Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, th... more Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, then manipulating the scaffold either mechanically, using bioreactors, or chemically, using growth factors, in an attempt to tailor the mechanical and biological properties of the engineered tissue. The material composition of the scaffold gives the construct its initial strength; then the scaffold either remodels or dissolves when implanted in the body. An ideal tissue replacement scaffold would be biocompatible, biodegradable, implantable, and would match the strength of the tissue it is replacing, and would remodel by natural mechanisms [1]. Finding or creating scaffold materials that meet all these specifications while providing an environment for cell attachment and proliferation is one of the main goals of conventional tissue engineering. Popular current scaffold materials include poly-l-lactic acid (PLLA) [2] and collagen [3]. Typically, the utilization of scaffolds in tissue engineering employs a top-down approach in which cells are seeded homogenously into the scaffold, then incubated in vitro prior to implantation. Scaffold properties, such as geometric dimensions (e.g., thickness) and cellular in-growth, are limited by the diffusion of nutrients, since these scaffolds do not incorporate vascular structures to transport nutrients and remove wastes deep into the scaffold as in native tissue [4]. Although seeded scaffolds have proven successful in some cases, there remains the need to have greater control of cell placement as well as the placement of additional features such as vascular structures, multiple cell types, growth factors, and extracellular matrix proteins that will aid in the fabrication of the next generation of engineered tissues.
The treatment of injured tendon is an ever-increasing clinical and financial burden, for which ti... more The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain reaction to measure the expression of tendon-specific makers, scleraxis and tenomodulin. Fiber peak stress, elastic modulus, toughness, and nuclear aspect ratio increased with the presence and duration of loading, while failure strain, toe-in strain, and nuclear area were unchanged. These biomechanical results suggest that cyclic strain promotes matrix deposition in a manner that increases the fiber resistance to stretch, but preserves fiber extensibility over the 7-day loading period. Over 7 days of loading, the scleraxis and tenomodulin expression increased drastically. Histologically, while there was no immediate difference in nuclear area with the addition of loading, nuclear aspect ratio significantly increased with loading duration, such that nuclei became progressively more elongated to the long axis of the fiber. Together with our biomechanical findings, such nuclear deformation suggests that cyclic strain elicits a mechanotransductive response, particularly one that modulates gene expression to promote matrix deposition during fiber development.
Volume 1A: Abdominal Aortic Aneurysms; Active and Reactive Soft Matter; Atherosclerosis; BioFluid Mechanics; Education; Biotransport Phenomena; Bone, Joint and Spine Mechanics; Brain Injury; Cardiac Mechanics; Cardiovascular Devices, Fluids and Imaging; Cartilage and Disc Mechanics; Cell and Tiss..., 2013
Microbeads are becoming popular tools in tissue engineering as 3D microstructure hydrogels. The g... more Microbeads are becoming popular tools in tissue engineering as 3D microstructure hydrogels. The gel nature of microbeads enables them to sequester soluble factors and mammalian cells, and their high surface area-to-volume ratio allows diffusion between the bead and the environment [1,2]. Microbeads are thus good systems for drug delivery and can serve as 3D microenvironments for cells. To fully maximize their potential as delivery systems and microenvironments, it is highly desirable to create spatially-precise hybrid cultures of microbeads and mammalian cells. Precise placement of microbeads in proximity to patterned cells will allow the study of spatial cellular interactions, paracrine signaling, and drug delivery.
The treatment of injured tendon is an ever-increasing clinical and financial burden, for which ti... more The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain...
Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, th... more Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, then manipulating the scaffold either mechanically, using bioreactors, or chemically, using growth factors, in an attempt to tailor the mechanical and biological properties of the engineered tissue. The material composition of the scaffold gives the construct its initial strength; then the scaffold either remodels or dissolves when implanted in the body. An ideal tissue replacement scaffold would be biocompatible, biodegradable, implantable, and would match the strength of the tissue it is replacing, and would remodel by natural mechanisms [1]. Finding or creating scaffold materials that meet all these specifications while providing an environment for cell attachment and proliferation is one of the main goals of conventional tissue engineering. Popular current scaffold materials include poly-l-lactic acid (PLLA) [2] and collagen [3]. Typically, the utilization of scaffolds in tissue engineering employs a top-down approach in which cells are seeded homogenously into the scaffold, then incubated in vitro prior to implantation. Scaffold properties, such as geometric dimensions (e.g., thickness) and cellular in-growth, are limited by the diffusion of nutrients, since these scaffolds do not incorporate vascular structures to transport nutrients and remove wastes deep into the scaffold as in native tissue [4]. Although seeded scaffolds have proven successful in some cases, there remains the need to have greater control of cell placement as well as the placement of additional features such as vascular structures, multiple cell types, growth factors, and extracellular matrix proteins that will aid in the fabrication of the next generation of engineered tissues.
Skeletal muscle loss, through injuries, myopathies, and interventional medicine, presents major c... more Skeletal muscle loss, through injuries, myopathies, and interventional medicine, presents major challenges in physiological function and clinical interventions [1]. Autologous tissue transplantation necessitates tissue loss from the donor site, and autologous grafts do not attain the strength of the original tissue. Exogenous tissue grafting faces similar strength issues, as well as the added challenge of immunorejection [2,3]. In vitro skeletal muscle tissue engineering holds promise for addressing these issues. However, these tissues have not yet shown proper dynamic response when compared to physiological muscle [2]. Mechanical and electrical stimulation have shown promise in improving construct properties [4], but mainly limited to 2D and scaffold-based constructs.
ASME 2011 Summer Bioengineering Conference, Parts A and B, 2011
ABSTRACT The clinical demand for tendon replacements following injury, surgical excision, or dise... more ABSTRACT The clinical demand for tendon replacements following injury, surgical excision, or disease drives current tissue engineering endeavors. Great strides have been made in producing functional tissues, but none have gained clinical acceptance. Scaffold-free and cell-based engineered tissue constructs allow the use of autologous cells and avoid potential scaffold-based complications such as immune rejection and breakdown byproducts. However, scaffold-free approaches have yet to replicate the mechanical properties of tendon [1,2]. In an effort to mimic some key aspects of in vivo embryonic tendon development, such as high cellularity and subsequent cell-to-cell contact, we have utilized a cell-based and scaffold-free method to direct fibroblast cell growth through geometric constraint to form single fibers [3,4]. Early application of mechanical cues (within hours of cell attachment) is essential for cell and collagen fiber alignment, as well as tissue maturation through matrix protein synthesis, and perhaps most importantly, these structural changes will result in altered mechanical properties. We recently established a method to apply mechanical stimulation to developing scaffold-free, cell-based fibers with the goal of replicating tenogenic development cues [5]. As an important step towards scaffold-free tendon replacements, the objective of this study was to demonstrate the influence of dynamic mechanical cues on growing fibers, which can ultimately be optimized to achieve tendon-like structure and mechanical properties.
ABSTRACT Normal organ development, function, and repair are coordinated by interactions between t... more ABSTRACT Normal organ development, function, and repair are coordinated by interactions between the epithelium and the surrounding stromal cell populations. Cellular function and homeostasis are controlled by an array of chemical and physical cues originating from the cells themselves and from the surrounding extracellular matrix (ECM). Both the endogenous cell population and ECM modulate and rely on the maintenance of basal level of tension within the tissue as a cue for growth and differentiation [1]. Furthermore, the loss of this tensional homeostasis is synonymous with many pathological conditions including; cancer, wound healing, and degenerative diseases [2].
Langmuir : the ACS journal of surfaces and colloids, 2016
Laser-induced forward transfer-based laser printing has been being implemented as a promising ori... more Laser-induced forward transfer-based laser printing has been being implemented as a promising orifice-free direct-write strategy for different printing applications. The printing quality during laser printing is largely affected by the jet and droplet formation process and subsequential impingement. The objective of this study is to investigate the impingement-based printing type and resulting printing quality during the laser printing of viscoelastic alginate solutions, which are representative inks for soft structure printing such as bioprinting. Three printing types are identified: droplet-impingement printing, jet-impingement printing with multiple breakups, and jet-impingement printing with a single breakup. Printing quality, in terms of printed droplet morphology and size, has been investigated as a function of alginate concentration, laser fluence, and direct-writing height based on a time-resolved imaging approach and microarrays of printed droplets. Of these, the best print...
Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast ca... more Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.
The present study investigated changes in rate of free radical production, antioxidant enzyme act... more The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury ( time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect ( P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase ( P = 0.015) and glutathione reductase ( P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione ( P = 0.008) and total glutathione ( P = 0.015). Morphological analysis...
Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, th... more Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, then manipulating the scaffold either mechanically, using bioreactors, or chemically, using growth factors, in an attempt to tailor the mechanical and biological properties of the engineered tissue. The material composition of the scaffold gives the construct its initial strength; then the scaffold either remodels or dissolves when implanted in the body. An ideal tissue replacement scaffold would be biocompatible, biodegradable, implantable, and would match the strength of the tissue it is replacing, and would remodel by natural mechanisms [1]. Finding or creating scaffold materials that meet all these specifications while providing an environment for cell attachment and proliferation is one of the main goals of conventional tissue engineering. Popular current scaffold materials include poly-l-lactic acid (PLLA) [2] and collagen [3]. Typically, the utilization of scaffolds in tissue engineering employs a top-down approach in which cells are seeded homogenously into the scaffold, then incubated in vitro prior to implantation. Scaffold properties, such as geometric dimensions (e.g., thickness) and cellular in-growth, are limited by the diffusion of nutrients, since these scaffolds do not incorporate vascular structures to transport nutrients and remove wastes deep into the scaffold as in native tissue [4]. Although seeded scaffolds have proven successful in some cases, there remains the need to have greater control of cell placement as well as the placement of additional features such as vascular structures, multiple cell types, growth factors, and extracellular matrix proteins that will aid in the fabrication of the next generation of engineered tissues.
The treatment of injured tendon is an ever-increasing clinical and financial burden, for which ti... more The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain reaction to measure the expression of tendon-specific makers, scleraxis and tenomodulin. Fiber peak stress, elastic modulus, toughness, and nuclear aspect ratio increased with the presence and duration of loading, while failure strain, toe-in strain, and nuclear area were unchanged. These biomechanical results suggest that cyclic strain promotes matrix deposition in a manner that increases the fiber resistance to stretch, but preserves fiber extensibility over the 7-day loading period. Over 7 days of loading, the scleraxis and tenomodulin expression increased drastically. Histologically, while there was no immediate difference in nuclear area with the addition of loading, nuclear aspect ratio significantly increased with loading duration, such that nuclei became progressively more elongated to the long axis of the fiber. Together with our biomechanical findings, such nuclear deformation suggests that cyclic strain elicits a mechanotransductive response, particularly one that modulates gene expression to promote matrix deposition during fiber development.
Volume 1A: Abdominal Aortic Aneurysms; Active and Reactive Soft Matter; Atherosclerosis; BioFluid Mechanics; Education; Biotransport Phenomena; Bone, Joint and Spine Mechanics; Brain Injury; Cardiac Mechanics; Cardiovascular Devices, Fluids and Imaging; Cartilage and Disc Mechanics; Cell and Tiss..., 2013
Microbeads are becoming popular tools in tissue engineering as 3D microstructure hydrogels. The g... more Microbeads are becoming popular tools in tissue engineering as 3D microstructure hydrogels. The gel nature of microbeads enables them to sequester soluble factors and mammalian cells, and their high surface area-to-volume ratio allows diffusion between the bead and the environment [1,2]. Microbeads are thus good systems for drug delivery and can serve as 3D microenvironments for cells. To fully maximize their potential as delivery systems and microenvironments, it is highly desirable to create spatially-precise hybrid cultures of microbeads and mammalian cells. Precise placement of microbeads in proximity to patterned cells will allow the study of spatial cellular interactions, paracrine signaling, and drug delivery.
The treatment of injured tendon is an ever-increasing clinical and financial burden, for which ti... more The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain...
Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, th... more Conventional tissue engineering typically involves homogenously seeding cells into a scaffold, then manipulating the scaffold either mechanically, using bioreactors, or chemically, using growth factors, in an attempt to tailor the mechanical and biological properties of the engineered tissue. The material composition of the scaffold gives the construct its initial strength; then the scaffold either remodels or dissolves when implanted in the body. An ideal tissue replacement scaffold would be biocompatible, biodegradable, implantable, and would match the strength of the tissue it is replacing, and would remodel by natural mechanisms [1]. Finding or creating scaffold materials that meet all these specifications while providing an environment for cell attachment and proliferation is one of the main goals of conventional tissue engineering. Popular current scaffold materials include poly-l-lactic acid (PLLA) [2] and collagen [3]. Typically, the utilization of scaffolds in tissue engineering employs a top-down approach in which cells are seeded homogenously into the scaffold, then incubated in vitro prior to implantation. Scaffold properties, such as geometric dimensions (e.g., thickness) and cellular in-growth, are limited by the diffusion of nutrients, since these scaffolds do not incorporate vascular structures to transport nutrients and remove wastes deep into the scaffold as in native tissue [4]. Although seeded scaffolds have proven successful in some cases, there remains the need to have greater control of cell placement as well as the placement of additional features such as vascular structures, multiple cell types, growth factors, and extracellular matrix proteins that will aid in the fabrication of the next generation of engineered tissues.
Skeletal muscle loss, through injuries, myopathies, and interventional medicine, presents major c... more Skeletal muscle loss, through injuries, myopathies, and interventional medicine, presents major challenges in physiological function and clinical interventions [1]. Autologous tissue transplantation necessitates tissue loss from the donor site, and autologous grafts do not attain the strength of the original tissue. Exogenous tissue grafting faces similar strength issues, as well as the added challenge of immunorejection [2,3]. In vitro skeletal muscle tissue engineering holds promise for addressing these issues. However, these tissues have not yet shown proper dynamic response when compared to physiological muscle [2]. Mechanical and electrical stimulation have shown promise in improving construct properties [4], but mainly limited to 2D and scaffold-based constructs.
ASME 2011 Summer Bioengineering Conference, Parts A and B, 2011
ABSTRACT The clinical demand for tendon replacements following injury, surgical excision, or dise... more ABSTRACT The clinical demand for tendon replacements following injury, surgical excision, or disease drives current tissue engineering endeavors. Great strides have been made in producing functional tissues, but none have gained clinical acceptance. Scaffold-free and cell-based engineered tissue constructs allow the use of autologous cells and avoid potential scaffold-based complications such as immune rejection and breakdown byproducts. However, scaffold-free approaches have yet to replicate the mechanical properties of tendon [1,2]. In an effort to mimic some key aspects of in vivo embryonic tendon development, such as high cellularity and subsequent cell-to-cell contact, we have utilized a cell-based and scaffold-free method to direct fibroblast cell growth through geometric constraint to form single fibers [3,4]. Early application of mechanical cues (within hours of cell attachment) is essential for cell and collagen fiber alignment, as well as tissue maturation through matrix protein synthesis, and perhaps most importantly, these structural changes will result in altered mechanical properties. We recently established a method to apply mechanical stimulation to developing scaffold-free, cell-based fibers with the goal of replicating tenogenic development cues [5]. As an important step towards scaffold-free tendon replacements, the objective of this study was to demonstrate the influence of dynamic mechanical cues on growing fibers, which can ultimately be optimized to achieve tendon-like structure and mechanical properties.
ABSTRACT Normal organ development, function, and repair are coordinated by interactions between t... more ABSTRACT Normal organ development, function, and repair are coordinated by interactions between the epithelium and the surrounding stromal cell populations. Cellular function and homeostasis are controlled by an array of chemical and physical cues originating from the cells themselves and from the surrounding extracellular matrix (ECM). Both the endogenous cell population and ECM modulate and rely on the maintenance of basal level of tension within the tissue as a cue for growth and differentiation [1]. Furthermore, the loss of this tensional homeostasis is synonymous with many pathological conditions including; cancer, wound healing, and degenerative diseases [2].
Langmuir : the ACS journal of surfaces and colloids, 2016
Laser-induced forward transfer-based laser printing has been being implemented as a promising ori... more Laser-induced forward transfer-based laser printing has been being implemented as a promising orifice-free direct-write strategy for different printing applications. The printing quality during laser printing is largely affected by the jet and droplet formation process and subsequential impingement. The objective of this study is to investigate the impingement-based printing type and resulting printing quality during the laser printing of viscoelastic alginate solutions, which are representative inks for soft structure printing such as bioprinting. Three printing types are identified: droplet-impingement printing, jet-impingement printing with multiple breakups, and jet-impingement printing with a single breakup. Printing quality, in terms of printed droplet morphology and size, has been investigated as a function of alginate concentration, laser fluence, and direct-writing height based on a time-resolved imaging approach and microarrays of printed droplets. Of these, the best print...
Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast ca... more Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.
The present study investigated changes in rate of free radical production, antioxidant enzyme act... more The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury ( time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect ( P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase ( P = 0.015) and glutathione reductase ( P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione ( P = 0.008) and total glutathione ( P = 0.015). Morphological analysis...
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Papers by David Corr