Protein Signature of Lung Cancer Tissues

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment

Assessing the oncological safety of autologous fat grafting for breast cancer

Orientadores: Aarão Mendes Pinto Neto, Luís Otávio Zanatta SarianTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Introdução: A lipoenxertia para reconstrução da mama é uma técnica efetiva, de baixa complexidade e baixo custo. Entretanto, a associação entre obesidade e câncer de mama, além de evidências laboratoriais de que as adipocinas podem estimular a proliferação celular e a diferenciação de células-tronco mesenquimais (ASC), suscitam dúvidas sobre a segurança oncológica do procedimento para a prática clínica. Objetivo: Avaliar o risco oncológico da lipoenxertia autóloga para o câncer de mama. Métodos: Esta tese trata de quatro estudos interligados. No primeiro, foi desenvolvido um modelo experimental para o estudo da lipoenxertia em ratas da raça Sprague-Dawley avaliando características adaptativas do tecido adiposo de omento pediculado por meio de avaliação macroscópica, histológica e morfologia de adipócitos. No segundo, este modelo foi aplicado para comparar o potencial oncogênico do tecido adiposo exposto a dieta hipercalórica com o procedimento de lipoenxertia, através de marcadores de inflamação crônica (CD68 em macrófagos), proliferação celular (Ki67) e níveis de PAI-1. No terceiro estudo comparamos mamas que receberam lipoenxertia de omento, de subcutâneo e as que não foram manipuladas, através de avaliação histológica, imunoistoquímica e PCR-RT de CD68, Ki67, PAI-1 e receptores de estrógeno (ER, marcador 1D5). O quarto estudo foi uma revisão sistemática da literatura sobre a utilização de tecido adiposo do omento para o tratamento das afecções mamárias. Resultados: No primeiro estudo, o tecido adiposo do omento pediculado sofreu processo adaptativo resultante de alterações no seu interstício, sem alteração morfológica dos adipócitos (p=0,27) e sem atipias celulares quando estimulado pela translocação. No segundo estudo, o tecido adiposo não manipulado exposto à dieta hipercalórica produziu proliferação celular (representada por elevação do Ki67, p=0,046) e elevação dos níveis de PAI-1 (p0,05. Na revisão sistemática foram identificados 60 estudos que envolveram 985 mulheres. Oito estudos analisaram o risco oncológico para recidiva de câncer de mama, sendo que em sete a enxertia foi realizada em estádio avançado e a recorrência local foi de 35,5% (143/403). Um destes estudos avaliou pacientes com câncer em estádio inicial (89 mulheres) e nenhuma recidiva foi identificada em follow-up médio superior a cinco anos após a reconstrução. Conclusão: Pelos estudos experimentais in vivo, identificamos o potencial oncogênico dos adipócitos nativos sem manipulação quando expostos a dieta hipercalórica. Entretanto, o tecido adiposo enxertado ou as células do sitio receptor (incluindo as células ductais) não foram capazes de mostrar qualquer alteração potencialmente oncogênica, quando analisado o processo de enxertia isoladamente. Nossos achados experimentais estão perfeitamente alinhados com as conclusões da literatura para lipoenxertia e de nossa revisão sistemática de estudos clínicos em tecido adiposo de omento, os quais sustentam a segurança oncológica da lipoenxertia para reconstrução da mamaAbstract: Introduction: Lipofilling for breast reconstruction is an effective procedure, with low complexity and low cost. However, its oncological safety was questioned when obesity was identified as a risk factor for breast cancer along with some in vitro and preclinical studies that identified the oncogenic potential of adipocytes to cellular proliferation and differentiation of adipose stromal cells (ASC). Objective: To assess of the oncological safety of autologous fat grafting for breast cancer. Method: The present research is composed by four studies. In the first one, it was proposed an experimental model for lipofilling using adipose tissue of pedicle omental flap in Sprague-Dawley rats to assess its adaptative properties through histopathological analyses and adipocyte morphology. In the second, this model was used to compare the oncogenic potential of samples of native unmanipulated adipose tissue when exposed to high-energy diet to samples of fat exposed only to the lipofilling procedure through markers of chronic inflammation (CD68 in macrophages), cellular proliferation (Ki67), and levels of PAI-1. In the third, lipofilled breasts (grafted with subcutaneous or omental fat tissue) were compared with unmanipulated breasts (control) through histopathological analyses, immunohistochemical and PCR-RT tests of CD68, Ki67, PAI-1 and 1D5 markers. The forth study was a systematic review of treatment of breast disorders with adipose tissue of pedicle omental flap. Results: In the first study, the adipose tissue of transposed pedicle omental flap underwent an adaptative transformation with intertitial changes, without changes of adipocyte morphology (p=0.27) e without cellular atypia. In the second, the unmanipulated fat exposed to high-energy diet showed cellular proliferation (overexpression of Ki67, p=0.046) and increased levels of PAI-1 (p0.05. The systematic review identified 60 studies with 985 women. Eight studies analyzed the oncologic risk for breast cancer relapse, seven of them described breast lipofilling after treatment of advanced disease and their recurrence rate was 35.5% (143/403). The eighth study in this series, analyzed 89 women treated for non-advanced disease and found no cancer recurrence during a mean follow-up longer than five years. Conclusion: Our experimental studies in vivo identified the oncogenic potential of native unmanipulated fat when exposed to a high-energy diet. However, this potential was not oberved in grafted fat or in its host microenvironment (including the ductal lobular cells) when the procedure of lipofilling was analyzed alone. Our experimental results are in accordance to clinical results reported in literature for breast lipofilling and to the clinical data identified in our systematic review for adipose tissue of pedicled omental flap. Thus, they sustain the oncological safety of fat grafting for breast reconstructionDoutoradoOncologia Ginecológica e MamáriaDoutor em Ciências da Saúd

INTEGRATION OF MULTI-PLATFORM HIGH-DIMENSIONAL OMIC DATA

The development of high-throughput biotechnologies have made data accessible from different platforms, including RNA sequencing, copy number variation, DNA methylation, protein lysate arrays, etc. The high-dimensional omic data derived from different technological platforms have been extensively used to facilitate comprehensive understanding of disease mechanisms and to determine personalized health treatments. Although vital to the progress of clinical research, the high dimensional multi-platform data impose new challenges for data analysis. Numerous studies have been proposed to integrate multi-platform omic data; however, few have efficiently and simultaneously addressed the problems that arise from high dimensionality and complex correlations. In my dissertation, I propose a statistical framework of shared informative factor model (SIFORM) that can jointly analyze multi-platform omic data and explore their associations with a disease phenotype. The common disease- associated sample characteristics across different data types can be captured through the shared structure space, while the corresponding weights of genetic variables directly index the strengths of their association with the phenotype. I compare the performance of the proposed method with several popular regularized regression methods and canonical correlation analysis (CCA)-based methods through extensive simulation studies and two lung adenocarcinoma applications. The two lung adenocarcinoma applications jointly explore the associations of mRNA expression and protein expression with smoking status and survival using The Cancer Genome Atlas (TCGA) datasets. The simulation studies demonstrate the superior performance of SIFORM in terms of biomarker detection accuracy. In lung cancer applications, SIFORM identifies many biomarkers that belong to key pathways for lung tumorigenesis. It also discovers potential prognostic biomarkers for lung cancer patients survival and some biomarkers that reveal different tumorigenesis mechanisms between light smokers and heavy smokers. To improve the prediction accuracy and interpretability of the proposed model, I extend it to PSIFORM by incorporating existing biological pathway information to current statistical framework. I adopt a network-based regularization to ensure that the neighboring genes in the same pathway tend to be selected (or eliminated) simultaneously. Through simulation studies and a TCGA kidney cancer application, I show that PSIFORM outperforms its competitors in both variable selection and prediction. The statistical framework of PSIFORM also has a great potential in incorporating the hierarchical order across the multi-platform omic measurements

Responses of respiratory system cells in vitro and in vivo to petrochemical combustion-derived ultrafine particles

Environmental contamination with airborne particles has been a human health concern for many years. Epidemiologic studies in urban communities have linked ambient particle exposure to various health effects, including chronic obstructive pulmonary disease, lung cancer, and several cardiovascular disease conditions. The pathogenesis of these conditions with respect to ambient particle exposure is complex because ambient particles are complex in composition. The particles vary greatly in origin, size, surface area, and elemental composition; and a given particle type, such as those generated by petrochemical (gasoline, diesel, industrial substrate) combustion, may be coated with many other compounds, including polynuclear aromatic hydrocarbons (PAHs). Our laboratory group had previously characterized the generation of PAHs from incomplete combustion of the high volume petrochemical 1,3-butadiene (BD) and briefly described the biological effects of BD’s incomplete combustion product, butadiene soot (BDS), in vitro. The studies presented here represent a continuation of these initial studies, where we first characterize BDS with respect to particle size distribution and assembly, PAH composition, and elemental content of BDS ultrafine particles. We also describe in vitro assays demonstrating that BDS ultrafine particles can transport and transfer adsorbed organic constituents directly to target respiratory cells, without uptake of the particles by the cells. Next, we demonstrate that combustion-derived PAHs adsorbed onto BDS particles are concentrated in lipid droplets of respiratory system cells and that, in vitro, these PAHs activate xenobiotic metabolism pathways. We also present an in vivo analysis of bronchoalveolar lavage fluid (BALF) with inflammatory cell infiltrates, histopathological evidence of inflammation and particle retention, and gene expression analysis revealing upregulation of several cytokines and AhR-responsive biotransformation enzymes. Finally, we present ultrastructural evidence that BDS particles can be internalized by bronchoepithelial cells in vitro and phagocytosed by alveolar macrophages in vivo. These studies were designed to characterize and promote BDS as both a model mixture and a real-life example of a petrochemical product of incomplete combustion with the potential both for environmental contamination and for contributing to health problems

Liquid biopsies of solid tumors: non-small-cell lung and pancreatic cancer

Doctor of PhilosophyDepartment of ChemistryStefan H. BossmannCancer is a group of diseases that are characterized by uncontrolled growth and spread of cells. In order to treat cancer successfully, it is important to diagnose cancers in their early stages, because survival often depends on the stage of cancer detection. For that purpose, highly sensitive and selective methods must be developed, taking advantage of suitable biomarkers. The expression levels of proteases differ from one cancer type to the other, because different cancers arise from different cell types. According to the literature, there are significant differences between the protease expression levels of cancer patients and healthy people, because solid tumors rely on proteases for survival, angiogenesis and metastasis. Development of fluorescence-based nanobiosensors for the early detection of pancreatic cancer and non-small-cell lung cancer is discussed in this thesis. The nanobiosensors are capable of detecting protease/arginase activities in serum samples over a broad range. The functionality of the nanobiosensor is based on Förster resonance energy transfer and surface energy transfer mechanisms. The nanobiosensors for protease detection feature dopamine-coated Fe/Fe₃O₄ nanoparticles, consensus (cleavage) peptide sequences, meso-tetra(4-carboxyphenyl)porphine (TCPP), and cyanine 5.5. The consensus peptide sequences were synthesized by solid-supported peptide synthesis. In this thesis, improved consensus sequences were used, which permit faster synthesis and higher signal intensities. TCPP, which is the fluorophore of the nanoplatform, was connected to the N-terminal end of the oligopeptides while it was still on the resin. After the addition of TCPP, the TCPP-oligopeptide was cleaved off the resin and linked to the primary amine groups of Fe/Fe₃O₄-bound via a stable amide bond. In the presence of a particular protease, the consensus sequences attached to the nanoparticle can be cleaved and release TCPP to the aqueous medium. Upon releasing the dye, the emission intensity increases significantly and can be detected by fluorescence spectroscopy or, similarly, by using a fluorescence plate reader. In sensing of arginase, posttranslational modification of the peptide sequence will occur, transforming arginine to ornithine. This changes the conformational dynamics of the oligopeptide tether, leading to the increase of the TCPP signal. This is a highly selective technology, which has a very low limit of detection (LOD) of 1 x 10⁻¹⁶ molL⁻¹ for proteases and arginase. The potential of this nanobiosensor technology to detect early pancreatic and lung cancer was demonstrated by using serum samples, which were collected from patients who have been diagnosed with pancreatic cancer and non-small cell lung cancer at the South Eastern Nebraska Cancer Center (lung cancer) and the University of Kansas Cancer Center (pancreatic cancer). As controls, serum samples collected from healthy volunteers were analyzed. In pancreatic cancer detection, the protease/arginase signature for the detection of pancreatic adenocarcinomas in serum was identified. It comprises arginase, MMPs -1, - 3, and -9, cathepsins -B and -E, urokinase plasminogen activator, and neutrophil elastase. For lung cancer detection, the specificity and sensitivity of the nanobiosensors permit the accurate measurements of the activities of nine signature proteases in serum samples. Cathepsin -L and MMPs-1, -3, and -7 permit detecting non-small-cell lung-cancer at stage 1