed in the diet. The goal of the current research was to determine the impact of fish oil supplementation on bone mass, density, formation, and resorption in an aged ovariectomized rat model. Twelvemonth-old female retired breeder Sprague-Dawley rats were fed a control (Control) or fish oil (Fish) diet. Two weeks following the introduction of the diets, the rats were either sham-operated (Sham) or bilaterally ovariectomized (OVX). Ten weeks after surgery, indices of bone mass and bone histomorphometry were measured. Bone mineral content (BMC) of the whole femur was significantly higher in the Fish/OVX than in the Control/OVX, and the differences were most pronounced in the distal and proximal ends of the femur. However, the Fish/Sham and the Control/Sham did not differ in the measures of BMC. Although the Control/OVX had significantly lower cortical area and greater endosteal perimeter compared with the Control/Sham, the differences were not significant between the Fish/Sham and the Fish/OVX. In addition, the Fish/OVX had a significantly larger percent double-labeled surface and mineral apposition rate at the endocortical surface than the Control/OVX. Our findings suggest that fish oil supplementation has a positive effect on bone metabolism and might be a possible intervention to slow the loss of bone observed following menopause.

Elemental and morphological identification of third-body particulate and calcium stearate inclusions in polyethylene components

Third-body particulate such as human bone chips, hydroxyapatite, and bone cement are considered contributing factors in accelerated wear in total joint replacement. Particulate wear debris is now considered the major contributing factor in aseptic loosening of total joint replacements. The ability to distinguish between different third-body particulate is necessary to better understand wear mechanisms when conducting implant retrieval analysis. The objective of this investigation is to demonstrate that backscattered electron imaging with correlated energy dispersive X-ray analysis can accurately identify third-body particulate in retrieved polyethylene components. It is important that this technique can also distinguish between third-body particulate and normal inclusions in the polyethylene such as calcium stearate, based on the distinct morphology and elemental composition of each material. Therefore, the ability to distinguish third-body particulate from calcium stearate inclusions is essential in gaining a better understanding of the contributing factors associated with coating separation and accelerated wear observed in clinically retrieved polyethylene components.