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He, we show that combined use of the EZH2 inhibitor GSK126 and the CDK4/6 inhibitor abemaciclib synergistically enhances antitumoral effects in preclinical GBM models. Dual blockade led to HIF1α upregulation and CalR translocation, accompanied by massive impairment of mitochondrial function. Basal oxygen consumption rate, ATP synthesis, and maximal mitochondrial respiration decreased, confirming disrupted endoplasmic reticulum-mitochondrial homeostasis. This was paralleled by mitochondrial depolarization and upregulation of the UPR sensors PERK, ATF6α, and IRE1α. Notably, dual EZH2/CDK4/6 blockade also reduced 3D-spheroid invasion, partially inhibited tumor growth in ovo, and led to impaired viability of patient-derived organoids. Mechanistically, this was due to transcriptional changes in genes involved in mitotic aberrations/spindle assembly (Rb, PLK1, RRM2, PRC1, CENPF, TPX2), histone modification (HIST1H1B, HIST1H3G), DNA damage/replication stress events (TOP2A, ATF4), immuno-oncology (DEPDC1), EMT-counterregulation (PCDH1) and a shift in the stemness profile towards a more differentiated state. We propose a dual EZH2/CDK4/6 blockade for further investigation.
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The number of multidrug-resistant strains of bacteria is increasing rapidly, while the number of new antibiotic discoveries has stagnated. This trend has caused a surge in interest in bacteriophages as anti-bacterial therapeutics, in part because there is near limitless diversity of phages to harness. While this diversity provides an opportunity, it also creates the dilemma of having to decide which criteria to use to select phages. Here we test whether a phage's ability to coevolve with its host (evolvability) should be considered and how this property compares to two previously proposed criteria: fast reproduction and thermostability. To do this, we compared the suppressiveness of three phages that vary by a single amino acid yet differ in these traits such that each strain maximized two of three characteristics. Our studies revealed that both evolvability and reproductive rate are independently important. The phage most able to suppress bacterial populations was the strain with high evolvability and reproductive rate, yet this phage was unstable. Phages varied due to differences in the types of resistance evolved against them and their ability to counteract resistance. When conditions were shifted to exaggerate the importance of thermostability, one of the stable phages was most suppressive in the short-term, but not over the long-term. Our results demonstrate the utility of biological therapeutics' capacities to evolve and adjust in action to resolve complications like resistance evolution. Furthermore, evolvability is a property that can be engineered into phage therapeutics to enhance their effectiveness.
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The microvasculature is integral to nearly every tissue in the body, providing not only perfusion to and from the tissue, but also homing sites for immune cells, cellular niches for tissue dynamics, and cooperative interactions with other tissue elements. As a microtissue itself, the microvasculature is a composite of multiple cell types exquisitely organized into structures (individual vessel segments and extensive vessel networks) capable of considerable dynamics and plasticity. Consequently, it has been challenging to include a functional microvasculature in assembled or fabricated tissues. Isolated fragments of intact microvessels, which retain the cellular composition and structures of native microvessels, are proving effective in a variety of vascularization applications including tissue in vitro disease modeling, vascular biology, mechanistic discovery, and tissue prevascularization in regenerative therapeutics and grafting. In this review, we will discuss the importance of recapitulating native tissue biology and the successful vascularization applications of isolated microvessels.
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Microvasos , Neovascularização Fisiológica , Humanos , Microvasos/fisiologia , AnimaisRESUMO
When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity. We explored this question in a bacteriophage receptor-binding protein during host-range evolution. We studied the properties of the receptor-binding protein of bacteriophage λ before and after host-range evolution and demonstrated that the evolved protein is relatively unstable and may exist in multiple conformations with unique receptor preferences. Through a combination of structural modeling and in vitro oligomeric state analysis, we found that the instability arises from mutations that interfere with trimer formation. This study raises the intriguing possibility that protein instability might play a previously unrecognized role in mediating host-range expansions in viruses.
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Evolução Molecular , Receptores Virais , Mutação , Receptores Virais/genética , Receptores Virais/metabolismo , Ligação ProteicaRESUMO
Despite advances in the treatment and care of severe physical injuries, trauma remains one of the main reasons for disability-adjusted life years worldwide. Trauma patients often suffer from disturbances in energy utilization and metabolic dysfunction, including hyperglycemia and increased insulin resistance. White adipose tissue plays an essential role in the regulation of energy homeostasis and is frequently implicated in traumatic injury due to its ubiquitous body distribution but remains poorly studied. Initial triggers of the trauma response are mainly damage-associated molecular patterns (DAMPs) such as histones. We hypothesized that DAMP-induced adipose tissue inflammation contributes to metabolic dysfunction in trauma patients. Therefore, we investigated whether histone release during traumatic injury affects adipose tissue. Making use of a murine polytrauma model with hemorrhagic shock, we found increased serum levels of histones accompanied by an inflammatory response in white adipose tissue. In vitro, extracellular histones induced an inflammatory response in human adipocytes. On the molecular level, this inflammatory response was mediated via a MYD88-IRAK1-ERK signaling axis as demonstrated by pharmacological and genetic inhibition. Histones also induced lytic cell death executed independently of caspases and RIPK1 activity. Importantly, we detected increased histone levels in the bloodstream of patients after polytrauma. Such patients might benefit from a therapy consisting of activated protein C and the FDA-approved ERK inhibitor trametinib, as this combination effectively prevented histone-mediated effects on both, inflammatory gene activation and cell death in adipocytes. Preventing adipose tissue inflammation and adipocyte death in patients with polytrauma could help minimize posttraumatic metabolic dysfunction.
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Adipócitos , Histonas , Inflamação , Fator 88 de Diferenciação Mieloide , Humanos , Animais , Histonas/metabolismo , Adipócitos/metabolismo , Adipócitos/efeitos dos fármacos , Inflamação/patologia , Inflamação/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Morte Celular/efeitos dos fármacos , Masculino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ferimentos e Lesões/complicações , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Glioblastoma represents a brain tumor with a notoriously poor prognosis. First-line therapy may include adjunctive Tumor Treating Fields (TTFields) which are electric fields that are continuously delivered to the brain through non-invasive arrays. On a different note, CUSP9v3 represents a drug repurposing strategy that includes 9 repurposed drugs plus metronomic temozolomide. Here, we examined whether TTFields enhance the antineoplastic activity of CUSP9v3 against this disease. METHODS: We performed preclinical testing of a multimodal approach of TTFields and CUSP9v3 in different glioblastoma models. RESULTS: TTFields had predominantly synergistic inhibitory effects on the cell viability of glioblastoma cells and non-directed movement was significantly impaired when combined with CUSP9v3. TTFields plus CUSP9v3 significantly enhanced apoptosis, which was associated with a decreased mitochondrial outer membrane potential (MOMP), enhanced cleavage of effector caspase 3 and reduced expression of Bcl-2 and Mcl-1. Moreover, oxidative phosphorylation and expression of respiratory chain complexes I, III and IV was markedly reduced. CONCLUSION: TTFields strongly enhance the CUSP9v3-mediated anti-glioblastoma activity. TTFields are currently widely used for the treatment of glioblastoma patients and CUSP9v3 was shown to have a favorable safety profile in a phase Ib/IIa trial (NCT02770378) which facilitates transition of this multimodal approach to the clinical setting.
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Antineoplásicos , Neoplasias Encefálicas , Terapia por Estimulação Elétrica , Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Reposicionamento de Medicamentos , Reprogramação Metabólica , Temozolomida/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Terapia CombinadaRESUMO
The formation of new vascular networks via angiogenesis is a crucial biological mechanism to balance tissue metabolic needs, yet the coordination of factors that influence the guidance of growing neovessels remain unclear. This study investigated the influence of extracellular cues within the immediate environment of sprouting tips over multiple hours and obtained quantitative relationships describing their effects on the growth trajectories of angiogenic neovessels. Three distinct microenvironmental cues-fibril tracks, ECM density, and the presence of nearby cell bodies-were extracted from 3D time series image data. The prominence of each cue was quantified along potential sprout trajectories to predict the response to multiple microenvironmental factors simultaneously. Sprout trajectories significantly correlated with the identified microenvironmental cues. Specifically, ECM density and nearby cellular bodies were the strongest predictors of the trajectories taken by neovessels (p < 0.001 and p = 0.016). Notwithstanding, direction changing trajectories, deviating from the initial neovessel orientation, were significantly correlated with fibril tracks (p = 0.003). Direction changes also occurred more frequently with strong microenvironmental cues. This provides evidence for the first time that local matrix fibril alignment influences changes in sprout trajectories but does not materially contribute to persistent sprouting. Together, our results suggest the microenvironmental cues significantly contribute to guidance of sprouting trajectories. Further, the presented methods quantitatively distinguish the influence of individual microenvironmental stimuli during guidance.
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Sinais (Psicologia) , Neovascularização Fisiológica , Morfogênese , Fenômenos Fisiológicos Cardiovasculares , Imageamento Tridimensional , Matriz Extracelular/fisiologiaRESUMO
Tissue organoids hold enormous potential as tools for a variety of applications, including disease modeling and drug screening. To effectively mimic the native tissue environment, it is critical to integrate a microvasculature with the parenchyma and stroma. In addition to providing a means to physiologically perfuse the organoids, the microvasculature also contributes to the cellular dynamics of the tissue model via the cells of the perivascular niche, thereby further modulating tissue function. In this review, we discuss current and developing strategies for vascularizing organoids, consider tissue-specific vascularization approaches, discuss the importance of perfusion, and provide perspectives on the state of the field.
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Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.
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Predicting the evolution of virus host range has proven to be extremely difficult, in part because of the sheer diversity of viruses, each with unique biology and ecological interactions. We have not solved this problem, but to make the problem more tractable, we narrowed our focus to three traits intrinsic to all viruses that may play a role in host-range evolvability: mutation rate, recombination rate, and phenotypic heterogeneity. Although each trait should increase evolvability, they cannot do so unbounded because fitness trade-offs limit the ability of all three traits to maximize evolvability. By examining these constraints, we can begin to identify groups of viruses with suites of traits that make them especially concerning, as well as ecological and environmental conditions that might push evolution toward accelerating host-range expansion.
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Especificidade de Hospedeiro , Vírus , Vírus/genéticaRESUMO
INTRODUCTION: Death due to cancer is mostly associated with therapy ineffectiveness, i.e. tumor cells no longer responding to treatment. The underlying dynamics that facilitate this mutational escape from selective pressure are well studied in several other fields and several interesting approaches exist to combat this phenomenon, for example in the context of antibiotic-resistance in bacteria. AREAS COVERED: Ninety percent of all cancer-related deaths are associated with treatment failure. Here, we discuss the common treatment modalities and prior attempts to overcome acquired resistance to therapy. The underlying molecular mechanisms are discussed and the implications of emerging resistance in other systems, such as bacteria, are discussed in the context of cancer. EXPERT OPINION: Reevaluating emerging therapy resistance in tumors as an evolutionary mechanism to survive in a rapidly and drastically altering fitness landscape leads to novel treatment strategies and distinct requirements for new drugs. Here, we propose a scheme of considerations that need to be applied prior to the discovery of novel therapeutic drugs.
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Desenho de Fármacos , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Mutação , BactériasRESUMO
During the struggle for survival, populations occasionally evolve new functions that give them access to untapped ecological opportunities. Theory suggests that coevolution between species can promote the evolution of such innovations by deforming fitness landscapes in ways that open new adaptive pathways. We directly tested this idea by using high-throughput gene editing-phenotyping technology (MAGE-Seq) to measure the fitness landscape of a virus, bacteriophage λ, as it coevolved with its host, the bacterium Escherichia coli. An analysis of the empirical fitness landscape revealed mutation-by-mutation-by-host-genotype interactions that demonstrate coevolution modified the contours of λ's landscape. Computer simulations of λ's evolution on a static versus shifting fitness landscape showed that the changes in contours increased λ's chances of evolving the ability to use a new host receptor. By coupling sequencing and pairwise competition experiments, we demonstrated that the first mutation λ evolved en route to the innovation would only evolve in the presence of the ancestral host, whereas later steps in λ's evolution required the shift to a resistant host. When time-shift replays of the coevolution experiment were run where host evolution was artificially accelerated, λ did not innovate to use the new receptor. This study provides direct evidence for the role of coevolution in driving evolutionary novelty and provides a quantitative framework for predicting evolution in coevolving ecological communities.
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Parasitos , Animais , Evolução Biológica , Escherichia coli/genética , Genótipo , MutaçãoRESUMO
Advanced in vitro tissue models better reflect healthy and disease tissue conditions in the body. However, complex tissue models are often manufactured using custom solutions and can be challenging to manufacture to scale. Here, we describe the automated fabrication of a cell-dense, thick (≤ 1 cm), human vascularized liver tissue model using a robotic biomanufacturing platform and off-the-shelf components to build, culture, and sample liver tissues hands-free without compromising tissue health or function. Fabrication of the tissue involved 3D bioprinting and incorporation of primary human hepatocytes, primary human non-parenchymal cells, and isolated fragments of intact human microvessels as vascular precursors. No differences were observed in select assessments of the liver tissues fabricated by hand or via automation. Furthermore, constant media exchange, via perfusion, improved urea output and elevated tissue metabolism. Interestingly, inclusion of adipose-derived human microvessels enhanced functional gene expression, including an enhanced response to a drug challenge. Our results describe the fabrication of a thick liver tissue environment useful for a variety of applications including liver disease modeling, infectious agent studies, and cancer investigations. We expect the automated fabrication of the vascularized liver tissue, at the point of use and using off-the-shelf platforms, eases fabrication of the complex model and increases its utility.
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Bioimpressão , Engenharia Tecidual , Bioimpressão/métodos , Hepatócitos/metabolismo , Humanos , Fígado , Microvasos , Engenharia Tecidual/métodosRESUMO
Angiogenesis is necessary for wound healing, tumorigenesis, implant inosculation, and homeostasis. In each situation, matrix structure and mechanics play a role in determining whether new vasculatures can establish transport to new or hypoxic tissues. Neovessel growth and directional guidance are sensitive to three-dimensional (3-D) matrix anisotropy and density, although the individual and integrated roles of these matrix features have not been fully recapitulated in vitro. We developed a tension-based method to align 3-D collagen constructs seeded with microvessel fragments in matrices of three levels of collagen fibril anisotropy and two levels of collagen density. The extent and direction of neovessel growth from the parent microvessel fragments increased with matrix anisotropy and decreased with density. The proangiogenic effects of anisotropy were attenuated at higher matrix densities. We also examined the impact of matrix anisotropy in an experimental model of neovessel invasion across a tissue interface. Matrix density was found to dictate the success of interface crossing, whereas interface curvature and fibril alignment were found to control directional guidance. Our findings indicate that complex configurations of matrix density and alignment can facilitate or complicate the establishment or maintenance of vascular networks in pathological and homeostatic angiogenesis. Furthermore, we extend preexisting methods for tuning collagen anisotropy in thick constructs. This approach addresses gaps in tissue engineering and cell culture by supporting the inclusion of large multicellular structures in prealigned constructs.NEW & NOTEWORTHY Matrix anisotropy and density have a considerable effect on angiogenic vessel growth and directional guidance. However, the current literature relies on 2-D and simplified models of angiogenesis (e.g., tubulogenesis and vasculogenesis). We present a method to align 3-D collagen scaffolds embedded with microvessel fragments to different levels of anisotropy. Neovessel growth increases with anisotropy and decreases with density, which may guide angiogenic neovessels across tissue interfaces such as during implant inosculation and tumorigenesis.
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Colágeno , Neovascularização Fisiológica , Anisotropia , Carcinogênese , Matriz Extracelular/química , Humanos , Morfogênese , Neovascularização PatológicaRESUMO
Tissue organoids or "mini organs" can be invaluable tools for understanding health and disease biology, modeling tissue dynamics, or screening potential drug candidates. Effective vascularization of these models is critical for truly representing the in vivo tissue environment. Not only is the formation of a vascular network, and ultimately a microcirculation, essential for proper distribution and exchange of oxygen and nutrients throughout larger organoids, but vascular cells dynamically communicate with other cells to modulate overall tissue behavior. Additionally, interstitial fluid flow, mediated by a perfused microvasculature, can have profound influences on tissue biology. Thus, a truly functionally and biologically relevant organoid requires a vasculature. Here, we review existing strategies for fabricating and incorporating vascular elements and perfusion within tissue organoids.
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Organoides , Oxigênio , PerfusãoRESUMO
Viruses are highly evolvable, but what traits endow this property? The high mutation rates of viruses certainly play a role, but factors that act above the genetic code, like protein thermostability, are also expected to contribute. We studied how the thermostability of a model virus, bacteriophage λ, affects its ability to evolve to use a new receptor, a key evolutionary transition that can cause host-range evolution. Using directed evolution and synthetic biology techniques we generated a library of host-recognition protein variants with altered stabilities and then tested their capacity to evolve to use a new receptor. Variants fell within three stability classes: stable, unstable, and catastrophically unstable. The most evolvable were the two unstable variants, whereas seven of eight stable variants were significantly less evolvable, and the two catastrophically unstable variants could not grow. The slowly evolving stable variants were delayed because they required an additional destabilizing mutation. These results are particularly noteworthy because they contradict a widely supported contention that thermostabilizing mutations enhance evolvability of proteins by increasing mutational robustness. Our work suggests that the relationship between thermostability and evolvability is more complex than previously thought, provides evidence for a new molecular model of host-range expansion evolution, and identifies instability as a potential predictor of viral host-range evolution.
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Proteínas Virais , Vírus , Especificidade de Hospedeiro , Mutação , Fenótipo , Proteínas Virais/genéticaRESUMO
Medulloblastoma (MB) is the most common solid tumour in children and, despite current treatment with a rather aggressive combination therapy, accounts for 10% of all deaths associated with paediatric cancer. Breaking the tumour cells' intrinsic resistance to therapy-induced cell death should lead to less aggressive and more effective treatment options. In other tumour entities, this has been achieved by modulating the balance between the various pro- and anti-apoptotic members of the Bcl-2 family with small molecule inhibitors. To evaluate the therapeutic benefits of ABT-199 (Venetoclax), a Bcl-2 inhibitor, and ABT-263 (Navitoclax), a dual Bcl-XL/Bcl-2 inhibitor, increasingly more relevant model systems were investigated. Starting from established MB cell lines, progressing to primary patient-derived material and finally an experimental tumour system imbedded in an organic environment were chosen. Assessment of the metabolic activity (a surrogate readout for population viability), the induction of DNA fragmentation (apoptosis) and changes in cell number (the combined effect of alterations in proliferation and cell death induction) revealed that ABT-263, but not ABT-199, is a promising candidate for combination therapy, synergizing with cell death-inducing stimuli. Interestingly, in the experimental tumour setting, the sensitizing effect of ABT-263 seems to be predominantly mediated via an anti-proliferative and not a pro-apoptotic effect, opening a future line of investigation. Our data show that modulation of specific members of the Bcl-2 family might be a promising therapeutic addition for the treatment of MB.
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Angiogenesis, the formation of new vessel elements from existing vessels, is important in homeostasis and tissue repair. Dysfunctional angiogenesis can contribute to numerous pathologies, including cancer, ischemia, and chronic wounds. In many instances, growing vessels must navigate along or across tissue-associated boundaries and interfaces tissue interfaces. To understand this dynamic, we developed a new model for studying angiogenesis at tissue interfaces utilizing intact microvessel fragments isolated from adipose tissue. Isolated microvessels retain their native structural and cellular complexity. When embedded in a 3D matrix, microvessels, sprout, grow, and connect to form a neovasculature. Here, we discuss and describe methodology for one application of our microvessel-based angiogenesis model, studying neovessel behavior at tissue interfaces.
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Microvasos , Neovascularização Fisiológica , Tecido Adiposo , Humanos , Morfogênese , Neovascularização PatológicaRESUMO
Cancers in animals present a large, underutilized reservoir of biomedical information with critical implication for human oncology and medicine in general. Discussing two distinct areas of tumour biology in non-human hosts, we highlight the importance of these findings for our current understanding of cancer, before proposing a coordinated strategy to harvest biomedical information from non-human resources and translate it into a clinical setting. First, infectious cancers that can be transmitted as allografts between individual hosts, have been identified in four distinct, unrelated groups, dogs, Tasmanian devils, Syrian hamsters and, surprisingly, marine bivalves. These malignancies might hold the key to improving our understanding of the interaction between tumour cell and immune system and, thus, allow us to devise novel treatment strategies that enhance anti-cancer immunosurveillance, as well as suggesting more effective organ and stem cell transplantation strategies. The existence of these malignancies also highlights the need for increased scrutiny when considering the existence of infectious cancers in humans. Second, it has long been understood that no linear relationship exists between the number of cells within an organism and the cancer incidence rate. To resolve what is known as Peto's Paradox, additional anticancer strategies within different species have to be postulated. These naturally occurring idiosyncrasies to avoid carcinogenesis represent novel potential therapeutic strategies.
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Transmissão de Doença Infecciosa , Metabolismo Energético/fisiologia , Neoplasias/etiologia , Neoplasias/virologia , Animais , Bivalves , Carcinogênese , Cricetinae , Modelos Animais de Doenças , Cães , Humanos , Marsupiais , Neoplasias/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Tumores Venéreos VeterináriosRESUMO
Given the considerable research efforts in understanding and manipulating the vasculature in tissue health and function, making effective measurements of vascular density is critical for a variety of biomedical applications. However, because the vasculature is a heterogeneous collection of vessel segments, arranged in a complex three-dimensional architecture, which is dynamic in form and function, it is difficult to effectively measure. Here, we developed a semi-automated method that leverages machine learning to identify and quantify vascular metrics in an angiogenesis model imaged with different modalities. This software, BioSegment, is designed to make high throughput vascular density measurements of fluorescent or phase contrast images. Furthermore, the rapidity of assessments makes it an ideal tool for incorporation in tissue manufacturing workflows, where engineered tissue constructs may require frequent monitoring, to ensure that vascular growth benchmarks are met.