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.

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.

[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.

Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo.

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.

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.

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.

Immunomorphologic classification of spontaneous lymphoid cell neoplasms occurring in female BALB/c mice.

WIth the use of a recently proposed, combined immunomorphologic classification (Pattengale-Taylor, 1981) for murine lymphomas and related leukemias, 70 spontaneously occurring lymphoid cell neoplasms from female BALB/cStCrl mice were evaluated and classified and then compared to the earlier Dunn classification (Dunn, 1954). The predominant lesion (i.e., 60% total incidence) in female BALB/c mice [previously called "reticulum cell sarcomas (neoplasm)", type B, by Dunn] was a lymphoid cell neoplasm derived from follicular center cells (i.e., follicular center cell [FCC] lymphoma). The B-cell nature was further confirmed in the majority (76%) of these FCC lymphomas by means of immunoperoxidase techniques that demonstrated the presence of cytoplasmic immunoglobulin (Clg). Smaller percentages of B-lymphoblastic (10% total incidence) and B-immunoblastic (7% total incidence) lymphomas were also observed. In addition, FCC, B-lymphoblastic, and B-immunoblastic lymphomas occurred in female BALB/c mice over 20 months of age. In contrast, Clg-negative lymphoblastic lymphomas involving the anterior mediastinum (and presumably T-cell in origin) occurred with a bimodal incidence in both young (13% total incidence at a mean age less than 6 mo) and old (10% total incidence at a mean age greater than 21 mo) female mice.

Alloantigenic phenotype of radiation-induced thymomas in the mouse.

The alloantigenic phenotype of 21 radiation-induced thymomas is described. Monoclonal antibodies and conventional antisera, absorbed to remove contaminating antibodies, were used to test the thymomas directly for the presence of H-2, Ia, Ly-1, Ly-2, Ly-4, Ly-5, Ly-6, Ly-9, Ly-15, Thy-1, TL, and Qa-2 antigens, and for surface immunoglobulin. The phenotypes obtained by direct tests were also confirmed by absorption studies. The tumors were all of T-cell origin (Thy-1+Ig-) and showed a restricted heterogeneity of their cell surface phenotype, concordant with the known alloantigenic phenotypes of functional T-cells. Thus the thymomas could be classified into 7 groups based on the differing expressions of Ia, Ly-1, Ly-2, Ly-4, and Ly-6 specificities; all tumors were H-2+, Ly-5+, Ly-9+, and Ly-15+. Thus a wide variety of phenotypically diverse tumors could be detected; if these represent the clonal expansion of different functional subsets, they could provide a valuable set of functional T-cells.

A classification of transplantable tumors in Nb rats controlled by estrogen from dormancy to autonomy.

Transplantable tumor lines were previously established from a variety of estrogen-induced tumors in Nb rats, including tumors of the adrenal cervix, salivary gland, and pancreas, a lymphoma, and a liposarcoma. Spontaneous tumors, however, were found to arise in untreated females and showed the same characteristics. Tumor growth was dependent upon or influenced by estrogen when assessed in estrogenized and unconditioned hosts. Intermitten estrogenization was effective, but tumor growth took place more slowly. The type of response observed led to a new classification of five types of hormone-responsive tumors including tumors inhibited by estrogen. Estrogen-dependent tumor cells might remain dormant indefinitely and not grow in unconditioned animals until stimulated to grow by estrogen. The growth rate of hormone-dependent adrenal carcinomas was related to the amount of estrogen. Tumor growth started more rapidly in the presence of low estrogen dose levels in old rats used as hosts than it did in young rats. Breast carcinomas required the largest amount of estrogen for growth, whereas ovarian thecomas would grow in normal females but not in males. The growth rate in conditioned hosts of most transplanted tumors (some have maintained hormone dependency over 10 years) increased over successive generations. Progression, however, towards a more autonomous state after repeated transplantations was remarkably slow, and a sudden change to autonomy was rarely noted. In contrast, transplants of 9,12-dimethylbenz(a)anthracene-induced mammary carcinomas progressed rapidly to autonomy. Fould's concept of progression (2, 3) has been discussed but the described classification of tumors under hormone influence apparently allows a more detailed analysis of definition of different types of progression.

Morphological classification of mouse liver tumors based on biological characteristics.

Examination of three strains of inbred mice suggested that specific morphological types of hepatic tumors are the result of genetic predisposition while other types of tumors are associated with exposure to chemical carcinogens. A simple classification system is proposed. Additional studies indicated that a putative promoting agent, such as phenobarbital, increased the incidence of those tumor types usually associated with spontaneous appearance and only in strains with a genetic predisposition to spontaneous tumorigenesis. Carcinogens induce an increase in all types of tumors.

Classification of animal lymphomas: the implications of applying Rappaport's classification for human lymphomas to experimental tumors.

One hundred and seventy animal lymphomas (species ranging from molluses to monkeys) were reclassified histologically according to the modified Rappaport classification for human lymphomas. The results were correlated with the etiology of the lymphomas, their clinical course, and in selected cases with their immunological type. The study stresses the value of such a procedure for comparative reasons, allowing a more adequate selection of animal models for human lymphomas.

Classification of malignant lymphomas of the mouse using morphological, immunological, and cytochemical methods: a working proposal.

We review techniques used to classify malignant lymphomas of the laboratory mouse. Besides an initial morphologic classification according to the Rappaport scheme for human lymphomas, individual tumor types were further subclassified by use of immunocytological T- and B-cell determinations as well as by histochemical procedures. The results, which allow a certain appraisal of the maturation stage of lymphoma cells, are discussed taking as examples six different experimental mouse lymphomas.

Differentiation by TICAS analysis of cell populations of tracheal aspirates from hamsters with squamous-cell carcinoma.

Individual cells from the tracheal aspirates of hamsters exposed to benzo-a-pyrene were scanned at .5 mum in three colors. Features relating to size, shape, and color were extracted and calculated by computer. The single cells were then classified by these features into separate populations with varying degrees of atypia, extending up to frank cancer cells. A high degree of accuracy was attained in classification by these methods.

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.

Carcinogenicity of methyl-tertiary butyl ether in gasoline.

Methyl tertiary butyl ether (MTBE) was added to gasoline on a nationwide scale in 1992 without prior testing of adverse, toxic, or carcinogenic effects. Since that time, numerous reports have appeared describing adverse health effects of individuals exposed to MTBE, both from inhalation of fumes in the workplace and while pumping gasoline. Leakage of MTBE, a highly water-soluble compound, from underground storage tanks has led to contamination of the water supply in many areas of the United States. Legislation has been passed by many states to prohibit the addition of MTBE to gasoline. The addition of MTBE to gasoline has not accomplished its stated goal of decreasing air pollution, and it has posed serious health risks to a large portion of the population, particularly the elderly and those with respiratory problems, asthma, and skin sensitivity. Reports of animal studies of carcinogenicity of MTBE began to appear in the 1990s, prior to the widespread introduction of MTBE into gasoline. These reports were largely ignored. In ensuing years, further studies have shown that MTBE causes various types of malignant tumors in mice and rats. The National Toxicology Program (NTP) Board of Scientific Counselors' Report on Carcinogens Subcommittee met in December 1998 to consider listing MTBE as "reasonably anticipated to be a human carcinogen." In spite of recommendations from Dr. Bailer, the primary reviewer, and other scientists on the committee, the motion to list MTBE in the report was defeated by a six to five vote, with one abstention. On the basis of animal studies, it is widely accepted that if a chemical is carcinogenic in appropriate laboratory animal test systems, it must be treated as though it were carcinogenic in humans. In the face of compelling evidence, NTP Committee members who voted not to list MTBE as "reasonably anticipated to be a human carcinogen" did a disservice to the general public; this action may cause needless exposure of many to health risks and possibly cancers.