Nonmonotone invasion landscape by noise-aware control of metastasis activator levels.

A major pharmacological assumption is that lowering disease-promoting protein levels is generally beneficial. For example, inhibiting metastasis activator BACH1 is proposed to decrease cancer metastases. Testing such assumptions requires approaches to measure disease phenotypes while precisely adjusting disease-promoting protein levels. Here we developed a two-step strategy to integrate protein-level tuning, noise-aware synthetic gene circuits into a well-defined human genomic safe harbor locus. Unexpectedly, engineered MDA-MB-231 metastatic human breast cancer cells become more, then less and then more invasive as we tune BACH1 levels up, irrespective of the native BACH1. BACH1 expression shifts in invading cells, and expression of BACH1's transcriptional targets confirm BACH1's nonmonotone phenotypic and regulatory effects. Thus, chemical inhibition of BACH1 could have unwanted effects on invasion. Additionally, BACH1's expression variability aids invasion at high BACH1 expression. Overall, precisely engineered, noise-aware protein-level control is necessary and important to unravel disease effects of genes to improve clinical drug efficacy.

Challenges in Ras therapeutics in pancreatic cancer.

Pancreatic cancer is considered among the most aggressive and the least curable of all human malignancies. It is usually characterized by multiple aberrations in tumor suppressor genes and oncogenes, most notably activating mutations in KRAS. This review examines the various attempts that have been made to inhibit Kras and its downstream signaling pathways in pancreatic cancer with an emphasis on challenges related to clinical trials. Attempts include preventing the localization of Ras protein to the plasma membrane, inhibiting downstream oncogenic signaling by targeting Kras effectors such as MEK1/2, Erk1/2 or Akt singly or in combination, and directly inhibiting Kras protein. Most clinical trials have focused on inhibiting downstream effector pathways and clinical benefit has been limited due to compensatory mechanisms and toxicity associated with small therapeutic windows. Additionally, genetic screens have been conducted to identify gene or genes that could provide therapeutic vulnerabilities in mutant KRAS cells and provide a way to target mutant Kras protein only. We also discuss how potentially transforming clinical trials have failed in the past and what new strategies are on-going in clinical trials for pancreas cancer. For long-term success in targeting Kras, future efforts should focus on combinatorial strategies to more effectively block Kras pathways at multiple points, and improve translational application of pre-clinical data to the clinic.

PI3K regulation of RAC1 is required for KRAS-induced pancreatic tumorigenesis in mice.

BACKGROUND & AIMS: New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. METHODS: Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110ß (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. RESULTS: Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. CONCLUSIONS: KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

Cardiac cellular coupling and the spread of early instabilities in intracellular Ca2+.

Recent experimental and modeling studies demonstrate the fine spatial scale, complex nature, and independent contribution of Ca(2+) dynamics as a proarrhythmic factor in the heart. The mechanism of progression of cell-level Ca(2+) instabilities, known as alternans, to tissue-level arrhythmias is not well understood. Because gap junction coupling dictates cardiac syncytial properties, we set out to elucidate its role in the spatiotemporal evolution of Ca(2+) instabilities. We experimentally perturbed cellular coupling in cardiac syncytium in vitro. Coupling was quantified by fluorescence recovery after photobleaching, and related to function, including subtle fine-scale Ca(2+) alternans, captured by optical mapping. Conduction velocity and threshold for alternans monotonically increased with coupling. Lower coupling enhanced Ca(2+) alternans amplitude, but the spatial spread of early (<2 Hz) alternation was the greatest under intermediate (not low) coupling. This nonmonotonic relationship was closely matched by the percent of samples exhibiting large-scale alternans at higher pacing rates. Computer modeling corroborated these experimental findings for strong but not weak electromechanical (voltage-Ca(2+)) coupling, and offered mechanistic insight. In conclusion, using experimental and modeling approaches, we reveal a general mechanism for the spatial spread of subtle cellular Ca(2+) alternans that relies on a combination of gap-junctional and voltage-Ca(2+) coupling.

Hypertrophic phenotype in cardiac cell assemblies solely by structural cues and ensuing self-organization.

In vitro models of cardiac hypertrophy focus exclusively on applying "external" dynamic signals (electrical, mechanical, and chemical) to achieve a hypertrophic state. In contrast, here we set out to demonstrate the role of "self-organized" cellular architecture and activity in reprogramming cardiac cell/tissue function toward a hypertrophic phenotype. We report that in neonatal rat cardiomyocyte culture, subtle out-of-plane microtopographic cues alter cell attachment, increase biomechanical stresses, and induce not only structural remodeling, but also yield essential molecular and electrophysiological signatures of hypertrophy. Increased cell size and cell binucleation, molecular up-regulation of released atrial natriuretic peptide, altered expression of classic hypertrophy markers, ion channel remodeling, and corresponding changes in electrophysiological function indicate a state of hypertrophy on par with other in vitro and in vivo models. Clinically used antihypertrophic pharmacological treatments partially reversed hypertrophic behavior in this in vitro model. Partial least-squares regression analysis, combining gene expression and functional data, yielded clear separation of phenotypes (control: cells grown on flat surfaces; hypertrophic: cells grown on quasi-3-dimensional surfaces and treated). In summary, structural surface features can guide cardiac cell attachment, and the subsequent syncytial behavior can facilitate trophic signals, unexpectedly on par with externally applied mechanical, electrical, and chemical stimulation.

Loss of function STK11 alterations and poor outcomes in non-small-cell lung cancer: Literature and case series of US Veterans.

Emerging evidence suggests that STK11 alterations, frequently found in non-small-cell lung cancers, may be prognostic and/or predictive of response to therapy, particularly immunotherapy. STK11 affects multiple important cellular pathways, and mutations lead to tumor growth by creating an immunosuppressive and altered metabolic environment through changes in AMPK, STING, and vascular endothelial growth factor pathways. We illustrate the questions surrounding STK11 genomic alteration in NSCLC with a case series comprising six United States Veterans from a single institution. We discuss the history of STK11, review studies on its clinical impact, and describe putative mechanisms of how loss of STK11 might engender resistance to immunotherapy or other therapies. While the exact impact of STK11 alteration in non-small-cell lung cancer remain to be fully elucidated, future research and ongoing clinical trials will help us better understand its role in cancer development and devise more effective treatment strategies.

Novel biomarkers in NSCLC: Radiomic analysis, kinetic analysis, and circulating tumor DNA.

Current radiographic methods of measuring treatment response for patients with nonsmall cell lung cancer have significant limitations. Recently, new modalities using standard of care images or minimally invasive blood-based DNA tests have gained interest as methods of evaluating treatment response. This article highlights three emerging modalities: radiomic analysis, kinetic analysis and serum-based measurement of circulating tumor DNA, with a focus on the clinical evidence supporting these methods. Additionally, we discuss the possibility of combining these modalities to develop a robust biomarker with strong correlation to clinically meaningful outcomes that could impact clinical trial design and patient care. At Last, we focus on how these methods specifically apply to a Veteran population.

Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution.

Optical mapping of cardiac excitation using voltage- and calcium-sensitive dyes has allowed a unique view into excitation wave dynamics, and facilitated scientific discovery in the cardiovascular field. At the same time, the structural complexity of the native heart has prompted the design of simplified experimental models of cardiac tissue using cultured cell networks. Such reduced experimental models form a natural bridge between single cells and tissue/organ level experimental systems to validate and advance theoretical concepts of cardiac propagation and arrhythmias. Macroscopic mapping (over >1cm(2) areas) of transmembrane potentials and intracellular calcium in these cultured cardiomyocyte networks is a relatively new development and lags behind whole heart imaging due to technical challenges. In this paper, we review the state-of-the-art technology in the field, examine specific aspects of such measurements and outline a rational system design approach. Particular attention is given to recent developments of sensitive detectors allowing mapping with ultra-high spatiotemporal resolution (>5 megapixels/s). Their interfacing with computer platforms to match the high data throughput, unique for this new generation of detectors, is discussed here. This critical review is intended to guide basic science researchers in assembling optical mapping systems for optimized macroscopic imaging with high resolution in a cultured cell setting. The tools and analysis are not limited to cardiac preparations, but are applicable for dynamic fluorescence imaging in networks of any excitable media.

The role of cardiac tissue alignment in modulating electrical function.

INTRODUCTION: Most cardiac arrhythmias are associated with pathology-triggered ion channel remodeling. However, multicellular effects, for example, exaggerated anisotropy and altered cell-to-cell coupling, can also indirectly affect action potential morphology and electrical stability via changed electrotonus. These changes are particularly relevant in structural heart disease, including hypertrophy and infarction. Recent computational studies showed that electrotonus factors into stability by altering dynamic properties (restitution). We experimentally address the question of how cell alignment and connectivity alter tissue function and whether these effects depend on the direction of wave propagation. METHODS AND RESULTS: We show that cardiac cell arrangement can alter electrical stability in an in vitro cardiac tissue model by mechanisms both dependent and independent of the direction of wave propagation, and local structural remodeling can be felt beyond a space constant. Notably, restitution of action potential duration (APD) and conduction velocity was significantly steepened in the direction of cell alignment. Furthermore, prolongation of APD and calcium transient duration was found in highly anisotropic cell networks, both for longitudinal and transverse propagation. This is in contrast to expected correlation between wave propagation direction and APD based on electrotonic effects only, but is consistent with our findings of increased cell size and secretion of atrial natriuretic factor, a hypertrophy marker, in the aligned structures. CONCLUSION: Our results show that anisotropic structure is a potent modulator of electrical stability via electrotonus and molecular signaling. Tissue alignment must be taken into account in experimental and computational models of arrhythmia generation and in designing effective treatment therapies.

Lenses and effective spatial resolution in macroscopic optical mapping.

Optical mapping of excitation dynamically tracks electrical waves travelling through cardiac or brain tissue by the use of fluorescent dyes. There are several characteristics that set optical mapping apart from other imaging modalities: dynamically changing signals requiring short exposure times, dim fluorescence demanding sensitive sensors and wide fields of view (low magnification) resulting in poor optical performance. These conditions necessitate the use of optics with good light gathering ability, i.e. lenses having high numerical aperture. Previous optical mapping studies often used sensor resolution to estimate the minimum spatial feature resolvable, assuming perfect optics and infinite contrast. We examine here the influence of finite contrast and real optics on the effective spatial resolution in optical mapping under broad-field illumination for both lateral (in-plane) resolution and axial (depth) resolution of collected fluorescence signals.

Extra-lymphatic solid tumor growth is not immunologically ignored and results in early induction of antigen-specific T-cell anergy: dominant role of cross-tolerance to tumor antigens.

A better understanding of how solid malignancies arise in an immunocompetent host, avoid immune recognition, and ultimately progress to widely disseminated cancer is essential to effectively harness the immune system against solid tumors. Because of their extra-lymphatic localization, it has been proposed that solid malignancies are just ignored by the immune system, thereby allowing their uncontrolled growth and dissemination. Alternatively, as most of the solid tumors are unable to express costimulatory molecules, the "signal one without signal two" model of tolerance induction has been frequently evoked to account for the failure of the immune system to reject antigenic tumors in vivo. In this study, we showed, however, that the extra-lymphatic growth of solid tumors is not immunologically ignored by the lymphoid compartment, resulting instead in the early induction of antigen-specific CD4(+) T-cell tolerance. Furthermore, analysis of parent-into-F1 bone marrow (BM) chimeras demonstrates that presentation of tumor antigens by BM-derived antigen-presenting cells represents the dominant mechanism in solid tumor-induced CD4(+) T-cell tolerance. Our findings of early development of antigen-specific T-cell unresponsiveness mediated by BM-derived antigen-presenting cells, not only provides a plausible explanation for the failure of the immune system to reject antigenic solid tumors in vivo, but more importantly, they have identified a barrier that, if appropriately manipulated, may lead to approaches to effectively harness the immune system against solid malignancies.

Liposomal Irinotecan in the Treatment of Refractory Pancreatic Cancer.

Effective therapies against metastatic pancreatic cancer remain limited, and despite treatment, many will ultimately progress. Previously, few options were available for second line therapy in metastatic pancreatic cancer. Liposomal encapsulated irinotecan, in combination with leucovorin-modulated fluorouracil, was found to significantly increase overall survival in patients who have progressed after gemcitabine- based therapy in a large, international, randomized clinical trial (NAPOLI-1). We reviewed the background of systemic therapy for metastatic pancreatic cancer, examined putative mechanisms for the success of encapsulated drugs, and identified recent patent applications on the use of liposomal irinotecan in pancreatic cancer. The landmark NAPOLI-1 trial established a second-line option for those with metastatic pancreatic cancer refractory to gemcitabine chemotherapy, but effective therapies with long duration of response are still lacking. Alternative techniques targeting key driver genes in pancreatic cancer and novel methods of early detection and targeting drugs are currently being explored. How liposomal irinotecan can be integrated into chemotherapy regimens, including neoadjuvant or first line combinations, are currently being tested in clinical trials and covered by several new patent applications.

OptoDyCE as an automated system for high-throughput all-optical dynamic cardiac electrophysiology.

The improvement of preclinical cardiotoxicity testing, discovery of new ion-channel-targeted drugs, and phenotyping and use of stem cell-derived cardiomyocytes and other biologics all necessitate high-throughput (HT), cellular-level electrophysiological interrogation tools. Optical techniques for actuation and sensing provide instant parallelism, enabling contactless dynamic HT testing of cells and small-tissue constructs, not affordable by other means. Here we show, computationally and experimentally, the limits of all-optical electrophysiology when applied to drug testing, then implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We validate optical actuation by virally introducing optogenetic drivers in rat and human cardiomyocytes or through the modular use of dedicated light-sensitive somatic 'spark' cells. We show that this automated all-optical approach provides HT means of cellular interrogation, that is, allows for dynamic testing of >600 multicellular samples or compounds per hour, and yields high-content information about the action of a drug over time, space and doses.

Acoustic micromachining of three-dimensional surfaces for biological applications.

We present the use of an accessible micromachining technique (acoustic micromachining) for manufacturing micron-feature surfaces with non-discretely varying depth. Acoustic micromachining allows for non-photolithographic production of metal templates with programmable spatial patterns and involves the use of standard acoustic, cutting and electroplating equipment for mass production of vinyl records. Simple 3D patterns were transferred from an acoustic signal into working nickel templates, from which elastic polymer molds were obtained, featuring deep surface grooves and non-discrete (smooth) variations in the z-dimension. Versatility and applicability of the method is demonstrated in obtaining microfluidics structures, manufacturing high-surface area wavy polymer fibers, assembly of cell networks on scaffolds with 3D topography, and microcontact printing of proteins and cells.

Electrospun fine-textured scaffolds for heart tissue constructs.

The structural and functional effects of fine-textured matrices with sub-micron features on the growth of cardiac myocytes were examined. Electrospinning was used to fabricate biodegradable non-woven poly(lactide)- and poly(glycolide)-based (PLGA) scaffolds for cardiac tissue engineering applications. Post-processing was applied to achieve macro-scale fiber orientation (anisotropy). In vitro studies confirmed a dose-response effect of the poly(glycolide) concentration on the degradation rate and the pH value changes. Different formulations were examined to assess scaffold effects on cell attachment, structure and function. Primary cardiomyocytes (CMs) were cultured on the electrospun scaffolds to form tissue-like constructs. Scanning electron microscopy (SEM) revealed that the fine fiber architecture of the non-woven matrix allowed the cardiomyocytes to make extensive use of provided external cues for isotropic or anisotropic growth, and to some extent to crawl inside and pull on fibers. Structural analysis by confocal microscopy indicated that cardiomyocytes had a preference for relatively hydrophobic surfaces. CMs on electrospun poly(L-lactide) (PLLA) scaffolds developed mature contractile machinery (sarcomeres). Functionality (excitability) of the engineered constructs was confirmed by optical imaging of electrical activity using voltage-sensitive dyes. We conclude that engineered cardiac tissue structure and function can be modulated by the chemistry and geometry of the provided nano- and micro-textured surfaces. Electrospinning is a versatile manufacturing technique for design of biomaterials with potentially reorganizable architecture for cell and tissue growth.

Functional cardiac cell constructs on cellulose-based scaffolding.

Cellulose and its derivatives have been successfully employed as biomaterials in various applications, including dialysis membranes, diffusion-limiting membranes in biosensors, in vitro hollow fibers perfusion systems, surfaces for cell expansion, etc. In this study, we tested the potential of cellulose acetate (CA) and regenerated cellulose (RC) scaffolds for growing functional cardiac cell constructs in culture. Specifically, we demonstrate that CA and RC surfaces are promoting cardiac cell growth, enhancing cell connectivity (gap junctions) and electrical functionality. Being optically clear and essentially non-autofluorescent, CA scaffolds did not interfere with functional optical measurements in the cell constructs. Molding to follow fine details or complex three-dimensional shapes are additional important characteristics for scaffold design in tissue engineering. Biodegradability can be controlled by hydrolysis, de-acetylization of CA and cytocompatible enzyme (cellulase) action, with glucose as a final product. Culturing of cardiac cells and growth of tissue-like cardiac constructs in vitro could benefit from the versatility and accessibility of cellulose scaffolds, combining good adhesion (comparable to the standard tissue-culture treated polystyrene), molding capabilities down to the nanoscale (comparable to the current favorite in soft lithography-polydimethylsiloxane) with controlled biodegradability.

Stimulating cardiac muscle by light: cardiac optogenetics by cell delivery.

BACKGROUND: After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before. METHODS AND RESULTS: We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves. CONCLUSIONS: Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.

Detecting space-time alternating biological signals close to the bifurcation point.

Time-alternating biological signals, i.e., alternans, arise in variety of physiological states marked by dynamic instabilities, e.g., period doubling. Normally, a sequence of large-small-large transients, they can exhibit variable patterns over time and space, including spatial discordance. Capture of the early formation of such alternating regions is challenging because of the spatiotemporal similarities between noise and the small-amplitude alternating signals close to the bifurcation point. We present a new approach for automatic detection of alternating signals in large noisy spatiotemporal datasets by exploiting quantitative measures of alternans evolution, e.g., temporal persistence, and by preserving phase information. The technique specifically targets low amplitude, relatively short alternating sequences and is validated by combinatorics-derived probabilities and empirical datasets with white noise. Using high-resolution optical mapping in live cardiomyocyte networks, exhibiting calcium alternans, we reveal for the first time early fine-scale alternans, close to the noise level, which are linked to the later formation of larger regions and evolution of spatially discordant alternans. This robust method aims at quantification and better understanding of the onset of cardiac arrhythmias and can be applied to general analysis of space-time alternating signals, including the vicinity of the bifurcation point.

Mechanical and spatial determinants of cytoskeletal geodesic dome formation in cardiac fibroblasts.

This study tests the hypothesis that the cell cytoskeletal (CSK) network can rearrange from geodesic dome type structures to stress fibers in response to microenvironmental cues. The CSK geodesic domes are highly organized actin microarchitectures within the cell, consisting of ordered polygonal elements. We studied primary neonatal rat cardiac fibroblasts. The cues used to trigger the interconversion between the two CSK architectures (geodesic domes and stress fibers) included factors affecting spatial order and the degree of CSK tension in the cells. Microfabricated three-dimensional substrates with micrometre sized grooves and peaks were used to alter the spatial order of cell growth in culture. CSK tension was modified by 2,3-butanedione 2-monoxime (BDM), cytochalasin D and the hyphae of Candida albicans. CSK geodesic domes occurred spontaneously in about 20% of the neonatal rat cardiac fibroblasts used in this study. Microfabricated structured surfaces produced anisotropy in the cell CSK and effectively converted geodesic domes into stress fibers in a dose-dependent manner (dependence on the period of the features). Affectors of actin structure, inhibitors of CSK tension and cell motility, e.g. BDM, cytochalasin D and the hyphae of C. albicans, suppressed or eliminated the geodesic domes. Our data suggest that the geodesic domes, similar to actin stress fibers, require maintenance of CSK integrity and tension. However, microenvironments that promote structural anisotropy in tensed cells cause the transformation of the geodesic domes into stress fibers, consistent with topographic cell guidance and some previous CSK model predictions.