Development of Exosomal Protein Detection Assays for Cancer Diagnostics Using Nanomaterials in Conjunction with Optical Spectroscopy and Imaging

Cases of cancer are on the rise, and cancer continues to be the major cause of death in the world. It has been known for years that the survival rate and possible recovery depend on early diagnosis and personalized treatment. However, tumors are in most cases almost undetectable until cancer has already invaded the surrounding tissue and begins to metastasize to distant organs at which point the treatment is significantly less effective or completely ineffective. And even if the tumor is detected, its analysis requires a tissue biopsy, which in many instances is a risky invasive procedure that does not allow regular monitoring of the effectiveness of treatment. Therefore, any strategy for early cancer identification will be based on the correct identification of cancer detection markers found in body fluids in various forms such as proteins, RNAs, and DNA. Emerging evidence points to extracellular vesicles, more precisely their subgroup - exosomes, as an abundant source in proteins and nucleic acids that reflects the state of the parental cell. In this dissertation, we summarize the exosomal biogenesis and composition, the influence of exosomes on cancer development and progression with emphasis on breast cancer, and major analytical methods applied to exosomal protein detection. Further, we report our take on exosomal protein detection as a form of novel bulk detection and single vesicle profiling techniques, which are designed for liquid biopsy in a clinical environment. Our approaches are based on optical spectroscopy and imaging such as surface enhanced Raman scattering (SERS), fluorescence, and dark-field light scattering imaging, and are designed to operate with small amounts (8-50 L) of already diluted samples. We demonstrated the potential of 3D printing and its applicability to create a miniaturized device that made it possible to customize detection conditions for nanosized exosomes and microvolume samples. Additionally, we developed a simple, fast, and inexpensive bulk method for detection of exosomal surface proteins using quantum dots in conjunction with fluorescent spectroscopy and we demonstrated its clinical potential on detection of HER2 cancer marker in plasma samples from a breast cancer patient. Lastly, we report single vesicle technology (SVT) based on dual fluorescent and dark-field imaging to achieve protein profiles at a single exosome level. Our SVT can overcome many obstacles that bulk technologies cannot and can bring long-sought-after early cancer detection into the clinical setting

Development of Extracellular Vesicle Isolation and Model Systems Toward Early Ovarian Cancer Diagnostics

Ovarian cancer (OC) is characterized by late stage discovery and low survivability. However, when diagnosed early (Stages I or II) the 5-year survival rate is 92% up from 29%.5 The extreme dichotomy in survivability is what makes OC a prime candidate for early diagnosis techniques. Exosomes, a subtype of extracellular vesicles, may bridge the gap between early and late diagnosis, but are lacking consistent isolation and detection technologies. Here poly(ethylene terephthalate) (PET) capillary channeled polymer (C-CP) fibers employing an HIC protocol are investigated as a novel exosome isolation method and a quick, inexpensive, and easy-to-use platform for OC diagnosis. The cell model system, immunoaffinity protocols, and biomarker identification tools developed here will aid in the refinement of a selective PET C-CP exosome isolation. The exosome isolation and diagnostic technique developed as a result of these investigations will allow for earlier and routine diagnosis of OC and save many women from one of the deadliest cancers

Tipping the Balance: Factors That Influence the ER Signaling Network in Breast Cancer

<p>The estrogen receptor(ER) is a master transcriptional regulator of the breast where it plays key roles in the development and maintenance of normal breast epithelium but is also critical to the growth of luminal breast cancers. ER is also a well-defined molecular therapeutic target and anti-estrogens, such as tamoxifen, are used clinically to inhibit the mitogenic activity of ER and delay disease progression. However, despite the initial benefits to tamoxifen therapy, nearly one third of luminal breast cancer tumors eventually become resistant, limiting the therapeutic utility the drug. Mechanisms of resistance can be attributed to circumvention of ER and reliance on alternative growth pathways, or through upregulation of pathways that converge with ER to allow reactivation. Understanding the molecular determinants of resistance is a critical endeavor that demands attention in order to shape new drug developments and extend the therapeutic efficacy of anti-estrogens.</p><p> A major challenge in elucidating mechanisms of resistance is in understanding the complexities of the ER signaling program in respect to receptor occupancy and the coordinated relationship with chromatin architecture and collaborating transcription factors. This work therefore integrates the relationship between accessible chromatin, as measured by DNase-Seq, with ER occupancy and ER-mediated transcription in an in vivo derived tamoxifen resistant cell line (TamR) and a comparator group of two closely related tamoxifen sensitive cell lines. Cumulatively, these data demonstrate an enhanced role for FOXA1 in tamoxifen resistance. Specifically, FOXA1 occupancy is greatly enriched at differential DNase hypersensitive loci in TamR cells, and FOXA1 target genes are dramatically upregulated. Furthermore, expression of these target genes can be restored to MCF7 levels with siRNA directed against FOXA1. The TamR cells also have increased ER occupancy at FOXA1 overlapping sites, where ER is engaged to chromatin in a ligand-independent manner and results in enhanced activation of nearby target genes that can be repressed with the pure anti-estrogen, ICI. The increased role of FOXA1 is not due to an increase in total protein levels however and instead is manifested through increased activity. </p><p>Other clinical associations of resistance have been elucidated for which there is little to no mechanistic evidence currently available. HOXB13 has been shown to associate with tamoxifen therapy failure from differential microarray expression profiling of patients who relapsed compared to those that remained disease-free at the five year follow-up. The outcome of our studies reveals HOXB13 to downregulate GATA3 levels, which in turn leads to loss of ER function and parallel activation of inflammatory pathways. </p><p>The present study also makes use of publicly available clinical datasets to generate an integrative database of 4885 patients from 25 independent studies. Furthermore, analytical methods and functions were also developed to allow efficient use and application of the data. Access to the breast cancer meta-set and functions are made available to end users via a web interface, GeneAnalytics. Together, the breast cancer meta-set and associated access through the GeneAnalytics web sites provides novel opportunities for researchers to integrate functional studies with tumor derived expression data to further our understanding of cancer related processes.</p><p>Collectively, our findings demonstrate that the ER signaling program is modified as tumors progress to resistance by an increased role of FOXA1 to facilitate ER binding and reprogramming, and by HOXB13 to suppress the actions of ER and promote inflammatory pathways. These mechanisms highlight distinct methods of resistance and provide rational for new therapeutic approaches to extend the utility of current anti-estrogens.</p>Dissertatio

Extracellular vesicles from induced neurons trigger epigenetic silencing of a brain neurotransmitter.

Introduction: Antithrombin (AT) is a glycoprotein involved in the regulation of blood coagulation. It belongs to the family of serine-protease inhibitors and acts as the most important antagonist of different clot- ting factors. Two types of inherited AT deficiency can be distinguished: Type I (quantitative deficit), and Type II (qualitative deficit). The latter is characterized by an impaired inhibitory activity related to dysfunc- tional domains of the protein. Three Type II subtypes can be defined: Type IIa (reactive site defect), Type IIb (heparin binding site defect) and Type IIc (pleiotropic defect). This classification has clinical importance since these subtypes have a different thrombotic risk. No functional routine diagnostic assay, however, can be assumed to detect all forms of Type II deficiencies since false-negative results may hamper the diagnosis. Methods: We analysed the biochemical/biophysical association of ATT to EVs. We separated EVs from plasma of healthy or Type II affected patients or from cultured hepatocytes through differential ultracentrifu- gation followed by sucrose density gradient and/or immunoprecipitation. We next combined dot blot ana- lysis, WB, 2D electrophoresis and enzymatic assays to reveal the nature of ATT association to EVs. Results: We evidenced that ATT is associated to the external leaflet of EVs. We also found that specific ATT isoforms are enriched in EV preparations in respect to total plasma and that those isoforms are selectively associated to EVs when comparing healthy or ATT type II deficient patients. Summary/Conclusion: ATT selective association pat- tern to EVs might be related either to mutations in the primary sequence of the protein or alterations in the glycosylation process, hence experiments are ongoing to reveal the nature of this phenomenon. Our findings suggest that analysis of ATT enriched in EV prepara- tions might be useful to gain insights into the patho- genesis and be of support in the diagnostic algorithm of ATT deficiency. Funding: This work acknowledges FFABR (Fondo finanziamento attività Base di ricerca from MIUR, Ministry of Education, Universities and Research, Italy) for financial support

Characterization of the regulatory roles of histone H1 in the homeostasis of the mammalian genome

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-10-2019Esta tesis tiene embargado el acceso al texto completo hasta el 25-04-2021Histone H1 is a key component of chromatin, involved in the formation of condensed structures that are refractory to the binding of different factors, thus hindering the action of both the transcription and replication machineries. Previous studies in our group have demonstrated that lowering the total amounts of histone H1 in mouse embryonic stem cells has drastic consequences on their DNA replication dynamics, due to a lack of coordination with transcriptional processes. Along this work, we have continued this investigation, characterizing the mechanisms that account for the conflicts between transcription and replication in H1 deficient cells. We have found that the replication dynamics alterations are coupled to a perturbed DNA replication initiation landscape, which would also be compatible with massive replication fork stalling mediated by conflicts with the transcriptional machinery. Indeed, we have underscored that the lack of histone H1 produces numerous transcriptional alterations. The most remarkable of them would be the widespread accumulation of unstable non-coding transcripts in chromatin, including PROMPTs, lncRNAs and enhancer RNAs. These transcripts are not post-transcriptionally bound to DNA: their attachment to chromatin is mediated by RNA polymerase II molecules. Coding transcription is also affected, since protein-coding genes display elongation failures and transcriptional read-through. However, all these phenotypes do not seem to be directly related to a chromatin conformational change or any epigenetic alteration, but rather to a defect in RNA processing and metabolism. Finally, we have found that the general phenotype of H1 deficient cells resembles the mutation of several components of the nuclear exosome complex, suggesting a possible functional connection between the linker histone with RNA surveillance mechanisms. These findings have broad implications for our knowledge about the interplay between chromatin and basic cellular processes, and how histone H1 contributes to the maintenance of genome stability and cell homeostasisLa histona H1 es un componente esencial de la cromatina necesaria para la compactación de su estructura, haciéndola así refractaria a la unión de diferentes factores y obstaculizando la acción de las maquinarias de replicación y de transcripción. Estudios previos de nuestro grupo han demostrado que reducir la cantidad global de histona H1 en células embrionarias de ratón tiene consecuencias drásticas en la dinámica de replicación del DNA, debido a la falta de coordinación con procesos transcripcionales. A lo largo de este trabajo, hemos continuado esta investigación, caracterizando los mecanismos que explican los conflictos entre la replicación y la transcripción en células con cantidades reducidas de histona H1. Hemos descubierto que las alteraciones en la dinámica de replicación están asociadas a un profundo cambio en el paisaje de iniciación de la replicación del DNA. Este fenotipo sería compatible con un bloqueo masivo de las horquillas de replicación, causado por conflictos con la maquinaria transcripcional. En efecto, también hemos comprobado que la falta de histona H1 produce numerosas alteraciones transcripcionales. La más llamativa sería la acumulación en cromatina de transcritos inestables no codificantes, entre los que se encuentran PROMPTs, lncRNAs y “enhancer” RNAs. Estos transcritos no se unen a la cromatina post-transcripcionalmente, sino a través de la RNA polimerasa II. La transcripción codificante también está afectada: los genes codificantes presentan fallos en la elongación y “read-through” transcripcional. Sin embargo, todos estos fenotipos no parecen guardar una relación directa con un cambio conformacional de la cromatina ni con ninguna alteración epigenética, sino más bien con un defecto del procesamiento y el metabolismo del RNA. Finalmente, hemos descubierto que el fenotipo general de las células defectivas para H1 se asemeja al de mutantes en varios componentes del complejo exosoma, lo que sugiere una posible conexión funcional entre la histona H1 y los mecanismos de vigilancia del RNA. Estos hallazgos tienen importantes implicaciones para nuestro conocimiento de la relación entre la cromatina y procesos celulares básicos, y de cómo la histona H1 contribuye al mantenimiento de la estabilidad genómica y la homeostasis celular