The formation of gaps in the endothelium is a crucial process underlying both cancer and immune c... more The formation of gaps in the endothelium is a crucial process underlying both cancer and immune cell extravasation, contributing to the functioning of the immune system during infection, the unfavorable development of chronic inflammation and tumor metastasis. Here, we present a stochastic-mechanical multiscale model of an endothelial cell monolayer and show that the dynamic nature of the endothelium leads to spontaneous gap formation, even without intervention from the transmigrating cells. These gaps preferentially appear at the vertices between three endothelial cells, as opposed to the border between two cells. We quantify the frequency and lifetime of these gaps, and validate our predictions experimentally. Interestingly, we find experimentally that cancer cells also preferentially extravasate at vertices, even when they first arrest on borders. This suggests that extravasating cells, rather than initially signaling to the endothelium, might exploit the autonomously forming gap...
During cell migration, cells become polarized, change their shape, and move in response to variou... more During cell migration, cells become polarized, change their shape, and move in response to various internal and external cues. Cell polarization is defined through the spatio-temporal organization of molecules such as PI3K or small GTPases, and is determined by intracellular signaling networks. It results in directional forces through actin polymerization and myosin contractions. Many existing mathematical models of cell polarization are formulated in terms of reaction-diffusion systems of interacting molecules, and are often defined in one or two spatial dimensions. In this paper, we introduce a 3D reaction-diffusion model of interacting molecules in a single cell, and find that cell geometry has an important role affecting the capability of a cell to polarize, or change polarization when an external signal changes direction. Our results suggest a geometrical argument why more roundish cells can repolarize more effectively than cells which are elongated along the direction of the o...
The formation of gaps in the endothelium is a crucial process underlying both cancer and immune c... more The formation of gaps in the endothelium is a crucial process underlying both cancer and immune cell extravasation, contributing to the functioning of the immune system during infection, the unfavorable development of chronic inflammation and tumor metastasis. Here, we present a stochastic-mechanical multiscale model of an endothelial cell monolayer and show that the dynamic nature of the endothelium leads to spontaneous gap formation, even without intervention from the transmigrating cells. These gaps preferentially appear at the vertices between three endothelial cells, as opposed to the border between two cells. We quantify the frequency and lifetime of these gaps, and validate our predictions experimentally. Interestingly, we find experimentally that cancer cells also preferentially extravasate at vertices, even when they first arrest on borders. This suggests that extravasating cells, rather than initially signaling to the endothelium, might exploit the autonomously forming gap...
The disruption of structurally compromised coronary plaque is thought to be the primary event cau... more The disruption of structurally compromised coronary plaque is thought to be the primary event causing heart attack. We have developed a new method for characterizing these vulnerable plaques using OCT and elastography.
Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In ... more Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In particular, the inner blood-retinal barrier (iBRB) and blood–brain barrier (BBB) share common origins in development, and similar morphology and function in adult tissue, while barrier breakdown and leakage of neurotoxic molecules can be accompanied by neurodegeneration. Therefore, pre-clinical research requires human in vitro models that elucidate pathophysiological mechanisms and support drug discovery, to add to animal in vivo modeling that poorly predict patient responses. Advanced cellular models such as microphysiological systems (MPS) recapitulate tissue organization and function in many organ-specific contexts, providing physiological relevance, potential for customization to different population groups, and scalability for drug screening purposes. While human-based MPS have been developed for tissues such as lung, gut, brain and tumors, few comprehensive models exist for ocular ...
Tumor cell migration is essential for invasion and dissemination from primary solid tumors and fo... more Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of ...
The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeuti... more The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant in vitro human BBB models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. This protocol describes an in vitro model of the human BBB self-assembled within microfluidic devices from stem-cell-derived or primary brain endothelial cells, and primary brain pericytes and astrocytes. This protocol requires 1.5 d for device fabrication, 7 d for device culture and up to 5 d for downstream imaging, protein and gene expression analyses. Methodologies to measure the permeability of any molecule in the BBB model, which take 30 min per device, are also included. Compared with standard 2D assays, the BBB model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. These properties, coupled with a functional brain endothelial expression profile and the capability to easily test several repeats with low reagent consumption, make this BBB model highly suitable for widespread use in academic and industrial laboratories.
Advances in experimental medicine and biology, 2018
Three-dimensional complex biomechanical interactions occur from the initial steps of tumor format... more Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types.
In this paper, we have developed a novel in vitro angiogenesis on a chip to identify potential mo... more In this paper, we have developed a novel in vitro angiogenesis on a chip to identify potential molecular targets, secreted from cell encapsulated beads; augmenting, altering and blocking endothelial cell responses, and to enable preliminary screening before transplantation. We confirmed that growth factors secreted from the encapsulated cells in CE beads were able to induce ECs migration and form lumen-like structures in the collagen scaffold.
The Hp-fld is an essential protein that is being used as a target to develop new antimicrobials [... more The Hp-fld is an essential protein that is being used as a target to develop new antimicrobials [1]. The binding of FMN cofactor to Helicobacter pylori's flavodoxin (Hp-fld) is a very complex scenario. Some studies have been carried out about this issue, and several possible association mechanisms proposed [2-4]. Nevertheless, many aspects of that mechanisms are yet to be fully clarified as for example the roles of different anions often bound to FMN phosphate binding subsite [5,6]. In this work, we investigated from a computational point of view the anion/protein interactions of one chloride and one sulphate bound to the FMN binding pockets of the homologous apoflavodoxin structures 2BMV [5] and 1FTG [6] respectively. Molecular dynamics simulations performed for both anions (50 and 10 runs respectively). Meticulous anion/protein conformation, solvation, interaction forces and statistical analyses were carried out. The temperature factor was also evaluated. In the chloride case,...
The blood-brain barrier (BBB) is known to be one of the least permeable portions of the vascular ... more The blood-brain barrier (BBB) is known to be one of the least permeable portions of the vascular system, serving to protect the central nervous system from agents deleterious to the brain while also preventing or limiting the passage of drugs to treat neurological diseases. Despite this, the brain is recognized to be one of the primary sites of metastasis, especially for lung and breast cancers. While animal experiments have proven useful, realistic models of the BBB using human cells are limited and remain a subject of intense research. Work over the past several years has led to several promising systems, although it has proven difficult to achieve levels of permeability comparable to those observed in vivo.
The blood-brain barrier (BBB) is a selective barrier that help to maintain brain homeostasis, how... more The blood-brain barrier (BBB) is a selective barrier that help to maintain brain homeostasis, however it also creates an obstacle to drug delivery. For years, in vivo animal models have been widely used for BBB studies and drug evaluations. Although these techniques are considered the gold standard, 80% of drug candidates that were successful in animal models later failed in clinical trials. For that reason, a cost-effective in vitro BBB model that adequately reflects human in vivo conditions is required. Here we developed a 3D microfluidic model of the BBB by self-organized vascular network including (iPS)-derived endothelial cells, human brain pericytes, and astrocytes.
This work presents a microwell prototyping technique for generating multicellular cancer aggregat... more This work presents a microwell prototyping technique for generating multicellular cancer aggregates in suspension culture for epithelial-mesenchymal-transition (EMT) drug screening. Microwell and aggregate size and geometry were examined in PMMA, PDMS, polystyrene materials materials. Retrieved aggregates were further studied in terms of cell dispersion through drug screening, to identify the effective drug dosage for the inhibition of EMT. Two distinct cell phenotypic behaviors were discovered from Low-attachment-dish- and microwell-generated aggregates under different conditions. Future studies of the two types of aggregates may contribute to the understanding of cancer metastasis.
The formation of gaps in the endothelium is a crucial process underlying both cancer and immune c... more The formation of gaps in the endothelium is a crucial process underlying both cancer and immune cell extravasation, contributing to the functioning of the immune system during infection, the unfavorable development of chronic inflammation and tumor metastasis. Here, we present a stochastic-mechanical multiscale model of an endothelial cell monolayer and show that the dynamic nature of the endothelium leads to spontaneous gap formation, even without intervention from the transmigrating cells. These gaps preferentially appear at the vertices between three endothelial cells, as opposed to the border between two cells. We quantify the frequency and lifetime of these gaps, and validate our predictions experimentally. Interestingly, we find experimentally that cancer cells also preferentially extravasate at vertices, even when they first arrest on borders. This suggests that extravasating cells, rather than initially signaling to the endothelium, might exploit the autonomously forming gap...
During cell migration, cells become polarized, change their shape, and move in response to variou... more During cell migration, cells become polarized, change their shape, and move in response to various internal and external cues. Cell polarization is defined through the spatio-temporal organization of molecules such as PI3K or small GTPases, and is determined by intracellular signaling networks. It results in directional forces through actin polymerization and myosin contractions. Many existing mathematical models of cell polarization are formulated in terms of reaction-diffusion systems of interacting molecules, and are often defined in one or two spatial dimensions. In this paper, we introduce a 3D reaction-diffusion model of interacting molecules in a single cell, and find that cell geometry has an important role affecting the capability of a cell to polarize, or change polarization when an external signal changes direction. Our results suggest a geometrical argument why more roundish cells can repolarize more effectively than cells which are elongated along the direction of the o...
The formation of gaps in the endothelium is a crucial process underlying both cancer and immune c... more The formation of gaps in the endothelium is a crucial process underlying both cancer and immune cell extravasation, contributing to the functioning of the immune system during infection, the unfavorable development of chronic inflammation and tumor metastasis. Here, we present a stochastic-mechanical multiscale model of an endothelial cell monolayer and show that the dynamic nature of the endothelium leads to spontaneous gap formation, even without intervention from the transmigrating cells. These gaps preferentially appear at the vertices between three endothelial cells, as opposed to the border between two cells. We quantify the frequency and lifetime of these gaps, and validate our predictions experimentally. Interestingly, we find experimentally that cancer cells also preferentially extravasate at vertices, even when they first arrest on borders. This suggests that extravasating cells, rather than initially signaling to the endothelium, might exploit the autonomously forming gap...
The disruption of structurally compromised coronary plaque is thought to be the primary event cau... more The disruption of structurally compromised coronary plaque is thought to be the primary event causing heart attack. We have developed a new method for characterizing these vulnerable plaques using OCT and elastography.
Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In ... more Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In particular, the inner blood-retinal barrier (iBRB) and blood–brain barrier (BBB) share common origins in development, and similar morphology and function in adult tissue, while barrier breakdown and leakage of neurotoxic molecules can be accompanied by neurodegeneration. Therefore, pre-clinical research requires human in vitro models that elucidate pathophysiological mechanisms and support drug discovery, to add to animal in vivo modeling that poorly predict patient responses. Advanced cellular models such as microphysiological systems (MPS) recapitulate tissue organization and function in many organ-specific contexts, providing physiological relevance, potential for customization to different population groups, and scalability for drug screening purposes. While human-based MPS have been developed for tissues such as lung, gut, brain and tumors, few comprehensive models exist for ocular ...
Tumor cell migration is essential for invasion and dissemination from primary solid tumors and fo... more Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of ...
The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeuti... more The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant in vitro human BBB models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. This protocol describes an in vitro model of the human BBB self-assembled within microfluidic devices from stem-cell-derived or primary brain endothelial cells, and primary brain pericytes and astrocytes. This protocol requires 1.5 d for device fabrication, 7 d for device culture and up to 5 d for downstream imaging, protein and gene expression analyses. Methodologies to measure the permeability of any molecule in the BBB model, which take 30 min per device, are also included. Compared with standard 2D assays, the BBB model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. These properties, coupled with a functional brain endothelial expression profile and the capability to easily test several repeats with low reagent consumption, make this BBB model highly suitable for widespread use in academic and industrial laboratories.
Advances in experimental medicine and biology, 2018
Three-dimensional complex biomechanical interactions occur from the initial steps of tumor format... more Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types.
In this paper, we have developed a novel in vitro angiogenesis on a chip to identify potential mo... more In this paper, we have developed a novel in vitro angiogenesis on a chip to identify potential molecular targets, secreted from cell encapsulated beads; augmenting, altering and blocking endothelial cell responses, and to enable preliminary screening before transplantation. We confirmed that growth factors secreted from the encapsulated cells in CE beads were able to induce ECs migration and form lumen-like structures in the collagen scaffold.
The Hp-fld is an essential protein that is being used as a target to develop new antimicrobials [... more The Hp-fld is an essential protein that is being used as a target to develop new antimicrobials [1]. The binding of FMN cofactor to Helicobacter pylori's flavodoxin (Hp-fld) is a very complex scenario. Some studies have been carried out about this issue, and several possible association mechanisms proposed [2-4]. Nevertheless, many aspects of that mechanisms are yet to be fully clarified as for example the roles of different anions often bound to FMN phosphate binding subsite [5,6]. In this work, we investigated from a computational point of view the anion/protein interactions of one chloride and one sulphate bound to the FMN binding pockets of the homologous apoflavodoxin structures 2BMV [5] and 1FTG [6] respectively. Molecular dynamics simulations performed for both anions (50 and 10 runs respectively). Meticulous anion/protein conformation, solvation, interaction forces and statistical analyses were carried out. The temperature factor was also evaluated. In the chloride case,...
The blood-brain barrier (BBB) is known to be one of the least permeable portions of the vascular ... more The blood-brain barrier (BBB) is known to be one of the least permeable portions of the vascular system, serving to protect the central nervous system from agents deleterious to the brain while also preventing or limiting the passage of drugs to treat neurological diseases. Despite this, the brain is recognized to be one of the primary sites of metastasis, especially for lung and breast cancers. While animal experiments have proven useful, realistic models of the BBB using human cells are limited and remain a subject of intense research. Work over the past several years has led to several promising systems, although it has proven difficult to achieve levels of permeability comparable to those observed in vivo.
The blood-brain barrier (BBB) is a selective barrier that help to maintain brain homeostasis, how... more The blood-brain barrier (BBB) is a selective barrier that help to maintain brain homeostasis, however it also creates an obstacle to drug delivery. For years, in vivo animal models have been widely used for BBB studies and drug evaluations. Although these techniques are considered the gold standard, 80% of drug candidates that were successful in animal models later failed in clinical trials. For that reason, a cost-effective in vitro BBB model that adequately reflects human in vivo conditions is required. Here we developed a 3D microfluidic model of the BBB by self-organized vascular network including (iPS)-derived endothelial cells, human brain pericytes, and astrocytes.
This work presents a microwell prototyping technique for generating multicellular cancer aggregat... more This work presents a microwell prototyping technique for generating multicellular cancer aggregates in suspension culture for epithelial-mesenchymal-transition (EMT) drug screening. Microwell and aggregate size and geometry were examined in PMMA, PDMS, polystyrene materials materials. Retrieved aggregates were further studied in terms of cell dispersion through drug screening, to identify the effective drug dosage for the inhibition of EMT. Two distinct cell phenotypic behaviors were discovered from Low-attachment-dish- and microwell-generated aggregates under different conditions. Future studies of the two types of aggregates may contribute to the understanding of cancer metastasis.
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Papers by Roger D. Kamm