Tuning Hydrogel Adhesivity and Degradability to Model the Influence of Premetastatic Niche Matrix Properties on Breast Cancer Dormancy and Reactivation.

Dormant, disseminated tumor cells (DTCs) can persist for decades in secondary tissues before being reactivated to form tumors. The properties of the premetastatic niche can influence the DTC phenotype. To better understand how matrix properties of premetastatic niches influence DTC behavior, three hydrogel formulations are implemented to model a permissive niche and two nonpermissive niches. Poly(ethylene glycol) (PEG)-based hydrogels with varying adhesivity ([RGDS]) and degradability ([N-vinyl pyrrolidinone]) are implemented to mimic a permissive niche with high adhesivity and degradability and two nonpermissive niches, one with moderate adhesivity and degradability and one with no adhesivity and high degradability. The influence of matrix properties on estrogen receptor positive (ER+ ) breast cancer cells (MCF7s) is determined via a multimetric analysis. MCF7s cultured in the permissive niche adopted a growth state, while those in the nonpermissive niche with reduced adhesivity and degradability underwent tumor mass dormancy. Complete removal of adhesivity while maintaining high degradability induced single cell dormancy. The ability to mimic reactivation of dormant cells through a dynamic increase in [RGDS] is also demonstrated. This platform provides the capability of inducing growth, dormancy, and reactivation of ER+  breast cancer and can be useful in understanding how premetastatic niche properties influence cancer cell fate.

Secretion of the disulfide bond generating catalyst QSOX1 from pancreatic tumor cells into the extracellular matrix: association with extracellular vesicles and matrix proteins.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a disulfide bond generating catalyst that is overexpressed in solid tumors. Expression of QSOX1 is linked to cancer cell invasion, tumor grade, and extracellular matrix (ECM) protein deposition. While the secreted version of QSOX1 is known to be present in various fluids and secretory tissues, its presence in the ECM of cancer is less understood. To characterize secreted QSOX1, we separated conditioned media based on size and density. We discovered that the majority of secreted QSOX1 resides in the EV-depleted fraction and in the soluble protein fraction. Very little QSOX1 could be detected in the EVP fraction. We used immunofluorescence to image subpopulations of EVs and found QSOX1 in Golgi-derived vesicles and medium/large vesicles, but in general, most extracellular QSOX1 was not attributed to these vesicles. Next, we quantified QSOX1 co-localization with the EV marker Alix. For the medium/large EVs, ~98% contained QSOX1 when fibronectin was used as a coating. However, on collagen coatings, only ~60% of these vesicles contained QSOX1, suggesting differences in EV cargo based on ECM coated surfaces. About 10% of small EVs co-localized with QSOX1 on every ECM protein surface except for collagen (0.64%). We next investigated adhesion of QSOX1 to ECM proteins in vitro and in situ and found that QSOX1 preferentially adheres to fibronectin, laminins, and Matrigel compared to gelatin and collagen. This mechanism was found to be, in part, mediated by the formation of mixed disulfides between QSOX1 and cysteine-rich ECM proteins. In summary, we found that QSOX1 (1) is in subpopulations of medium/large EVs, (2) seems to interact with small Alix+ EVs, and (3) adheres to cysteine-rich ECM proteins, potentially through the formation of intermediate disulfides. These observations offer significant insight into how enzymes, such as QSOX1, can facilitate matrix remodeling events in solid tumor progression.

The Influence of Ligand Density and Degradability on Hydrogel Induced Breast Cancer Dormancy and Reactivation.

The role of hydrogel properties in regulating the phenotype of triple negative metastatic breast cancer is investigated using four cell lines: the MDA-MB-231 parental line and three organotropic sublines BoM-1833 (bone-tropic), LM2-4175 (lung-tropic), and BrM2a-831 (brain-tropic). Each line is encapsulated and cultured for 15 days in three poly(ethylene glycol) (PEG)-based hydrogel formulations composed of proteolytically degradable PEG, integrin-ligating RGDS, and the non-degradable crosslinker N-vinyl pyrrolidone. Dormancy-associated metrics including viable cell density, proliferation, metabolism, apoptosis, chemoresistance, phosphorylated-ERK and -p38, and morphological characteristics are quantified. A multimetric classification approach is implemented to categorize each hydrogel-induced phenotype as: 1) growth, 2) balanced tumor dormancy, 3) balanced cellular dormancy, or 4) restricted survival, cellular dormancy. Hydrogels with high adhesivity and degradability promote growth. Hydrogels with no adhesivity, but high degradability, induce restricted survival, cellular dormancy in the parental line and balanced cellular dormancy in the organotropic lines. Hydrogels with reduced adhesivity and degradability induce balanced cellular dormancy in the parental and lung-tropic lines and balanced tumor mass dormancy in bone- and brain-tropic lines. The ability to induce escape from dormancy via dynamic incorporation of RGDS is also presented. These results demonstrate that ECM properties and organ-tropism synergistically regulate cancer cell phenotype and dormancy.

Biofabrication Strategies and Engineered In Vitro Systems for Vascular Mechanobiology.

The vascular system is integral for maintaining organ-specific functions and homeostasis. Dysregulation in vascular architecture and function can lead to various chronic or acute disorders. Investigation of the role of the vascular system in health and disease has been accelerated through the development of tissue-engineered constructs and microphysiological on-chip platforms. These in vitro systems permit studies of biochemical regulation of vascular networks and parenchymal tissue and provide mechanistic insights into the biophysical and hemodynamic forces acting in organ-specific niches. Detailed understanding of these forces and the mechanotransductory pathways involved is necessary to develop preventative and therapeutic strategies targeting the vascular system. This review describes vascular structure and function, the role of hemodynamic forces in maintaining vascular homeostasis, and measurement approaches for cell and tissue level mechanical properties influencing vascular phenomena. State-of-the-art techniques for fabricating in vitro microvascular systems, with varying degrees of biological and engineering complexity, are summarized. Finally, the role of vascular mechanobiology in organ-specific niches and pathophysiological states, and efforts to recapitulate these events using in vitro microphysiological systems, are explored. It is hoped that this review will help readers appreciate the important, but understudied, role of vascular-parenchymal mechanotransduction in health and disease toward developing mechanotherapeutics for treatment strategies.

The Influence of Matrix-Induced Dormancy on Metastatic Breast Cancer Chemoresistance.

Metastasis remains the leading cause of cancer-associated death worldwide. Disseminated tumor cells can undergo dormancy upon infiltration of secondary organs, and chemotherapeutics fail to effectively eliminate dormant populations. Mechanistic understanding of dormancy-associated chemoresistance could lead to development of targeted therapeutic strategies. Toward this goal, we implemented three poly(ethylene glycol) (PEG)-based hydrogel formulations fabricated from proteolytically degradable PEG (PEG-PQ), integrin ligating PEG-RGDS, and the non-degradable cross-linker N-vinylpyrrolidone (NVP) to induce three distinct phenotypes in triple negative MDA-MB-231 breast cancer cells. With constant 5% w/v PEG-PQ, PEG-RGDS and NVP concentrations were tuned to induce (i) a growth state characterized by high proliferation, high metabolic activity, significant temporally increased cell density, and an invasive morphology; (ii) a balanced dormancy state characterized by a temporal balance (~1:1 ratio) in new live and dead cell density and a non-invasive morphology; and (iii) a cellular dormancy state characterized by rounded, solitary quiescent cells with low viability, proliferation, and metabolic activity. The cellular responses to doxorubicin (DOX), paclitaxel (PAC), and 5-fluorouracil (5-FU) in the three phenotypic states were quantified. Under DOX treatment, cells in dormant states demonstrated increased chemoresistance with a 1.4- to 1.8-fold increase in half maximal effective concentration (EC50) and 1.3- to 1.8-fold increase in half maximal inhibitory concentration (IC50) compared to cells in the growth state. PAC and 5-FU treatment led to similar results. To mechanistically investigate the role of dormancy in conferring DOX resistance, cytoplasmic and nuclear accumulation of DOX was measured. The results indicated comparable DOX accumulation between all three phenotypic states; however, the intracellular to intranuclear distribution indicated a ~1.5 fold increase in DOX nuclear accumulation in cells in the growth state compared to the two dormant states. These results further validate the utility of implementing engineered hydrogels as in vitro platforms of breast cancer dormancy for the development of anti-dormancy therapeutic strategies.

Fabrication, characterization, and implementation of engineered hydrogels for controlling breast cancer cell phenotype and dormancy.

A better understanding of how microenvironmental factors regulate cancer dormancy is needed for development of new therapeutic strategies to control metastatic recurrence and disease progression. Modeling cancer dormancy using engineered, in vitro platforms is necessary for investigation under well-defined and well-controlled microenvironments. We present methods and protocols to fabricate, characterize, and implement engineered hydrogels with well-defined biochemical and physical properties for control over breast cancer cell phenotype in three-dimensional (3D) culture. Changes in hydrogel adhesivity, crosslink density, and degradability induce a range of phenotypic behaviors in breast cancer cells including: (1) high growth, (2) moderate growth, (3) single cell, restricted survival dormancy, and (4) balanced dormancy. We describe a method of classifying hydrogel formulations that support each of these phenotypic states. We also describe a method to phenotypically switch cancer cells from single cell dormancy to high growth by dynamically modulating ligand density, thereby recapitulating reactivation and metastatic recurrence.

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

Accurate flow in augmented networks (AFAN): an approach to generating three-dimensional biomimetic microfluidic networks with controlled flow.

In vivo, microvasculature provides oxygen, nutrients, and soluble factors necessary for cell survival and function. The highly tortuous, densely-packed, and interconnected three-dimensional (3D) architecture of microvasculature ensures that cells receive these crucial components. The ability to duplicate microvascular architecture in tissue-engineered models could provide a means to generate large-volume constructs as well as advanced microphysiological systems. Similarly, the ability to induce realistic flow in engineered microvasculature is crucial to recapitulating in vivo-like flow and transport. Advanced biofabrication techniques are capable of generating 3D, biomimetic microfluidic networks in hydrogels, however, these models can exhibit systemic aberrations in flow due to incorrect boundary conditions. To overcome this problem, we developed an automated method for generating synthetic augmented channels that induce the desired flow properties within three-dimensional microfluidic networks. These augmented inlets and outlets enforce the appropriate boundary conditions for achieving specified flow properties and create a three-dimensional output useful for image-guided fabrication techniques to create biomimetic microvascular networks.

Datasets describing hydrogel properties and cellular metrics for modeling of tumor dormancy.

Breast cancer dormancy is an underlying challenge toward targeting and controlling metastatic recurrence and disease progression. Development of engineered, well-defined in vitro models is necessary to systematically recapitulate tumor dormancy and investigate potential therapeutic strategies. Toward this end, a set of sixteen hydrogel formulations with varying degrees of adhesivity and crosslink density was developed for encapsulation, three-dimensional (3D) culture, and phenotypic assessment of MDA-MB-231 breast cancer cells. The hydrogel adhesivity was regulated by incorporation of RGDS peptide conjugated to acrylate poly(ethylene glycol) (PEG-RGDS) and the crosslink density by incorporation of N-vinyl pyrrolidinone (NVP). Here, we present data concerning the characterization of hydrogel properties (PEG-RGDS incorporation, hydrogel crosslink density, and hydrogel diffusivity as a function of NVP concentration) and phenotypic metrics (viability, early apoptosis, proliferation, metabolic activity, viable cell density, and morphological features) of encapsulated MDA-MB-231s over 15 days in culture. Interpretation of this data can be found in a research article titled "Tunable Hydrogels for Controlling Phenotypic Cancer Cell States to Model Breast Cancer Dormancy and Reactivation" (Pradhan et al., 2019) [1].

Tunable hydrogels for controlling phenotypic cancer cell states to model breast cancer dormancy and reactivation.

During metastasis, disseminated tumor cells (DTCs) from the primary tumor infiltrate secondary organs and reside there for varying lengths of time prior to forming new tumors. The time delay between infiltration and active proliferation, known as dormancy, mediates the length of the latency period. DTCs may undergo one of four fates post-infiltration: death, cellular dormancy, dormant micrometastasis, or invasive growth which, is in part, mediated by extracellular matrix (ECM) properties. Recapitulation of these cell states using engineered hydrogels could facilitate the systematic and controlled investigation of the mechanisms by which ECM properties influence DTC fate. Toward this goal, we implemented a set of sixteen hydrogels with systematic variations in chemical (ligand (RGDS) density and enzymatic degradability) and mechanical (elasticity, swelling, mesh size) properties to investigate their influence on the fate of encapsulated metastatic breast cancer cells, MDA-MB-231. Cell viability, apoptosis, proliferation, metabolic activity, and morphological measurements were acquired at five-day intervals over fifteen days in culture. Analysis of the phenotypic metrics indicated the presence of four different cell states that were classified as: (1) high growth, (2) moderate growth, (3) single cell, restricted survival, dormancy, or (4) balanced dormancy. Correlating hydrogel properties with the resultant cancer cell state indicated that ligand (RGDS) density and enzymatic degradability likely had the most influence on cell fate. Furthermore, we demonstrate the ability to reactivate cells from the single cell, dormant state to the high growth state through a dynamic increase in ligand (RGDS) density after forty days in culture. This tunable engineered hydrogel platform offers insight into matrix properties regulating tumor dormancy, and the dormancy-proliferation switch, and may provide future translational benefits toward development of anti-dormancy therapeutic strategies.

Reference-Free Traction Force Microscopy Platform Fabricated via Two-Photon Laser Scanning Lithography Enables Facile Measurement of Cell-Generated Forces.

Cells sense and respond to the physical nature of their microenvironment by mechanically probing their surroundings via cytoskeletal contractions. The material response to these stresses can be measured via traction force microscopy (TFM). Traditional TFM platforms present several limitations including variable spatial resolution, difficulty in attaining the full three-dimensional (3D) deformation/stress profile, and the requirement to remove or relax the cells being measured to determine the zero-stress state. To overcome these limitations, we developed a two-photon, photochemical coupling approach to fabricate a new TFM platform that provides high-resolution control over the 3D placement of fluorescent fiducial markers for facile measurement of cell-generated shear and normal components of traction forces. The highly controlled placement of the 3D marker array provides a built-in, zero stress state eliminating the need to perturb the cells being measured while also providing increased throughput. Using this platform, we discovered that the magnitude of cell-generated shear and normal force components are linked both spatially and temporally. The facile nature and increased throughput of measuring cell-generated forces afforded by this new platform will be useful to the mechanotransduction community and others.

Brain Capillary Networks Across Species: A few Simple Organizational Requirements Are Sufficient to Reproduce Both Structure and Function.

Despite the key role of the capillaries in neurovascular function, a thorough characterization of cerebral capillary network properties is currently lacking. Here, we define a range of metrics (geometrical, topological, flow, mass transfer, and robustness) for quantification of structural differences between brain areas, organs, species, or patient populations and, in parallel, digitally generate synthetic networks that replicate the key organizational features of anatomical networks (isotropy, connectedness, space-filling nature, convexity of tissue domains, characteristic size). To reach these objectives, we first construct a database of the defined metrics for healthy capillary networks obtained from imaging of mouse and human brains. Results show that anatomical networks are topologically equivalent between the two species and that geometrical metrics only differ in scaling. Based on these results, we then devise a method which employs constrained Voronoi diagrams to generate 3D model synthetic cerebral capillary networks that are locally randomized but homogeneous at the network-scale. With appropriate choice of scaling, these networks have equivalent properties to the anatomical data, demonstrated by comparison of the defined metrics. The ability to synthetically replicate cerebral capillary networks opens a broad range of applications, ranging from systematic computational studies of structure-function relationships in healthy capillary networks to detailed analysis of pathological structural degeneration, or even to the development of templates for fabrication of 3D biomimetic vascular networks embedded in tissue-engineered constructs.

Engineered In Vitro Models of Tumor Dormancy and Reactivation.

Metastatic recurrence is a major hurdle to overcome for successful control of cancer-associated death. Residual tumor cells in the primary site, or disseminated tumor cells in secondary sites, can lie in a dormant state for long time periods, years to decades, before being reactivated into a proliferative growth state. The microenvironmental signals and biological mechanisms that mediate the fate of disseminated cancer cells with respect to cell death, single cell dormancy, tumor mass dormancy and metastatic growth, as well as the factors that induce reactivation, are discussed in this review. Emphasis is placed on engineered, in vitro, biomaterial-based approaches to model tumor dormancy and subsequent reactivation, with a focus on the roles of extracellular matrix, secondary cell types, biochemical signaling and drug treatment. A brief perspective of molecular targets and treatment approaches for dormant tumors is also presented. Advances in tissue-engineered platforms to induce, model, and monitor tumor dormancy and reactivation may provide much needed insight into the regulation of these processes and serve as drug discovery and testing platforms.

Fundamentals of Laser-Based Hydrogel Degradation and Applications in Cell and Tissue Engineering.

The cell and tissue engineering fields have profited immensely through the implementation of highly structured biomaterials. The development and implementation of advanced biofabrication techniques have established new avenues for generating biomimetic scaffolds for a multitude of cell and tissue engineering applications. Among these, laser-based degradation of biomaterials is implemented to achieve user-directed features and functionalities within biomimetic scaffolds. This review offers an overview of the physical mechanisms that govern laser-material interactions and specifically, laser-hydrogel interactions. The influences of both laser and material properties on efficient, high-resolution hydrogel degradation are discussed and the current application space in cell and tissue engineering is reviewed. This review aims to acquaint readers with the capability and uses of laser-based degradation of biomaterials, so that it may be easily and widely adopted.

Being beyond: The Black Legend and how we got over it.

We used to think it was the job of a historian of Spanish science to combat the negative evaluations of Hispanic cultures that came to be known as the Black Legend. Paradoxically, attempts to amend dominant narratives of the history of science (such as the Scientific Revolution) so that they might accommodate Spain bolstered the very stories we meant to dismantle. Caring about the Black Legend deformed the history we were trying to write and never convinced the people we hoped to sway. In this article, we provide an overview of the historiographic tendencies that most shaped our careers - responses to the Black Legend, such as contributionist history and bibliometrics - and explain why we have chosen to move on.

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

Fabrication of 3D Biomimetic Microfluidic Networks in Hydrogels.

A laser-based hydrogel degradation technique is developed that allows for local control over hydrogel porosity, fabrication of 3D vascular-derived, biomimetic, hydrogel-embedded microfluidic networks, and generation of two intertwining, yet independent, microfluidic networks in a single construct.

Biomimetic Surface Patterning Promotes Mesenchymal Stem Cell Differentiation.

Both chemical and mechanical stimuli can dramatically influence cell behavior. By optimizing the signals cells experience, it may be possible to control the behavior of therapeutic cell populations. In this work, biomimetic geometries of adhesive ligands, which recapitulate the morphology of mature cells, are used to direct human mesenchymal stem cell (HMSC) differentiation toward a desired lineage. Specifically, adipocytes cultured in 2D are imaged and used to develop biomimetic virtual masks used in laser scanning lithography to form patterned fibronectin surfaces. The impact of adipocyte-derived pattern geometry on HMSC differentiation is compared to the behavior of HMSCs cultured on square and circle geometries, as well as adipocyte-derived patterns modified to include high stress regions. HMSCs on adipocyte mimetic geometries demonstrate greater adipogenesis than HMSCs on the other patterns. Greater than 45% of all HMSCs cultured on adipocyte mimetic patterns underwent adipogenesis as compared to approximately 19% of cells on modified adipocyte patterns with higher stress regions. These results are attributed to variations in cytoskeletal tension experienced by cells on the different protein micropatterns. The effects of geometry on adipogenesis are mitigated by the incorporation of a cytoskeletal protein inhibitor; exposure to this inhibitor leads to increased adipogenesis on all patterns examined.

Progeny Clustering: A Method to Identify Biological Phenotypes.

Estimating the optimal number of clusters is a major challenge in applying cluster analysis to any type of dataset, especially to biomedical datasets, which are high-dimensional and complex. Here, we introduce an improved method, Progeny Clustering, which is stability-based and exceptionally efficient in computing, to find the ideal number of clusters. The algorithm employs a novel Progeny Sampling method to reconstruct cluster identity, a co-occurrence probability matrix to assess the clustering stability, and a set of reference datasets to overcome inherent biases in the algorithm and data space. Our method was shown successful and robust when applied to two synthetic datasets (datasets of two-dimensions and ten-dimensions containing eight dimensions of pure noise), two standard biological datasets (the Iris dataset and Rat CNS dataset) and two biological datasets (a cell phenotype dataset and an acute myeloid leukemia (AML) reverse phase protein array (RPPA) dataset). Progeny Clustering outperformed some popular clustering evaluation methods in the ten-dimensional synthetic dataset as well as in the cell phenotype dataset, and it was the only method that successfully discovered clinically meaningful patient groupings in the AML RPPA dataset.

Recapitulation and Modulation of the Cellular Architecture of a User-Chosen Cell of Interest Using Cell-Derived, Biomimetic Patterning.

Heterogeneity of cell populations can confound population-averaged measurements and obscure important findings or foster inaccurate conclusions. The ability to generate a homogeneous cell population, at least with respect to a chosen trait, could significantly aid basic biological research and development of high-throughput assays. Accordingly, we developed a high-resolution, image-based patterning strategy to produce arrays of single-cell patterns derived from the morphology or adhesion site arrangement of user-chosen cells of interest (COIs). Cells cultured on both cell-derived patterns displayed a cellular architecture defined by their morphology, adhesive state, cytoskeletal organization, and nuclear properties that quantitatively recapitulated the COIs that defined the patterns. Furthermore, slight modifications to pattern design allowed for suppression of specific actin stress fibers and direct modulation of adhesion site dynamics. This approach to patterning provides a strategy to produce a more homogeneous cell population, decouple the influences of cytoskeletal structure, adhesion dynamics, and intracellular tension on mechanotransduction-mediated processes, and a platform for high-throughput cellular assays.