T lymphocytes are a key regulatory component of the adaptive immune system. Understanding how the... more T lymphocytes are a key regulatory component of the adaptive immune system. Understanding how the micro- and nano-scale details of the extracellular environment influence T cell activation may have wide impact on the use of T cells for therapeutic purposes. In this article, we examine how the micro- and nano-scale presentation of ligands to cell surface receptors, including microscale organization and nanoscale mobility, influences the activation of T cells. We extend these studies to include the role of cell-generated forces, and the rigidity of the microenvironment, on T cell activation. These approaches enable delivery of defined signals to T cells, a step toward understanding the cell-cell communication in the immune system, and developing micro/nano- and material- engineered systems for tailoring immune responses for adoptive T cell therapies.
Multicellular spheroids are an important 3-dimensional cell culture model that reflects many key ... more Multicellular spheroids are an important 3-dimensional cell culture model that reflects many key aspects of in vivo microenvironments. This paper presents a scalable, self-assembly based approach for fabricating microcavity substrates for multicellular spheroid cell culture. Hydrophobic glass microbeads were self-assembled into a tightly packed monolayer through the combined actions of surface tension, gravity, and lateral capillary forces at the water-air interface of a polymer solution. The packed bead monolayer was subsequently embedded in the dried polymer layer. The surface was used as a template for replicating microcavity substrates with perfect spherical shapes. We demonstrated the use of the substrate in monitoring the formation process of tumor spheroids, a proof-of-concept scale-up fabrication procedure into standard microplate formats, and its application in testing cancer drug responses in the context of bone marrow stromal cells. The presented technique offers a simple...
Hair cycling is a prime example of stem cell dependent tissue regeneration and replenishment, and... more Hair cycling is a prime example of stem cell dependent tissue regeneration and replenishment, and its regulatory mechanisms remain poorly understood. In the present study, we evaluated the effect of a blockage in terminal keratinocytic lineage differentiation in the Foxn1(-/-) nude phenotype on the epithelial progeny. Most notably we found a constitutive upregulation of LIM homeobox protein 2 (Lhx2), a marker gene of epithelial stem cellness indispensible for hair cycle progression. However, histological evidence along with an erratic, acyclic rise of otherwise suppressed CyclinD1 levels along with several key markers of keratinocyte lineage differentiation indicate a frustrated expansion of epithelial stem cell niches in skin. In addition, CD49f/CD34/CD200-based profiling demonstrated highly significant shifts in subpopulations of epithelial progeny. Intriguingly this appeared to include the expansion of Oct4+ stem cells in dermal fractions of skin isolates in the Foxn1 knock-out o...
It is increasingly recognized that cell signaling, as a chemical process, must be considered at t... more It is increasingly recognized that cell signaling, as a chemical process, must be considered at the local, micrometer scale. Micro- and nanofabrication techniques provide access to these dimensions, with the potential to capture and manipulate the spatial complexity of intracellular signaling in experimental models. This review focuses on recent advances in adapting surface engineering for use with biomolecular systems that interface with cell signaling, particularly with respect to surfaces that interact with multiple receptor systems on individual cells. The utility of this conceptual and experimental approach is demonstrated in the context of epithelial cells and T lymphocytes, two systems whose ability to perform their physiological function is dramatically impacted by the convergence and balance of multiple signaling pathways.
The ability to pattern proteins and other biomolecules onto substrates is important for capturing... more The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (http://gmwgroup.harvard.edu/) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning. An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.
Tumour-stromal interactions are a determining factor in cancer progression. In vivo, the interact... more Tumour-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumour-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyse bulk and interfacial tumour-stromal signalling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human oestrogen receptor-positive (ER+) breast cancer samples, and heterogeneous drug activity relative to the tumour-stroma interface. Specifically, an unknown mechanism of reversine is shown in targeting tumour-stromal interfacial interactions using ER+ MCF-7 breast cancer and bone marrow-derived stromal cells. Reversine suppresses MCF-7 tumour growth and bone metastasis in vivo by reducing tumour stromalization including collagen deposition and recruitment of activated stromal cells. This...
Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative ... more Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis. Key message: Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131. ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor. Oxidative stress and inflammation promote cell surface expression of CD131. ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes. Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.
Supported lipid bilayers capture the fluidity and chemical properties of cellular membranes. In t... more Supported lipid bilayers capture the fluidity and chemical properties of cellular membranes. In this report, we introduce a method for creating surfaces that contain multiple, aligned regions of supported membranes of different compositions at scales of micrometers and smaller. This method uses the design of a diffusional barrier to increase the resolution that can be achieved directly using traditional bilayer patterning techniques, such as laminar flow. We demonstrate the use of this platform for presenting ligands to the T Cell Receptor and LFA-1 that are tethered to separate, closely juxtaposed regions of bilayer, capturing an important aspect of the natural organization observed between T cells and Antigen Presenting Cells. Our results present a novel platform for the study of spatial separation of extracellular ligands and its impact on cell signals.
T lymphocytes are a key regulatory component of the adaptive immune system. Understanding how the... more T lymphocytes are a key regulatory component of the adaptive immune system. Understanding how the micro- and nano-scale details of the extracellular environment influence T cell activation may have wide impact on the use of T cells for therapeutic purposes. In this article, we examine how the micro- and nano-scale presentation of ligands to cell surface receptors, including microscale organization and nanoscale mobility, influences the activation of T cells. We extend these studies to include the role of cell-generated forces, and the rigidity of the microenvironment, on T cell activation. These approaches enable delivery of defined signals to T cells, a step toward understanding the cell-cell communication in the immune system, and developing micro/nano- and material- engineered systems for tailoring immune responses for adoptive T cell therapies.
Multicellular spheroids are an important 3-dimensional cell culture model that reflects many key ... more Multicellular spheroids are an important 3-dimensional cell culture model that reflects many key aspects of in vivo microenvironments. This paper presents a scalable, self-assembly based approach for fabricating microcavity substrates for multicellular spheroid cell culture. Hydrophobic glass microbeads were self-assembled into a tightly packed monolayer through the combined actions of surface tension, gravity, and lateral capillary forces at the water-air interface of a polymer solution. The packed bead monolayer was subsequently embedded in the dried polymer layer. The surface was used as a template for replicating microcavity substrates with perfect spherical shapes. We demonstrated the use of the substrate in monitoring the formation process of tumor spheroids, a proof-of-concept scale-up fabrication procedure into standard microplate formats, and its application in testing cancer drug responses in the context of bone marrow stromal cells. The presented technique offers a simple...
Hair cycling is a prime example of stem cell dependent tissue regeneration and replenishment, and... more Hair cycling is a prime example of stem cell dependent tissue regeneration and replenishment, and its regulatory mechanisms remain poorly understood. In the present study, we evaluated the effect of a blockage in terminal keratinocytic lineage differentiation in the Foxn1(-/-) nude phenotype on the epithelial progeny. Most notably we found a constitutive upregulation of LIM homeobox protein 2 (Lhx2), a marker gene of epithelial stem cellness indispensible for hair cycle progression. However, histological evidence along with an erratic, acyclic rise of otherwise suppressed CyclinD1 levels along with several key markers of keratinocyte lineage differentiation indicate a frustrated expansion of epithelial stem cell niches in skin. In addition, CD49f/CD34/CD200-based profiling demonstrated highly significant shifts in subpopulations of epithelial progeny. Intriguingly this appeared to include the expansion of Oct4+ stem cells in dermal fractions of skin isolates in the Foxn1 knock-out o...
It is increasingly recognized that cell signaling, as a chemical process, must be considered at t... more It is increasingly recognized that cell signaling, as a chemical process, must be considered at the local, micrometer scale. Micro- and nanofabrication techniques provide access to these dimensions, with the potential to capture and manipulate the spatial complexity of intracellular signaling in experimental models. This review focuses on recent advances in adapting surface engineering for use with biomolecular systems that interface with cell signaling, particularly with respect to surfaces that interact with multiple receptor systems on individual cells. The utility of this conceptual and experimental approach is demonstrated in the context of epithelial cells and T lymphocytes, two systems whose ability to perform their physiological function is dramatically impacted by the convergence and balance of multiple signaling pathways.
The ability to pattern proteins and other biomolecules onto substrates is important for capturing... more The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (http://gmwgroup.harvard.edu/) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning. An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.
Tumour-stromal interactions are a determining factor in cancer progression. In vivo, the interact... more Tumour-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumour-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyse bulk and interfacial tumour-stromal signalling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human oestrogen receptor-positive (ER+) breast cancer samples, and heterogeneous drug activity relative to the tumour-stroma interface. Specifically, an unknown mechanism of reversine is shown in targeting tumour-stromal interfacial interactions using ER+ MCF-7 breast cancer and bone marrow-derived stromal cells. Reversine suppresses MCF-7 tumour growth and bone metastasis in vivo by reducing tumour stromalization including collagen deposition and recruitment of activated stromal cells. This...
Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative ... more Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis. Key message: Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131. ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor. Oxidative stress and inflammation promote cell surface expression of CD131. ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes. Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.
Supported lipid bilayers capture the fluidity and chemical properties of cellular membranes. In t... more Supported lipid bilayers capture the fluidity and chemical properties of cellular membranes. In this report, we introduce a method for creating surfaces that contain multiple, aligned regions of supported membranes of different compositions at scales of micrometers and smaller. This method uses the design of a diffusional barrier to increase the resolution that can be achieved directly using traditional bilayer patterning techniques, such as laminar flow. We demonstrate the use of this platform for presenting ligands to the T Cell Receptor and LFA-1 that are tethered to separate, closely juxtaposed regions of bilayer, capturing an important aspect of the natural organization observed between T cells and Antigen Presenting Cells. Our results present a novel platform for the study of spatial separation of extracellular ligands and its impact on cell signals.
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