Propranolol blocks osteosarcoma cell cycle progression, inhibits angiogenesis and slows xenograft growth in combination with cisplatin-based chemotherapy

Osteosarcoma is still associated with limited response to standard-of-care therapy and alarmingly elevated mortality rates, especially in low- and middle-income countries. Despite multiple efforts to repurpose β-blocker propranolol in oncology, its potential application in osteosarcoma management remains largely unexplored. Considering the unsatisfied clinical needs of this aggressive disease, we evaluated the antitumoral activity of propranolol using different in vitro and in vivo osteosarcoma preclinical models, alone or in addition to chemotherapy. Propranolol significantly impaired cellular growth in β2-adrenergic receptor-expressing MG-63 and U-2OS cells, and was capable of blocking growth-stimulating effects triggered by catecholamines. siRNA-mediated ADRB2 knockdown in MG-63 cells was associated with decreased cell survival and a significant attenuation of PPN anti-osteosarcoma activity. Direct cytostatic effects of propranolol were independent of apoptosis induction and were associated with reduced mitosis, G0/G1 cell cycle arrest and a significant down-regulation of cell cycle regulator Cyclin D1. Moreover, colony formation, 3D spheroid growth, cell chemotaxis and capillary-like tube formation were drastically impaired after propranolol treatment. Interestingly, anti-migratory activity of β-blocker was associated with altered actin cytoskeleton dynamics. In vivo, propranolol treatment (10 mg/kg/day i.p.) reduced the early angiogenic response triggered by MG-63 cells in nude mice. Synergistic effects were observed in vitro after combining propranolol with chemotherapeutic agent cisplatin. Sustained administration of propranolol (10 mg/kg/day i.p., five days a week), alone and especially in addition to low-dose metronomic cisplatin (2 mg/kg/day i.p., three times a week), markedly reduced xenograft progression. After histological analysis, propranolol and cisplatin combination resulted in low tumor mitotic index and increased tumor necrosis. β-blockade using propranolol seems to be an achievable and cost-effective therapeutic approach to modulate osteosarcoma aggressiveness. Further translational studies of propranolol repurposing in osteosarcoma are warranted.Fil: Solernó, Luisina María. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; ArgentinaFil: Sobol, Natasha Tatiana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gottardo, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; ArgentinaFil: Capobianco, Carla Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; ArgentinaFil: Ferrero, Maximiliano Ruben. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Vásquez, Liliana. Universidad de San Martín de Porres; PerúFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garona, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentin

Perturbing and imaging nuclear compartments to reveal mechanisms of transcription regulation and telomere maintenance

The cell nucleus is organized into functional domains that form around chromatin, which serves as a scaffold composed of DNA, proteins, and associated RNAs. On the 0.1-1 µm mesoscale these domains can form spatially defined compartments with distinct composition and properties that enrich specific genomic activities like transcription, chromatin modification or DNA repair. In addition, extrachromosomal DNA elements and RNAs can separate from the chromatin template and assemble with proteins into nuclear bodies. The resulting accumulations of proteins and nucleic acids in the nucleus modulate chromatin-templated processes and their organization. The assembly of these compartments occurs in a self-organizing manner via direct and indirect binding of proteins to DNA and/or RNA. Recently, it has been proposed that multivalent interactions drive compartmentalization by inducing phase separation with a non-stoichiometric accumulation of factors into biomolecular condensates. Despite the importance of compartments for genome regulation, insights into their structure and material properties and how these affect their function is limited. To address this issue, it is important to devise approaches that can perturb nuclear compartments in a targeted manner, while also measuring changes in genome activities within the same cell. In this thesis, the methodology to reveal the underlying structure-function relationships of nuclear compartments has been advanced and applied to compartments involved in activation and silencing of chromatin, and telomere maintenance in cancer cells. I first established a toolbox of chromatin effector constructs to probe and perturb properties of nuclear compartments in living cells that comprised different combinations of DNA binding, transcription activation and light-dependent interaction domains. In addition, I developed workflows to quantitatively assess relevant compartment features by fluorescence microscopy. These methods were employed to study the compaction mechanism of mouse pericentric heterochromatin (PCH) foci and to investigate the interplay between transcriptional co-activators, phase separation and transcription at an inducible reporter gene cluster. It revealed determinants of PCH compaction and identified differential co-activator usage and multivalent interactions as contributors to transcription factor (TF) strength. The results furthermore challenged the model of TF phase separation as a general positive driver of gene transcription. In the second part, I focused on exploiting the detection of compartments for measuring activity of the alternative lengthening of telomeres (ALT) pathway used by cancer cells to extend their telomeres in absence of telomerase. I developed ALT-FISH, a scalable and quantitative imaging assay that detects ALT pathway-specific compartments containing large amounts of single-stranded telomeric nucleic acids. I applied the method to cell line models from different cancer entities and to tumor tissue from leiomyosarcoma and neuroblastoma patients. By devising automated ALT-FISH data acquisition and analysis IV workflows, I implemented an approach, which enabled ALT activity measurements in hundreds of thousands of single cells. These technological advancements provided a quantitative description of ALT activity at single cell resolution and were used to characterize the spatial distribution of ALT activity in relation to other biological features and in response to perturbations. Finally, a novel approach for studying the regulation of ALT in tumors could be established by integrating the method with the spatially resolved detection of single cell transcriptomes. In summary, this thesis introduced and utilized several methods to establish connections between nuclear compartment organization, chromatin features, transcription regulation, and telomere maintenance. These perturbation and imaging techniques are versatile and may be applied to dissect nuclear activities related to other compartments and biological model systems. Furthermore, the detection of ALT activity has demonstrated that compartments can offer valuable biological insights into how phenotypic cellular heterogeneity is encoded and linked to diseases such as cancer

Development of Targeted Liposomal Formulation Approaches for Enhanced Colorectal Cancer Therapy

Colorectal cancer (CRC) is the 4th most commonly detected cancer in the USA. Despite promising advances, the 5-year survival rate for the metastatic disease remains dismal (40⁰C with ultrasound contrast agents and bacterial attachments can improve the real-time chemo-immunotherapy of CRC. Towards these goals, we investigated the following specific aims in murine models of colon cancer: 1) Develop echogenic-LTSL (E-LTSL) for real-time ultrasound-enhanced reporting of tumor temperature and doxorubicin delivery, 2) Utilize tumor homing Salmonella typhimurium for LTSL delivery and enhanced chemo-immunotherapy with High Intensity Focused Ultrasound (HIFU) tumor heating (~42°C), and 3) Investigate the ability of magnetic bacteria Magnetospirillim magneticum (AMB-1) to aid LTSL tumor drug delivery under magnetic guidance. Our data showed that intratumoral vascular contrast of E-LTSL as a function of temperature and doxorubicin delivery was strongly correlated, enabling robust estimation of temporal variation in colon tumor temperature and drug delivery. LTSL attachment didn’t impact Salmonella viability and improved chemo-immunotherapy outcomes in murine colon cancers by promoting the population of M1 macrophages with HIFU heating. Finally, the use of magnetic guidance for AMB-LTSL significantly reduced the colon cancer viability by enhancing cellular and tumor localizations of doxorubicin. In conclusion, we found that multifunctional LTSL formulations significantly improved the CRC treatment outcomes in murine models by aiding the real-time monitoring and removing the resistive and suppressive tumor microenvironment features

Optical Printing of Multiscale Hydrogel Structures

Hydrogel has been a promising candidate to recapitulate the chemical, physical and mechanical properties of natural extracellular matrix (ECM), and they have been widely used for tissue engineering, lab on a chip and biophotonics applications. A range of optical fabrication technologies such as photolithography, digital projection stereolithography and laser direct writing have been used to shape hydrogels into structurally complex functional devices and constructs. However, it is still greatly challenging for researchers to design and fabricate multiscale hydrogel structures using a single fabrication technology. To address this challenge, the goal of this work is the design and develop novel multimode optical 3D printing technology capable of printing hydrogels with multiscale features ranging from centimeter to micrometer sizes and in the process transforming simple hydrogels into functional devices for many biomedical applications. Chapter 2 presents a new multimode optical printing technology that synergistically combined large-scale additive manufacturing with small-scale additive/subtractive manufacturing. This multiscale fabrication capability was used to (i) align cells using laser induced densification in Chapter 3, (ii) develop diffractive optics based on changes in refractive indices in Chapter 4, (iii) print diffractive optical elements in Chapter 5, and (iv) digitally print complex microfluidic devices and other 3D constructs in Chapter 6. Overall, this work open doors to a new world of fabrication where multiscale functional hydrogel structures are possible for a range biomedical application

Label-free polarisation-resolved optical imaging of biological samples

Myelin is a biological structure present in all the gnathostomata. It is a highly- ordered structure, in which many lipid-enriched and densely compacted phospho- lipid bilayers are rolled up in a cylindrical symmetry around a subgroup of axons. The myelin sheath increases the electrical transverse resistance and reduces the ca- pacitance making the saltatory conduction of action potentials possible and therefore leading to a critically improved performance in terms of nervous impulse conduc- tion speeds and travel lengths. Myelin pathologies are a large group of neurological diseases that often result in death or disability. In order to investigate the main causes of myelin damage and its temporal progression many microscopy techniques are currently employed, such as electron microscopy and histochemistry or fluorescence imaging. However, electron microscopy and histochemistry imaging require complex sample prepara- tion and are therefore unsuitable for live imaging. Fluorescence imaging, as well as its derivatives, confocal and two-photon imaging, relies on the use of fluorescent probes to generate the image contrast but fluorophores and the associated sample processing, when applicable to living specimens, might nonetheless modify the bi- ological properties of the target molecule and perturb the whole biological process under investigation; moreover, fluorescent immunostaining still requires the fixation of the cells. Coherent anti-Stokes Raman Scattering (CARS) microscopy, on the other hand, is a powerful and innovative imaging modality that permits the study of liv- ing specimens with excellent chemical contrast and spatial resolution and without the confounding and often tedious use of chemical or biological probes. This is par- ticularly important in clinical settings, where the patient biopsy must be explanted in order to stain the tissue. In these cases it may be useful to resort to a set of label-free microscopy techniques. Among these, CARS microscopy is an ideal tool to investigate myelin morphology and structure, thanks to its abundance of CH2 bonds. The chemical selectivity of CARS microscopy is based on the properties of the contrast-generating CARS process. This is a nonlinear process in which the energy difference of a pair of incoming photons (\u201cpump\u201d and \u201cStokes\u201d) matches the energy of one of the vibrational modes of a molecular bond of interest. This vibrational excited state is coherently probed by a third photon (\u201cprobe\u201d) and anti-Stokes radi- ation is emitted. In this thesis I shall discuss the development of a multimodal nonlinear opti- cal setup implementing CARS microscopy together with general Four-Wave Mix- ing, Second Harmonic Generation and Sum Frequency Generation microscopies. Moreover, I shall present a novel polarisation-resolved imaging scheme based on the CARS process, which I named Rotating-Polarisation (RP) CARS microscopy and implemented in the same setup. This technique, using a freely-rotating pump-and- probe-beam-polarisation plane, exploits the CARS polarisation-dependent rules in order to probe the degree of anisotropy of the chemical-bond spatial orientations inside the excitation point-spread function and their average orientation, allowing at the same time the acquisition of large-field-of-view images with minimal polarisa- tion distortions. I shall show that RP-CARS is an ideal tool to investigate the highly- ordered structure of myelinated nervous fibres thanks to the strong anisotropy and symmetry properties of the myelin molecular architecture. I shall also demonstrate that this technique allows the fully label-free assessment of the myelin health status both in a chemical model of myelin damage (lysophos- phatidylcholine-exposed mouse nerve) and in a genetic model (twitcher mouse) of a human leukodystrophy (Krabbe disease) while giving useful insights into the pathogenic mechanisms underlying the demyelination process. I shall also discuss the promises of this technique for applications in optical tractography of the nerve fibres in the central nervous system and for the investigation of the effects of ageing on the peripheral nervous system. Moreover, I shall demonstrate by means of numer- ical simulations that RP-CARS microscopy is extremely robust against the presence of scatterers (such as lipid vesicles, commonly found in the peripheral nervous sys- tem). Finally, I shall discuss the results of the exploitation of my multimodal setup in a different area at the boundary of biophysics and nanomedicine: the observation of the internalization of different kinds of nanoparticles (boron-nitride nanotubes, barium-titanate nanoparticles and barium-titanate-core/gold-shell nanoparticles) by cultured cells and the demonstration of the nanopatterned nature of a structure built with two-photon lithography

Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53

The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses

The mutational landscape of normal urothelium

Genetic heterogeneity exists between cells within an individual. The accumulation of these somatic mutations can ultimately result in oncogenic transformation. Cancer development is often a multi-step process, the earliest stages of which remain incompletely understood. Recent technological advances have enabled detection of somatic mutations in healthy tissues, revealing that as we age many of our tissues are converted into a patchwork of mutant clones, despite retaining their histologically normal appearance. These clonal expansions are commonly driven by mutations in known oncogenes and tumour suppressor genes, with the driver landscape in a given tissue resembling its associated cancer. Bladder cancer exhibits complex landscapes of clonal selection and somatic mutagenesis. In order to characterise these features in normal urothelium, we developed an approach to sequence laser-dissected microbiopsies derived from transplant donors with no history of cancer and cystectomy specimens from bladder cancer patients. Using a combination of targeted and exome sequencing, we demonstrate that the driver landscape is dominated by chromatin remodellers, with mutations occurring less frequently in other classes of canonical bladder cancer genes, and is extensively heterogeneous between individuals. Whole-genome sequencing reveals that APOBEC mutagenesis, which accounts for the majority of mutations in bladder cancer, occurs in normal urothelium, with variable exposure levels between clones. Additionally, we identify three novel mutational signatures, one of which is associated with smoking, a major risk factor for bladder cancer the molecular basis for which was previously unknown. Overall, this dissertation provides a tantalising glimpse into the rich and diverse mutational landscape of normal urothelium. These findings may potentially contribute to the development of personalised risk models and tools for the early detection of cancer