Daniel M. Lewis

Intratumoral oxygen gradients mediate sarcoma cell invasion

Significance Previous works demonstrated the role of hypoxia in tumor development and metastasis. However, the understanding how oxygen (O2) gradients regulate early stages of tumor metastasis is lacking. Leveraging our O2-controlling hydrogel, we generated a 3D in vitro model that enables us to analyze cancer cell responses to O2 gradients and small-molecule inhibitors. Using this approach, we present a previously unidentified concept in which O2 acts as a 3D physicotactic agent during sarcoma tumor invasion, findings that are important for the understanding of the metastatic process. Through this concept, we also establish the 3D in vitro model as a platform for testing therapeutic targets and interventions for the treatment of sarcoma and potentially other cancers. Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm3) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1–6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.

Endothelial progenitor cell recruitment in a microfluidic vascular model

During vessel injury, endothelial progenitors cells (EPCs) are recruited from bone marrow and directed to the hypoxic injury site. The hypoxic conditions in the damaged blood vessel promote TNF-α, which upregulates intercellular adhesion molecule-1 (ICAM-1). EPCs attach to endothelial cell lining using ICAM-1. Here we aimed to examine EPC attachment to ECs in an injured-blood vessel conditions. We first determined ICAM-1 expression in stimulated HUVECs. We stimulated HUVECs with 21% oxygen (atmospheric), atmospheric with TNF-α-supplemented media, 1% oxygen (hypoxia), and hypoxia with TNF-α-supplemented media and found the highest ECFC attachment on HUVECs stimulated with TNF-α and hypoxia, correlating with the highest ICAM-1 expression. We next designed, fabricated and tested a three-dimensional microbioreactor (3D MBR) system with precise control and monitoring of dissolve oxygen and media flow rate in the cellular environment. We utilized a step-wise seeding approach, producing monolayer of HUVECs on all four walls. When stimulated with both TNF-α and hypoxia, ECFC retention on HUVECs was significantly increased under low shear stress compared to static controls. Overall, the 3D MBR system mimics the pathological oxygen tension and shear stress in the damaged vasculature, providing a platform to model vascular-related disorders.

Bioinspired Hydrogels to Engineer Cancer Microenvironments

Recent research has demonstrated that tumor microenvironments play pivotal roles in tumor development and metastasis through various physical, chemical, and biological factors, including extracellular matrix (ECM) composition, matrix remodeling, oxygen tension, pH, cytokines, and matrix stiffness. An emerging trend in cancer research involves the creation of engineered three-dimensional tumor models using bioinspired hydrogels that accurately recapitulate the native tumor microenvironment. With recent advances in materials engineering, many researchers are developing engineered tumor models, which are promising platforms for the study of cancer biology and for screening of therapeutic agents for better clinical outcomes. In this review, we discuss the development and use of polymeric hydrogel materials to engineer native tumor ECMs for cancer research, focusing on emerging technologies in cancer engineering that aim to accelerate clinical outcomes.

Sex differences in the mitochondrial bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension

ABSTRACT Biological sex is thought to influence mitochondrial bioenergetic function. Previous respiration measurements examining brain mitochondrial sex differences were made at atmospheric oxygen using isolated brain mitochondria. Oxygen is 160 mm Hg (21%) in the atmosphere, while the oxygen tension in the brain generally ranges from ˜5 to 45 mm Hg (˜1–6% O2). This study tested the hypothesis that sex and/or brain physiological oxygen tension influence the mitochondrial bioenergetic properties of primary rat cortical astrocytes and microglia. Oxygen consumption was measured with a Seahorse XF24 cell respirometer in an oxygen‐controlled environmental chamber. Strikingly, male astrocytes had a higher maximal respiration than female astrocytes when cultured and assayed at 3% O2. Three percent O2 yielded a low physiological dissolved O2 level of ˜1.2% (9.1 mm Hg) at the cell monolayer during culture and 1.2–3.0% O2 during assays. No differences in bioenergetic parameters were observed between male and female astrocytes at 21% O2 (dissolved O2 of ˜19.7%, 150 mm Hg during culture) or between either of these cell populations and female astrocytes at 3% O2. In contrast to astrocytes, microglia showed no sex differences in mitochondrial bioenergetic parameters at either oxygen level, regardless of whether they were non‐stimulated or activated to a proinflammatory state. There were also no O2‐ or sex‐dependent differences in proinflammatory TNF‐&agr; or IL‐1&bgr; cytokine secretion measured at 18 h activation. Overall, results reveal an intriguing sex variance in astrocytic maximal respiration that requires additional investigation. Findings also demonstrate that sex differences can be masked by conducting experiments at non‐physiological O2. HIGHLIGHTSMale astrocytes have greater respiratory capacity than female at a physiological O2.No sex differences in astrocyte respiration are seen at atmospheric O2.No sex differences in microglial respiration are observed at either O2.Respiration is more inhibited when microglia are activated at a physiological O2.

O2-controllable hydrogels for studying cellular responses to hypoxic gradients in three dimensions in vitro and in vivo

Oxygen (O2) acts as a potent upstream regulator of cell function. In both physiological and pathophysiological microenvironments, the O2 concentration is not uniformly distributed but instead follows a gradient that depends on distance from oxygen-carrying blood vessels. Such gradients have a particularly important role in development, tissue regeneration, and tumor growth. In this protocol, we describe how to use our previously reported gelatin-based O2-controllable hydrogels that can provide hypoxic microenvironments in vitro. The hydrogel polymeric network is formed via a laccase-mediated cross-linking reaction. In this reaction, laccase catalyzes diferulic acid (diFA) formation to form hydrogels with an O2-consuming reaction. Cells, such as cancer or endothelial cells, as well as tumor/tissue grafts, can be encapsulated in the hydrogels during hydrogel formation and then analyzed for cellular responses to 3D hypoxic gradients and to elucidate the underlying mechanisms governing these responses. Importantly, oxygen gradients can be precisely controlled in standard cell/tissue culture conditions and in vivo. This platform has been applied to study vascular morphogenesis in response to hypoxia and to understand how oxygen gradients mediate cancer cell behavior. Herein, we describe the means to validate the assay from polymer synthesis and characterization—which take 1–2 weeks and include verification of ferulic acid (FA) conjugation, rheological measurements, and O2 monitoring—to the study of cellular responses and use in rodent models. Time courses for biological experiments using this hydrogel are variable, and thus they may range from hours to weeks, depending on the application and user end goal.

Microfluidic platform for the real time measurement and observation of endothelial barrier function under shear stress

Electric cell-substrate impedance sensing (ECIS) is a quickly advancing field to measure the barrier function of endothelial cells. Most ECIS systems that are commercially available use gold electrodes, which are opaque and do not allow for real-time imaging of cellular responses. In addition, most ECIS systems have a traditional tissue culture Petri-dish set up. This conventional set-up does not allow the introduction of physiologically relevant shear stress, which is crucial for the endothelial cell barrier function. Here, we created a new ECIS micro-bioreactor (MBR) that incorporates a clear electrode made of indium tin oxide in a microfluidic device. Using this device, we demonstrate the ability to monitor the barrier function along culture of cells under varying flow rates. We show that while two cell types align in the direction of flow in responses to high shear stress, they differ in the barrier function. Additionally, we observe a change in the barrier function in response to chemical perturbation. Following exposure to EDTA that disrupts cell-to-cell junctions, we could not observe distinct morphological changes but measured a loss of impedance that could be recovered with EDTA washout. High magnification imaging further demonstrates the loss and recovery of the barrier structure. Overall, we establish an ECIS MBR capable of real-time monitoring of the barrier function and cell morphology under shear stress and allowing high-resolution analysis of the barrier structure.

Dual Cross-Linked Biofunctional and Self-Healing Networks to Generate User-Defined Modular Gradient Hydrogel Constructs

Gradient hydrogels have been developed to mimic the spatiotemporal differences of multiple gradient cues in tissues. Current approaches used to generate such hydrogels are restricted to a single gradient shape and distribution. Here, a hydrogel is designed that includes two chemical cross-linking networks, biofunctional, and self-healing networks, enabling the customizable formation of modular gradient hydrogel construct with various gradient distributions and flexible shapes. The biofunctional networks are formed via Michael addition between the acrylates of oxidized acrylated hyaluronic acid (OAHA) and the dithiol of matrix metalloproteinase (MMP)-sensitive cross-linker and RGD peptides. The self-healing networks are formed via dynamic Schiff base reaction between N-carboxyethyl chitosan (CEC) and OAHA, which drives the modular gradient units to self-heal into an integral modular gradient hydrogel. The CEC-OAHA-MMP hydrogel exhibits excellent flowability at 37 °C under shear stress, enabling its injection to generate gradient distributions and shapes. Furthermore, encapsulated sarcoma cells respond to the gradient cues of RGD peptides and MMP-sensitive cross-linkers in the hydrogel. With these superior properties, the dual cross-linked CEC-OAHA-MMP hydrogel holds significant potential for generating customizable gradient hydrogel constructs, to study and guide cellular responses to their microenvironment such as in tumor mimicking, tissue engineering, and stem cell differentiation and morphogenesis.

Abstract B61: Intratumoral oxygen gradients mediate sarcoma cell invasion

Hypoxia is a critical factor in the progression and metastasis of many cancers including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply leading to heterogeneous areas of O2 depletion. Here we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoms mouse tumors (>300mm3) contain a severely hypoxic core (≤0.1% pO2) whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1%-6% pO2). To analyze tumor invasion, we utilized O2-controllable hydrogels to recreate the physio-pathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer-distance in the hypoxic hydrogels compared to nonhypoxic hydrogels. To accurately model the effect of the O2 gradient, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through HIF1α activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physico-tactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets. Citation Format: Daniel M. Lewis, Kyung Min Park, T.S. Karin Eisinger-Mathason, Celeste Simon, Sharon Gerecht. Intratumoral oxygen gradients mediate sarcoma cell invasion. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B61.