Classification of prostate cancer using a protease activity nanosensor library.

Improved biomarkers are needed for prostate cancer, as the current gold standards have poor predictive value. Tests for circulating prostate-specific antigen (PSA) levels are susceptible to various noncancer comorbidities in the prostate and do not provide prognostic information, whereas physical biopsies are invasive, must be performed repeatedly, and only sample a fraction of the prostate. Injectable biosensors may provide a new paradigm for prostate cancer biomarkers by querying the status of the prostate via a noninvasive readout. Proteases are an important class of enzymes that play a role in every hallmark of cancer; their activities could be leveraged as biomarkers. We identified a panel of prostate cancer proteases through transcriptomic and proteomic analysis. Using this panel, we developed a nanosensor library that measures protease activity in vitro using fluorescence and in vivo using urinary readouts. In xenograft mouse models, we applied this nanosensor library to classify aggressive prostate cancer and to select predictive substrates. Last, we coformulated a subset of nanosensors with integrin-targeting ligands to increase sensitivity. These targeted nanosensors robustly classified prostate cancer aggressiveness and outperformed PSA. This activity-based nanosensor library could be useful throughout clinical management of prostate cancer, with both diagnostic and prognostic utility.

Molecular Insights Into Pathogenesis of Peripheral T Cell Lymphoma: a Review.

PURPOSE OF REVIEW: Peripheral T cell lymphoma (PTCL) is a heterogeneous group of lymphoproliferative neoplasms, with at least 29 distinct entities described in current WHO classification. Using present diagnostic approaches, more than a third of PTCL cases cannot be classified, hence designated as PTCL-not otherwise specified (PTCL-NOS). Herein, we summarize the current genomic findings and their role in the molecular pathogenesis in different PTCL entities. RECENT FINDINGS: Gene expression profiling (GEP) studies have identified distinct molecular signatures for accurate diagnosis and elucidated oncogenic pathways enriched in major PTCL entities. Furthermore, genomic characterization has identified recurrent somatic mutations and potential therapeutic targets. Further efforts are underway to develop genetically faithful murine models. GEP studies have identified molecular subgroups of PTCL, characterized by distinct genetic and epigenetic alterations. Understanding the molecular mechanisms of T cell lymphomagenesis using in vivo model will help to reveal novel therapeutic targets.

The Mouse Model of Pancreatic Cancer Atlas (MMPCA) for classification of pancreatic cancer lesions: A large histological investigation of the Ptf1aCre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading forms of cancer related deaths in the United States. With limited treatment options and unreliable diagnostic methods, long-term survival rates following a diagnosis of pancreatic cancer remain poor. Pancreatic intraepithelial neoplasia (PanIN) are precancerous lesions that precede progression towards PDAC. PanIN occur in increasing complexity as the disease progresses and the description of PanIN plays a critical role in describing, staging and diagnosing PDAC. Inconsistencies in PanIN classifications exist even amongst leading pathologists. This has led to debate and confusion among researchers and pathologists involved in pancreatic cancer research, diagnosis and treatment. We have sought to initiate a discussion with leading pathologists with a goal of increasing consensus in the interpretation of PanIN and associated structures within the precancerous pancreas. Toward achieving this goal, we are in the process of conducting an extensive study of over 1000 male and female pancreata in varying stages of PanIN progression isolated from the Ptf1aCre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer. Using this extensive database, we have established the Mouse Model of Pancreatic Cancer Atlas (MMPCA) to serve as a platform for meaningful and interactive discussion among researchers and pathologists who study pancreatic disease. We hope that the MMPCA will be an effective tool for promoting a more consistent and accurate consensus of PanIN classifications in the future.

Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency.

Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a 'classical' condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system's silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the 'stratified siRNA silencing vector' requires.

Pathology, genetics and precursors of human and experimental pancreatic neoplasms: An update.

Over the past decade, there have been substantial improvements in our knowledge of pancreatic neoplasms and their precursor lesions. Extensive genetic analyses, recently using high-throughput molecular techniques and next-generation sequencing methodologies, and the development of sophisticated genetically engineered mouse models closely recapitulating human disease, have improved our understanding of the genetic basis of pancreatic neoplasms. These advances are paving the way for refined, molecular-based classifications of pancreatic neoplasms with the potential to better predict prognosis and, possibly, response to therapy. Another major development resides in the identification of subsets of pancreatic exocrine and endocrine neoplasms which occur in the context of hereditary syndromes and whose genetic basis and tumor development have been at least partially defined. However, despite all molecular progress, correct and careful morphological characterization of tissue specimens both in the context of experimental and routine diagnostic pathology represents the basis for any further genetic investigation or clinical decision. This review focuses on the current and new concepts of classification and on the current models of tumor development, both in the field of exocrine and endocrine neoplasms, and underscores the importance of applying standardized terminology to allow adequate data interpretation and promote scientific exchange in the field of pancreas research.

Adaptation of semiautomated circulating tumor cell (CTC) assays for clinical and preclinical research applications.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

[Observation on the growth and metastasis of cross-strain transplanted tumors in different mouse strains].

OBJECTIVE: Mouse tumors were subcutaneously transplanted into different mouse strains and their growth and metastatic properties were checked, to explore the possibility of establishing animal tumor models in different mouse strains other than their normal host strains. METHODS: Seven mouse tumor cell lines: H22, S180, U14, FC, Ca761, SMG-A and DCS were transplanted into C57BL/6J, ICR or KM mice, and their tumorigenicity, growth and metastasis were recorded and analyzed. RESULTS: The tumor formation rate of H22 cells in both the C57BL/6J and ICR mice was 100%, but the growth of H22 tumors was significantly faster in the C57BL/6J (2.8 ± 0.4)g than in the ICR mice (1.5 ± 0.5)g at the 17th day after transplantation (P<0.001). The S180 tumors grew stably in C57BL/6J mice and the tumor formation rate was 100%. The U14 inoculated into C57BL/6J and KM mice showed both lymphatic and lung metastasis and formed significantly larger tumors in KM mice [(12.6 ± 3.4)g] than that in the C57BL/6J mice [(10.2 ± 2.2)g] on the 32rd day after transplantation (P = 0.002). Transplantation of FC, Ca761, and SMG-A did not form tumors or the tumors were completely regressed later in C57BL/6J mice. DCS cells formed tumors in C57BL/6J mice, but some of the tumors regressed. The retained tumors were passaged in C57BL/6J mice, and the substrain DCS-C57 cells was established which showed stable growth and had a 100% tumor formation rate and 100% lung metastasis rate in C57BL/6J mice. CONCLUSIONS: Cross-strain transplanted tumors can be successfully established by inoculation of poorly differentiated and highly malignant tumor cells into different mouse strains. Some highly immunogenic tumor cells may form tumor, however, the tumors are regressed later, and can not establish cross-strain transplanted tumors in other mouse strains. Stable transplanted tumor models can be obtained from the partially regressed tumors after continuous passages in vivo.

Proliferative and nonproliferative lesions of the rat and mouse mammary, Zymbal's, preputial, and clitoral glands.

The mammary gland of laboratory rodents is an important organ for the evaluation of effects of xenobiotics, especially those that perturb hormonal homeostasis or are potentially carcinogenic. Mammary gland cancer is a leading cause of human mortality and morbidity worldwide and is a subject of major research efforts utilizing rodent models. Zymbal's, preputial, and clitoral glands are standard tissues that are evaluated in animal models that enable human risk assessment of xenobiotics. A widely accepted and utilized international harmonization of nomenclature for mammary, Zymbal's, preputial, and clitoral gland lesions in laboratory animals will improve diagnostic alignment among regulatory and scientific research organizations and enrich international exchanges of information among toxicologists and pathologists.

Carcinogenic activity of benzo[a]pyrene in a 2 year oral study in Wistar rats.

Because of the relatively high human oral exposure to polycyclic aromatic hydrocarbons (PAHs) compared to the inhalation exposure, the known carcinogenicity of this type of compounds and the limited data from oral studies available with polycyclic aromatic hydrocarbons, an oral carcinogenicity study was performed using benzo[a]pyrene (B[a]P) as a PAH representative. Wistar rats, 52 animals per sex and group were exposed daily (5 days a week) to 0, 3, 10 or 30 mg B[a]P/kg bw/day by gavage for 104 weeks and were subject to gross- and histopathology. The main tumours observed were hepatocellular carcinomas and forestomach tumours. Other tumours induced in this study were tumours of the auditory canal, skin and appendages, oral cavity, small intestine, kidney, and soft tissue sarcomas. For hepatocellular carcinomas and forestomach tumours, the BMDL10 were 3 and 1 mg/kg bw/day, respectively. The incidence of altered hepatic foci was increased in the 3mg/kg bw/day group. The increase in liver tumours is considered the most relevant effect for human risk assessment in terms of pathogenesis and sensitivity, and is proposed as the basis for human cancer risk assessment for oral PAH exposure.

Quantitative metric profiles capture three-dimensional temporospatial architecture to discriminate cellular functional states.

BACKGROUND: Computational analysis of tissue structure reveals sub-visual differences in tissue functional states by extracting quantitative signature features that establish a diagnostic profile. Incomplete and/or inaccurate profiles contribute to misdiagnosis. METHODS: In order to create more complete tissue structure profiles, we adapted our cell-graph method for extracting quantitative features from histopathology images to now capture temporospatial traits of three-dimensional collagen hydrogel cell cultures. Cell-graphs were proposed to characterize the spatial organization between the cells in tissues by exploiting graph theory wherein the nuclei of the cells constitute the nodes and the approximate adjacency of cells are represented with edges. We chose 11 different cell types representing non-tumorigenic, pre-cancerous, and malignant states from multiple tissue origins. RESULTS: We built cell-graphs from the cellular hydrogel images and computed a large set of features describing the structural characteristics captured by the graphs over time. Using three-mode tensor analysis, we identified the five most significant features (metrics) that capture the compactness, clustering, and spatial uniformity of the 3D architectural changes for each cell type throughout the time course. Importantly, four of these metrics are also the discriminative features for our histopathology data from our previous studies. CONCLUSIONS: Together, these descriptive metrics provide rigorous quantitative representations of image information that other image analysis methods do not. Examining the changes in these five metrics allowed us to easily discriminate between all 11 cell types, whereas differences from visual examination of the images are not as apparent. These results demonstrate that application of the cell-graph technique to 3D image data yields discriminative metrics that have the potential to improve the accuracy of image-based tissue profiles, and thus improve the detection and diagnosis of disease.

In vivo molecular imaging to diagnose and subtype tumors through receptor-targeted optically labeled monoclonal antibodies.

Molecular imaging of cell surface receptors can potentially diagnose tumors based on their distinct expression profiles. Using multifilter spectrally resolved optical imaging with three fluorescently labeled antibodies, we simultaneously imaged three different cell surface receptors to distinguish tumor types noninvasively. We selected tumors overexpressing different subtypes of EGFR receptor: HER-1 (A431) and HER-2 (NIH3T3/HER2(+)), or interleukin-2 receptor alpha-subunit receptor (IL-2Ralpha; SP2/Tac). After tumor establishment, a cocktail of three fluorescently labeled monoclonal antibodies was injected: cetuximab-Cy5 (targetingHER-1), trastuzumab-Cy7(HER-2),anddaclizumab-AlexaFluor-700 (IL-2Ra). Optical fluorescence imaging was performed after 24 hours with both a red filter set and three successive filter sets (yellow, red, and deep red). Spectrally resolved imaging of 10 mice clearly distinguished A431, NIH3T3/HER2(+), and SP2-Tac tumors based on their distinct optical spectra. Three-filter sets significantly increased the signal-to-background ratio compared to a single-filter set by reducing the background signal, thus significantly improving the differentiation of each of the receptors targeted (P < .022). In conclusion, following multifilter spectrally resolved imaging, different tumor types can be simultaneously distinguished and diagnosed in vivo. Multiple filter sets increase the signal-to-noise ratio by substantially reducing the background signal, and may allow more optical dyes to be resolved within the narrow limits of the near-infrared spectrum.

Induction of sarcomas by a single subcutaneous injection of 7,12-dimethylbenz[a]anthracene into neonatal male Sprague-Dawley rats: histopathological and immunohistochemical analyses.

Animal experiments have shown that carcinogenicity of chemicals is higher in fetal or neonatal periods than adult. We investigated sensitivities to a carcinogen in peri-neonatal rats with a model of sarcomas-induction by a subcutaneous injection of chemo-carcinogen that has rarely done in neonatal rats. Neonatal male SD rats were injected with 7,12-DMBA 10, 100, and 500 microg, which resulted in sarcomas-induction in 0, 62, and 94% of rats. Male SD rats were injected with DMBA 500 microg at 0, 3, 7, 14, and 21 days, which resulted in sarcomas-induction in 94, 70, 64, 50, and 44% of rats. Although the induced sarcomas were occasionally in mixed morphological feature as previous reports for sarcomas of rat, each was immunohistochemically in almost monotonous pattern, and classification was feasible. The incidence of rhabdomyosarcomas was higher in rats neonatally injected with a higher dose of DMBA than a lower dose, and in rats injected at peri-neonatal periods than later periods. In histological observations for the site of injection before overt sarcomas develop, clusters of atypical mesenchymal cells emerged as previous studies, but also those were immunohistochemically differentiated into rhabdomyocytes and other mesenchymal cells. We consider these findings may contribute a little to elucidation of process of sarcomas-induction in rats.

Cancer risk assessment of toxaphene.

The primary purpose is to do cancer risk assessment of toxaphene by using four steps of risk assessment proposed by the United States National Academy of Sciences/National Research Council (NAS/NRC). Four steps of risk assessment including hazard identification, dose-response relationship, exposure assessment, and risk characterization were used to evaluate cancer risk of toxaphene. Toxaphene was the most heavily used insecticide in many parts of the world before it was banned in 1982. It increased incidence of neoplasms of liver and uterus in mice and increased incidence of neoplasms of endocrine organs, thyroid, pituitary, adrenal, mammary glands, and reproductive systems in rats. From mice's and rats' study, slope factor for toxaphene is 0.8557 (mg/ kg/day)(-1). Lifetime average daily dose (LADD) of toxaphene from ambient air, surface water, soil, and fish were 1.08 x 10(-6), 5.71 x 10(-6), 3.43 x 10(-7), and 7.96 x 10(-5) mg/kg/day, respectively. Cancer risk of toxaphene for average exposure is 7.42 x 10(-5). From this study, toxaphene might have carcinogenic risk among humans.

Experimental cancer studies of chlorinated by-products.

Chlorinated drinking water contains a number of different by-products formed during the chlorination process from organic matter. The carcinogenicity of only a fraction of them have been evaluated in experimental animals. The focus has been on compounds and groups of compounds that are most abundant in chlorinated drinking water or the in vitro toxicity data have suggested genotoxic potential. From trihalomethanes, chloroform causes liver tumors in mice and female rats and renal tumors in male mice and rats. Tumor formation by chloroform is strongly associated with cytotoxicity and regenerative cell proliferation in tissues and that has been considered to be one determinant of its carcinogenicity. From halogenic acetic acids, dichloroacetic acid (DCA) and trichlotoacetic acid (TCA) are hepatocarcinogenic in mice and DCA in male rats. Their genotoxicity is equivocal and nongenotoxic mechanisms, such as peroxisome proliferation and hypomethylation of DNA in the liver, likely contribute to tumor development. From chlorinated furanones (CHFs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a multisite carcinogen in rats (e.g. in thyroid glands and liver) and it has caused DNA damage in vivo. MX may be a complete carcinogen because it also has promoter properties in vitro. Chlorinated drinking water may also contain brominated by-products providing the raw water contains bromide. At least some of them (bromodichloromethane, bromoform) have been shown to be carcinogenic in laboratory animals. Altogether, although several by-products have been shown to have carcinogenic potential in laboratory animals, it not yet possible to state which compounds or groups of by-products cause the cancer risk in chlorinated drinking water. The cellular mechanisms of their effects and these effects at low concentrations are still poorly understood. The few studies with mixtures of these by-products suggest that the mixture effects may be complex and unpredictable (inhibitory, additive, synergistic).

Life span and spontaneous tumors incidence of the Wistar Mishima (WM/MsNrs) rat.

The life spans and spontaneous tumors in a total of 1960 Wistar Mishima (WM/MsNrs) rats, inbred strain, from the 80-130th generations were examined. The average life span (mean +/- SD) was 731 +/- 173 days (n = 1053) in the males and 813 +/- 214 days (n = 907) in the females (p < 0.0001). The average life span of tumor-afflicted females was significantly longer than that of the non-tumor group (p < 0.0001), while no such difference was observed in males. Tumors were observed in 33 males (3.1%) and 246 females (27.1%). In the males, tumors were often observed under the skin (2.2%). Frequencies of tumors in lung and liver, bones and intestine were less than 0.5%. In the females, incidence of mammary tumor was 20.1%, and various organs such as ovaries, uterus, bones, lung, and liver had tumor incidence frequencies of less than 3.5%. It was concluded that WM/MsNrs rats might be suitable for life span and age-related studies because of their characteristics of length of longevity and the low incidence of spontaneous tumors in both sexes.

The effect of various chemotherapeutic agents given with mild hyperthermia on different types of tumours.

It has been shown that hyperthermia can enhance the cytotoxicity of some chemotherapeutics. However, the most effective agent(s) at elevated temperatures have yet to be determined. A previous study suggests that the drug of choice at elevated temperatures may be different from that at the physiological temperature, and that the alkylating agents may be most effective at elevated temperatures. To further investigate these possibilities, the effect of chemotherapeutic agents were compared. These agents were cyclophosphamide, ifosfamide, melphalan, cis-diamminedichloroplatinum (II), 5-fluorouracil, mitomycin C and bleomycin. Three tumours (mammary carcinoma, osteosarcoma and squamous cell carcinoma) were used. They were transplanted into the feet of C3H/He mice. When tumours reached 65 mm(3), a test agent was injected intraperitoneally. Tumours were immediately heated at 41.5 degrees C for 30 min, and the tumour growth (TG) time was studied for each tumour. Using the TG times, the TG-50 (the time required for one-half of the total number of the treated tumours to reach the volume of 800 mm(3) from 65 mm(3)) was calculated. Subsequently, the tumour growth delay time (GDT) and the thermal enhancement ratio (TER) were obtained. The GDT was the difference between the TG-50 of treated tumours and that of non-treated control tumours. The TER was the ratio of the GDT of a group treated with an agent at 41.5 degrees C to that of a group treated with the agent at room temperature. Results showed that the top three effective agents tested at 41.5 degrees C were solely alkylating agents--CY, IFO and L-PAM--for each kind of tumour. A GDT of cisplatin was smaller than those of the alkylating agents. The smallest TER, 1.1, was observed for 5-fluorouracil, which was given for mammary carcinoma, and for mitomycin C, which was given for squamous cell carcinoma. It could be concluded that the alkylating agents at elevated temperatures might be the drugs of choice for many types of tumours. The possible mechanisms of thermal enhancement associated with these agents are discussed.

Results of long-term experimental studies on the carcinogenicity of methyl alcohol and ethyl alcohol in rats.

Methyl alcohol was administered in drinking water supplied ad libitum at doses of 20,000, 5,000, 500, or 0 ppm to groups of male and female Sprague-Dawley rats 8 weeks old at the start of the experiment. Animals were kept under observation until spontaneous death. Ethyl alcohol was administered by ingestion in drinking water at a concentration of 10% or 0% supplied ad libitum to groups of male and female Sprague-Dawley rats; breeders and offspring were included in the experiment. Treatment started at 39 weeks of age (breeders), 7 days before mating, or from embryo life (offspring) and lasted until their spontaneous death. Under tested experimental conditions, methyl alcohol and ethyl alcohol were demonstrated to be carcinogenic for various organs and tissues. They must also be considered multipotential carcinogenic agents. In addition to causing other tumors, ethyl alcohol induced malignant tumors of the oral cavity, tongue, and lips. These sites have been shown to be target organs in man by epidemiologic studies.

Results of long-term carcinogenicity bioassays on tert-amyl-methyl-ether (TAME) and di-isopropyl-ether (DIPE) in rats.

Tert-amyl-methyl ether (TAME) was administered by gavage in extra virgin olive oil solution at concentrations of 750, 250, or 0 mg/kg bw to groups of 100 male and 100 female Sprague-Dawley rats 8 weeks old at the start of the experiment. Di-isopropyl ether (DIPE) was administered in the same manner at the doses of 1000, 250, or 0 mg/kg body weight to groups of 100 male and 100 female Sprague-Dawley rats. TAME and DIPE were each delivered in 1-mL solution 4 days a week for 78 weeks. Control animals received 1 mL of extra virgin olive oil without TAME or DIPE. At the end of the treatment period, all animals were kept under observation until spontaneous death. Under these test conditions, TAME and DIPE were found to be potential carcinogenic agents for various organs and tissues.

Results of long-term experimental studies on the carcinogenicity of ethylene-bis-dithiocarbamate (Mancozeb) in rats.

Mancozeb, an ethylene-bis-dithiocarbamate (EBDC), has been one of the most commonly used fungicides in commercial use for several decades. Nevertheless, up to now, no adequate published experimental studies on the carcinogenicity of Mancozeb have been published. Because of the importance of the compound and of the number of people potentially exposed (workers engaged in the production and use of the fungicide, people living in agricultural areas where the compound is sprayed, and people consuming polluted products), a long-term experimental study of Mancozeb was begun at the Cancer Research Center of the Ramazzini Foundation. Groups of 150 male and female Sprague-Dawley rats, 8 weeks old at the start of the treatment, were administered Mancozeb at the concentration of 1000, 500, 100, 10, and 0 ppm in feed supplied ad libitum for 104 weeks. At the end of the treatment, animals were kept under controlled conditions until spontaneous death. Mancozeb caused an increase in (1) total malignant tumors, (2) malignant mammary tumors, (3) Zymbal gland and ear duct carcinomas, (4) hepatocarcinomas, (5) malignant tumors of the pancreas, (6) malignant tumors of the thyroid gland, (7) osteosarcomas of the bones of the head, and (8) hemolymphoreticular neoplasias. On the basis of these data, Mancozeb must be considered a multipotent carcinogenic agent.