A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients

Rodenhizer, Darren; Gaude, Edoardo; Cojocari, Dan; Mahadevan, Radhakrishnan; Frezza, Christian; Wouters, Bradly G.; McGuigan, Alison P.

doi:10.1038/nmat4482

Cited by 119 publications

(142 citation statements)

References 29 publications

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“…27,28) and even O 2 gradients 29,30 . In addition, methods for control of O 2 tension and O 2 gradients compatible with 3D cell encapsulations have been developed 31–34 . Although these platforms may be preferred for some experimental setups and should be considered in an application-dependent manner, several general limitations exist.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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“…Cell-containing sheets have been analyzed with a number of different readouts, including: quantitative polymerase chain reaction (qPCR), fluorescence microscopy, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. 21–24 Due to the similarities between chemical environments in PBCs and avascular tissue, this platform has been used to investigate the invasion of breast and lung carcinomas, 21,23,25,26 cardiac ischemia, 27 and cancer metabolism. 24,28,29 …”

Section: Introductionmentioning

confidence: 99%

Assessing chemotherapeutic effectiveness using a paper-based tumor model

Boyce

LaBonia

Hummon

et al. 2017

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In vitro models for screening new cancer chemotherapeutics often rely on two-dimensional cultures to predict therapeutic potential. Unfortunately, the predictive power of these models is limited, as they fail to recapitulate the complex three-dimensional environments in tumors that promote the development of a chemoresistant phenotype. In this study, we describe the preparation and characterization of paper-based cultures (PBC) engineered to assess chemotherapeutic effectiveness in three dimensional, diffusion-limited environments. Similar environments are found within poorly vascularized tumors. These cultures, which are assembled by stacking together cell-laden paper scaffolds to yield thick tissue-like structures, generate monotonic gradients vertically through the culture and provide distinct chemical environments for each scaffold. After prolonged incubation, the scaffolds can simply be peeled apart and analyzed to relate cellular responses to the chemical environment experienced at that scaffold. Through fluorescence imaging, viable and proliferative cell populations were mapped within a stacked culture and found to be most abundant in scaffolds close to the nutrient-rich medium. By adjusting the cell density, we modulated the spatiotemporal evolution of oxygen gradients across the cultures and correlated these environmental changes with cellular sensitivity to SN-38 exposure. From these results, we showed that differences in the oxygen gradients produced cellular populations with significantly different chemosensitivities. Through this work, we highlight PBCs ability to serve as an analytical model capable of determining chemotherapeutic effectiveness under a range of chemical environments.

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“…During ischemia, succinate significantly accumulates in multiple organs and its oxidation is responsible for ROS generation during reperfusion [91]. Succinate was also shown to increase under hypoxic conditions, in cancer cells cultured in 3D scaffolds [92], consistent with its role as hypoxia sensing molecule. Other cues trigger oncometabolite accumulation.…”

Section: Environmental Cues Regulates Oncometabolite Productionmentioning

confidence: 75%

Oncometabolites: Unconventional triggers of oncogenic signalling cascades

Sciacovelli

Frezza

2016

Free Radical Biology and Medicine

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149

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Cancer is a complex and heterogeneous disease thought to be caused by multiple genetic lesions. The recent finding that enzymes of the tricarboxylic acid (TCA) cycle are mutated in cancer rekindled the hypothesis that altered metabolism might also have a role in cellular transformation. Attempts to link mitochondrial dysfunction to cancer uncovered the unexpected role of small molecule metabolites, now known as oncometabolites, in tumorigenesis. In this review, we describe how oncometabolites can contribute to tumorigenesis. We propose that lesions of oncogenes and tumour suppressors are only one of the possible routes to tumorigenesis, which include accumulation of oncometabolites triggered by environmental cues.

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A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients

Cited by 119 publications

References 29 publications

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

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

Assessing chemotherapeutic effectiveness using a paper-based tumor model

Oncometabolites: Unconventional triggers of oncogenic signalling cascades

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