Heterogeneity of Pancreatic Cancer Metastases in a Single Patient Revealed by Quantitative Proteomics

Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the administration of a combination of agents, with each agent targeted to the features of different subclones.Approximately half of the patients with pancreatic cancer are initially diagnosed with metastases to distal sites, with the commonest sites being the liver, lung, and peritoneum (1). Therapeutic strategies against metastases could help reduce the high mortality rates associated with this cancer (2). Understanding the nature of metastatic pancreatic cancer at a systems level can enable the discovery of potential targets for the development of targeted therapies.Pancreatic cancer has been shown to be a genetically evolving and heterogeneous disease (3–5). Clonal diversity and evolution of cancer genomes have also been demonstrated based on the isolation of distinct clonal populations purified directly from patient biopsies by means of flow cytometry followed by genomic characterization (6). A number of reports have documented the adoption of a proteomic approach for the discovery of potential biomarkers in pancreatic cancer (7, 8). However, these studies generally assume pancreatic cancers to be homogeneous, and the emphasis is placed on identifying molecules that are common across a broad array of tumors. There is a lack of studies systematically examining the proteomic changes or signaling pathways across pancreatic cancers to dissect the nature of the heterogeneity of each clone. An excellent setting in which the heterogeneity of tumors can be studied systematically is in a patient harboring metastases to several distant sites. To this end, we chose cells isolated from three metastatic pancreatic lesions of a single patient. The exomes of each tumor site were previously sequenced to study the progression of pancreatic cancer, and the results showed that all cell lines were identical for the genetic status of driver mutations (e.g. KRAS, TP53, and SMAD4) (9). Our hypothesis was that a better understanding of the proteomic consequences of the heterogeneity derived from genetic changes, and possibly other types of alterations, might provide additional opportunities to identify therapeutic targets.In order to precisely quantify differences across the proteomes of multiple metastatic pancreatic cancer lesions, we employed a SILAC-based¹ quantitative proteomics strategy combined with high-resolution mass spectrometry (10, 11). Based on changes observed at the whole-proteome level, we found that a class of cell surface receptors showed significant enrichment with the highest alteration of their expression among the three metastatic pancreatic cancer cell lines examined (i.e. peritoneum, lung, and liver). Because the total protein levels provide information about the static levels of proteins and not their activity per se, we decided to examine the activation of phosphorylation-driven pathways, many of which are activated by cell surface receptors. To globally examine tyrosine phosphorylation-based signaling pathways, we carried out mass spectrometric analysis of purified tyrosine phosphorylated peptides enriched using anti-phosphotyrosine antibodies. As a result, we observed differential activation of tyrosine kinases in the three different sites of metastases. For example, Axl receptor tyrosine kinase was found to be hyperphosphorylated in lung and liver metastases relative to peritoneal metastasis. Expression of Axl receptor tyrosine kinase in primary and matched pancreatic cancers on tissue microarrays was validated by immunohistochemistry. Given such unique patterns of activation of pathways, it was possible that tumors derived from different sites could show differences in their sensitivity to pathway inhibitors. To test this, we performed experiments in which we screened cell lines derived from each metastatic site against a panel of small molecule inhibitors. We observed that the three metastatic pancreatic cancers had differential sensitivities to different inhibitors. For example, cells derived from the peritoneal metastasis were highly sensitive to lapatinib, whereas greater sensitivity to the Axl inhibitor R428 was observed in the lung metastasis cell line. Finally, we showed that treatment of mice bearing xenografts from these different pancreatic cancer cell lines with R428, an inhibitor of Axl receptor tyrosine kinase, led to reduction of tumors with evidence of activation of Axl.     

Adaptive Evolution and Environmental Durability Jointly Structure Phylodynamic Patterns in Avian Influenza Viruses

Avian influenza viruses (AIVs) have been pivotal to the origination of human pandemic strains. Despite their scientific and public health significance, however, there remains much to be understood about the ecology and evolution of AIVs in wild birds, where major pools of genetic diversity are generated and maintained. Here, we present comparative phylodynamic analyses of human and AIVs in North America, demonstrating (i) significantly higher standing genetic diversity and (ii) phylogenetic trees with a weaker signature of immune escape in AIVs than in human viruses. To explain these differences, we performed statistical analyses to quantify the relative contribution of several potential explanations. We found that HA genetic diversity in avian viruses is determined by a combination of factors, predominantly subtype-specific differences in host immune selective pressure and the ecology of transmission (in particular, the durability of subtypes in aquatic environments). Extending this analysis using a computational model demonstrated that virus durability may lead to long-term, indirect chains of transmission that, when coupled with a short host lifespan, can generate and maintain the observed high levels of genetic diversity. Further evidence in support of this novel finding was found by demonstrating an association between subtype-specific environmental durability and predicted phylogenetic signatures: genetic diversity, variation in phylogenetic tree branch lengths, and tree height. The conclusion that environmental transmission plays an important role in the evolutionary biology of avian influenza viruses—a manifestation of the “storage effect”—highlights the potentially unpredictable impact of wildlife reservoirs for future human pandemics and the need for improved understanding of the natural ecology of these viruses.     

ARTIST: High-Resolution Genome-Wide Assessment of Fitness Using Transposon-Insertion Sequencing

Transposon-insertion sequencing (TIS) is a powerful approach for deciphering genetic requirements for bacterial growth in different conditions, as it enables simultaneous genome-wide analysis of the fitness of thousands of mutants. However, current methods for comparative analysis of TIS data do not adjust for stochastic experimental variation between datasets and are limited to interrogation of annotated genomic elements. Here, we present ARTIST, an accessible TIS analysis pipeline for identifying essential regions that are required for growth under optimal conditions as well as conditionally essential loci that participate in survival only under specific conditions. ARTIST uses simulation-based normalization to model and compensate for experimental noise, and thereby enhances the statistical power in conditional TIS analyses. ARTIST also employs a novel adaptation of the hidden Markov model to generate statistically robust, high-resolution, annotation-independent maps of fitness-linked loci across the entire genome. Using ARTIST, we sensitively and comprehensively define Mycobacterium tuberculosis and Vibrio cholerae loci required for host infection while limiting inclusion of false positive loci. ARTIST is applicable to a broad range of organisms and will facilitate TIS-based dissection of pathways required for microbial growth and survival under a multitude of conditions.     

Recent divergences and size decreases of eastern gorilla populations

Compared with other African apes, eastern gorillas (Gorilla beringei) have been little studied genetically. We used analysis of autosomal DNA genotypes obtained from non-invasively collected faecal samples to estimate the evolutionary histories of the two extant mountain gorilla populations and the closely related eastern lowland gorillas. Our results suggest that eastern lowland gorillas and mountain gorillas split beginning some 10 000 years ago, followed 5000 years ago by the split of the two mountain gorilla populations of Bwindi Impenetrable National Park and the Virungas Massif. All three populations have decreased in effective population size, with particularly substantial 10-fold decreases for the mountain gorillas. These dynamics probably reflect responses to habitat changes resulting from climate fluctuations over the past 20 000 years as well as increasing human influence in this densely populated region in the last several thousand years.     

Molecular analysis reveals lowbush blueberry pest predation rates depend on ground beetle (Coleoptera: Carabidae) species and pest density

Molecular gut-content analysis allows determination of pest predation by field-collected predators. Ground beetles (Coleoptera: Carabidae) common in lowbush blueberries may consume blueberry spanworm, Itame argillacearia (Packard) (Lepidoptera: Geometridae), and blueberry flea beetle, Altica sylvia Malloch (Coleoptera: Chrysomelidae), providing pest suppression. Using newly developed pest specific primers, laboratory feeding trials showed that the median detection time (MDT) for blueberry spanworm in the largest beetle, Carabus nemoralis O.F. Müller, was 3.7 h, whereas Poecilus lucublandus (Say) and Pterostichus mutus (Say) had MDTs between 27.1 and 31.6 h for both pests. At a field-site with high pest abundances, the probability of detecting blueberry spanworm and blueberry flea beetle DNA was greater in P. lucublandus, 26 and 39 % respectively, than in P.mutus, 8 and 20 % respectively. Only 0 and 1 % of P. lucublandus and P. mutus, respectively, tested positive for blueberry spanworm DNA at a second site with low abundance. At the first site, the probability of detecting pest DNA in both ground beetle species was positively related to pest density. Higher pest DNA detection rates and captures of ground beetles corresponded to field areas where significant pest reductions occurred from late May to early June. Conservation of predatory carabid beetles could lead to valuable biological control in lowbush blueberries.     

Analysis of stable states in global savannas: is the CART pulling the horse?

Multiple stable states, bifurcations and thresholds are fashionable concepts in the ecological literature, a recognition that complex ecosystems may at times exhibit the interesting dynamic behaviours predicted by relatively simple biomathematical models. Recently, several papers in Global Ecology and Biogeography, Proceedings of the National Academy of Sciences USA, Science and elsewhere have attempted to quantify the prevalence of alternate stable states in the savannas of Africa, Australia and South America, and the tundra–taiga–grassland transitions of the circum‐boreal region using satellite‐derived woody canopy cover. While we agree with the logic that basins of attraction can be inferred from the relative frequencies of ecosystem states observed in space and time, we caution that the statistical methodologies underlying the satellite product used in these studies may confound our ability to infer the presence of multiple stable states. We demonstrate this point using a uniformly distributed ‘pseudo‐tree cover’ database for Africa that we use to retrace the steps involved in creation of the satellite tree‐cover product and subsequent analysis. We show how classification and regression tree (CART)‐based products may impose discontinuities in satellite tree‐cover estimates even when such discontinuities are not present in reality. As regional and global remote sensing and geospatial data become more easily accessible for ecological studies, we recommend careful consideration of how error distributions in remote sensing products may interact with the data needs and theoretical expectations of the ecological process under study.     

Glass Delamination: a Comparison of the Inner Surface Performance of Vials and Pre-filled Syringes

The occurrence of glass delamination is a serious concern for parenteral drug products. Over the past several years, there has been a series of product recalls involving glass delamination in parenteral drugs stored in vials which has led to heightened industry and regulatory scrutiny. In this study, a two-pronged approach was employed to assess the inner surface durability of vials and pre-filled syringes. Non-siliconized syringes were used in order to directly compare glass to glass performance between vials and syringes. The vial and syringe performance was screened with pharmaceutically relevant formulation conditions. The influence of pH, buffer type, ionic strength, and glass type and source was evaluated. In addition, an aggressive but discriminating formulation condition (glutaric acid, pH 11) was used to ascertain the impact of syringe processing. Advanced analytical tools including inductively coupled plasma/mass spectrometry, scanning electron microscopy, atomic force microscopy, and dynamic secondary ion mass spectroscopy showed significant differences in glass performance between vials and syringes. Pre-filled syringes outperform vials for most tests and conditions. The manufacturing conditions for vials lead to glass defects, not found in pre-filled syringes, which result in a less chemically resistant surface. The screening methodology presented in this work can be applied to assess suitability of primary containers for specific drug applications.Key words: borosilicate vials, glass delamination, glass corrosion, hydrolytic resistance, pre-filled syringes     

A Simple,Quantitative Method Using Alginate Gel to Determine Rat Colonic Tumor Volume In Vivo

Many studies of the response of colonic tumors to therapeutics use tumor multiplicity as the endpoint to determine the effectiveness of the agent. These studies can be greatly enhanced by accurate measurements of tumor volume. Here we present a quantitative method to easily and accurately determine colonic tumor volume. This approach uses a biocompatible alginate to create a negative mold of a tumor-bearing colon; this mold is then used to make positive casts of dental stone that replicate the shape of each original tumor. The weight of the dental stone cast correlates highly with the weight of the dissected tumors. After refinement of the technique, overall error in tumor volume was 16.9% ± 7.9% and includes error from both the alginate and dental stone procedures. Because this technique is limited to molding of tumors in the colon, we utilized the Apc^Pirc/+ rat, which has a propensity for developing colonic tumors that reflect the location of the majority of human intestinal tumors. We have successfully used the described method to determine tumor volumes ranging from 4 to 196 mm³. Alginate molding combined with dental stone casting is a facile method for determining tumor volume in vivo without costly equipment or knowledge of analytic software. This broadly accessible method creates the opportunity to objectively study colonic tumors over time in living animals in conjunction with other experiments and without transferring animals from the facility where they are maintained.Colon cancer is the third leading cause of cancer in men and women, with more than 100,000 new cases diagnosed each year in the United States alone. This disease is not limited to humans—cancers of the colon and rectum also affect companion species, such as dogs, albeit less frequently than in humans.²⁰ Colorectal cancers generally develop from precancerous polyps, which can be detected and removed during colonoscopy screening before they become invasive cancers. However, not all precancers will become cancerous,²³ and a better understanding of early tumor growth dynamics in models of the disease can simultaneously increase the rate of detection of polyps destined to become cancerous and decrease the rate of unnecessary removal of benign polyps.Sizing of tumors creates an additional dimension beyond studies examining tumor multiplicity alone. Terminal sizing of tumors uses an eyepiece reticule under a dissection microscope to measure the maximal diameter of each tumor. However, this method likely misrepresents tumor volume for several reasons. First, tumors often are not symmetrical in shape, thereby limiting the interpretation of even multiple linear measurements. When volume calculations rely on the use of a formula, the irregular shape of solid tumors may require the testing of many different formulas to find the optimal one for that particular measurement and model.⁸ Second, if tumor sizing occurs after fixation, the original shape of the tumor can be affected. However, when tumor sizing occurs before fixation, the added time to size the tumors may result in degradation of the intestinal tissue, limiting further analysis. An alternate method of tumor sizing involves using the surrogate of tumor weight, the current ‘gold standard,’ for terminal studies. Tumor weight correlates closely with tumor size, although tumor density may vary depending on the tumor type. In addition, this technique is limited to use at the terminal time point. Methods that determine true tumor volume are powerful; those that can be applied in vivo to study the tumor longitudinally are even more compelling.It recently has been recognized that not all early colonic tumors grow; some remain static for years whereas a few spontaneously regress.²³ Importantly, the early growth profile of a tumor may correlate with its eventual fate.²³ This aspect of tumor biology is a newly emerging area that deserves deeper study. The current gold standard for determining longitudinal tumor volume is CT, given that tumor weight is available only through terminal experiments. In mice, microCT colonography can be used to detect a 16% change in tumor volume with 95% confidence in living animals.⁵ However, the cost of CT equipment limits this technology to shared facilities, and the pathogen status of these facilities may preclude returning animals to the place where they were original housed, limiting the opportunities for longitudinal study. Importantly, many institutions do not have access to microCT technology, and even if available, 3D renderings must be recreated to determine tumor volume, a process requiring specialized software and detailed computing knowledge. Furthermore, CT exposes animal subjects to radiation, which may interfere with the tumor biology. Although MRI can be used to determine tumor volume accurately in the absence of ionizing radiation, specialized scanners and software are required, and enemas or intravenous treatments are needed to visualize tumors clearly.²⁶Another imaging modality uses the surface area of signal due to proteins expressing a fluorescent marker, such as red fluorescent protein, as a surrogate for tumor volume.¹⁷ However, tumor volume measured by fluorescent surface area¹² may not accurately represent tumor volume in irregularly shaped tumors. In addition, this method necessitates a surgical procedure to orthotopically transplant fluorophore-expressing cells, raising questions of immune interactions between the recipient animal and the donor cells or to the surgery itself. If nude or immunocompromised animals are used in the procedure, the ability to study the immune aspect of tumor biology is reduced or eliminated.Alternatively, tumor volume can be estimated from endoscopic images. The study of tumors by colonoscopy has become routine for both mouse^6,10 and rat^1,15 models of the disease. In contrast to terminal assessments, colonoscopy allows tumors to be visualized in vivo over time, capturing the dynamics of tumor growth. Documentation of this aspect of tumor biology can greatly enrich studies evaluating chemopreventive or therapeutic agents.^6,15 Quantitative methods for determining tumor volume take this benefit a step further, allowing the investigation of the effects of background strain, therapeutic agents, environmental factors, or other modifiers of tumor growth pattern. One method to estimate tumor size uses the fraction of luminal cross-section occluded by tumor.² However, the colonic lumen expands as the animal grows, and its size often increases to accommodate the growing tumor, to prevent intestinal blockage. Optical methods to extrapolate tumor sizes from 2D images obtained in vivo during colonoscopy are achieved by inserting a flexible metal rod of known dimensions into the working channel of the endoscope.¹⁰ However, because colonic tumors can differ in shape (some are flat whereas others are pedunculated), area measurements may not translate accurately to tumor volume.To overcome these limitations and to add another tool to the growing cancer-research toolbox, we have developed a method using a biologically inert alginate to create negative molds of colonic tumors. These molds are filled with dental stone to achieve a positive cast of each tumor. A conversion factor then is used to calculate the volume of the original tumor from the dry weight of the dental stone cast. This procedure, which requires no specialized or expensive equipment and no complicated analytical methods, can be performed within the facility where the rats are housed and takes less than 15 min, including the 8 to 12 min during which the alginate sets. Therefore, our new method offers possibilities to study the dynamics of tumor growth in virtually any animal facility, regardless of the health status of subject animals or equipment availability.