bims-orenst 2021-06-27 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2021–06–27
eleven papers selected by
Joram Mooiweer, University of Groningen

The CCL2-CCR2 astrocyte-cancer cell axis in tumor extravasation at the brain.
Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.
A new microfluidic method enabling the generation of multi-layered tissues-on-chips using skin cells as a proof of concept.
Impairing flow-mediated endothelial remodeling reduces extravasation of tumor cells.
High-throughput microfluidic 3D biomimetic model enabling quantitative description of the human breast tumor microenvironment.
Biological gel-based microchamber array for tumor cell proliferation and migration studies in well-controlled biochemical gradients.
Mechanical Modulation of Tumor Nodules under Flow.
A microfluidic device enabling drug resistance analysis of leukemia cells via coupled dielectrophoretic detection and impedimetric counting.
Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration.
Reversible blood-brain barrier opening utilizing the membrane active peptide melittin in vitro and in vivo.
Patient-derived scaffolds as a drug-testing platform for endocrine therapies in breast cancer.

Sci Adv. 2021 Jun;pii: eabg8139. [Epub ahead of print]7(26):

The CCL2-CCR2 astrocyte-cancer cell axis in tumor extravasation at the brain.

Cynthia Hajal, Yoojin Shin, Leanne Li, Jean Carlos Serrano, Tyler Jacks, Roger D Kamm.

Although brain metastases are common in cancer patients, little is known about the mechanisms of cancer extravasation across the blood-brain barrier (BBB), a key step in the metastatic cascade that regulates the entry of cancer cells into the brain parenchyma. Here, we show, in a three-dimensional in vitro BBB microvascular model, that astrocytes promote cancer cell transmigration via their secretion of C-C motif chemokine ligand 2 (CCL2). We found that this chemokine, produced primarily by astrocytes, promoted the chemotaxis and chemokinesis of cancer cells via their C-C chemokine receptor type 2 (CCR2), with no notable changes in vascular permeability. These findings were validated in vivo, where CCR2-deficient cancer cells exhibited significantly reduced rates of arrest and transmigration in mouse brain capillaries. Our results reveal that the CCL2-CCR2 astrocyte-cancer cell axis plays a fundamental role in extravasation and, consequently, metastasis to the brain.

DOI: https://doi.org/10.1126/sciadv.abg8139
Sci Rep. 2021 Jun 23. 11(1): 13159

Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.

Victoria L Slaughter, John W Rumsey, Rachel Boone, Duaa Malik, Yunqing Cai, Narasimhan Narasimhan Sriram, Christopher J Long, Christopher W McAleer, Stephen Lambert, Michael L Shuler, J J Hickman.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

DOI: https://doi.org/10.1038/s41598-021-92264-2
Sci Rep. 2021 Jun 23. 11(1): 13160

A new microfluidic method enabling the generation of multi-layered tissues-on-chips using skin cells as a proof of concept.

L Valencia, V Canalejas-Tejero, M Clemente, I Fernaud, M Holgado, J L Jorcano, D Velasco.

Microfluidic-based tissues-on-chips (TOCs) have thus far been restricted to modelling simple epithelia as a single cell layer, but likely due to technical difficulties, no TOCs have been reported to include both an epithelial and a stromal component despite the biological importance of the stroma for the structure and function of human tissues. We present, for the first time, a novel approach to generate 3D multilayer tissue models in microfluidic platforms. As a proof of concept, we modelled skin, including a dermal and an epidermal compartment. To accomplish this, we developed a parallel flow method enabling the deposition of bilayer tissue in the upper chamber, which was subsequently maintained under dynamic nutrient flow conditions through the lower chamber, mimicking the function of a blood vessel. We also designed and built an inexpensive, easy-to-implement, versatile, and robust vinyl-based device that overcomes some of the drawbacks present in PDMS-based chips. Preliminary tests indicate that this biochip will allow the development and maintenance of multilayer tissues, which opens the possibility of better modelling of the complex cell-cell and cell-matrix interactions that exist in and between the epithelium and mesenchyme, allowing for better-grounded tissue modelling and drug screening.

DOI: https://doi.org/10.1038/s41598-021-91875-z
Sci Rep. 2021 Jun 23. 11(1): 13144

Impairing flow-mediated endothelial remodeling reduces extravasation of tumor cells.

Gautier Follain, Naël Osmani, Valentin Gensbittel, Nandini Asokan, Annabel Larnicol, Luc Mercier, Maria Jesus Garcia-Leon, Ignacio Busnelli, Angelique Pichot, Nicodème Paul, Raphaël Carapito, Seiamak Bahram, Olivier Lefebvre, Jacky G Goetz.

Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

DOI: https://doi.org/10.1038/s41598-021-92515-2
Acta Biomater. 2021 Jun 18. pii: S1742-7061(21)00399-8. [Epub ahead of print]

High-throughput microfluidic 3D biomimetic model enabling quantitative description of the human breast tumor microenvironment.

Ilana Berger Fridman, James Kostas, Michal Gregus, Somak Ray, Matthew R Sullivan, Alexander R Ivanov, Smadar Cohen, Tania Konry.

  Cancer is driven by both genetic aberrations in the tumor cells and fundamental changes in the tumor microenvironment (TME). These changes offer potential targets for novel therapeutics, yet lack of in vitro 3D models recapitulating this complex microenvironment impedes such progress. Here, we generated several tumor-stroma scaffolds reflecting the dynamic in vivo breast TME, using a high throughput microfluidic system. Alginate (Alg) or alginate-alginate sulfate (Alg/Alg-S) hydrogels were used as ECM-mimics, enabling the encapsulation and culture of tumor cells, fibroblasts and immune cells (macrophages and T cells, of the innate and adaptive immune systems, respectively). Specifically, Alg/Alg-S was shown capable of capturing and presenting growth factors and cytokines with binding affinity that is comparable to heparin. Viability and cytotoxicity were shown to strongly correlate with the dynamics of cellular milieu, as well as hydrogel type. Using on-chip immunofluorescence, production of reactive oxygen species and apoptosis were imaged and quantitatively analyzed. We then show how macrophages in our microfluidic system were shifted from a proinflammatory to an immunosuppressive phenotype when encapsulated in Alg/Alg-S, reflecting in vivo TME dynamics. LC-MS proteomic profiling of tumor cells sorted from the TME scaffolds revealed upregulation of proteins involved in cell-cell interactions and immunomodulation in Alg/Alg-S scaffolds, correlating with in vivo findings and demonstrating the appropriateness of Alg/Alg-S as an ECM biomimetic. Finally, we show the formation of large tumor-derived vesicles, formed exclusively in Alg/Alg-S scaffolds. Altogether, our system offers a robust platform for quantitative description of the breast TME that successfully recapitulates in vivo patterns. Statement of significance: Cancer progression is driven by profound changes in both tumor cells and surrounding stroma. Here, we present a high throughput microfluidic system for the generation and analysis of dynamic tumor-stroma scaffolds, that mimic the complex in vivo TME cell proportions and compositions, constructing robust in vitro models for the study of the TME. Utilizing Alg/Alg-S as a bioinspired ECM, mimicking heparin's in vivo capabilities of capturing and presenting signaling molecules, we show how Alg/Alg-S induces complex in vivo-like responses in our models. Alg/Alg-S is shown here to promote dynamic protein expression patterns, that can serve as potential therapeutic targets for breast cancer treatment. Formation of large tumor-derived vesicles, observed exclusively in the Alg/Alg-S scaffolds suggests a mechanism for tumor survival.

Keywords:  3D disease models; biomaterials; breast cancer; microfluidics; proteomic profiling; tumor microenvironment

DOI:  https://doi.org/10.1016/j.actbio.2021.06.025
Lab Chip. 2021 Jun 23.

Biological gel-based microchamber array for tumor cell proliferation and migration studies in well-controlled biochemical gradients.

Jingru Yao, Guoqiang Li, Yang Jiao, Yu Zheng, Yanping Liu, Gao Wang, Lianjie Zhou, Hongfei Zhang, Xianquan Zhang, Jianwei Shuai, Qihui Fan, Fangfu Ye, Silong Lou, Guo Chen, Kena Song, Yong Liao, Liyu Liu.

Breast cancer metastasis is a complex process controlled by multiple factors, including various cell-cell interactions, cell-environment coupling, and oxygen, nutrient and drug gradients that are intimately related to the heterogeneous breast tissue structure. In this study, we constructed a high-throughput in vitro biochip system containing an array of 642 microchambers arranged in a checkerboard configuration, with each chamber embedded in a composite extracellular matrix (ECM) composed of engineered collagen and Matrigel to mimic local heterogeneous environment in vivo. In addition, a controllable complex tetragonal chemical concentration profile can be achieved by imposing chemical compounds at the four boundaries of the chip, leading to distinct local nutrient and/or drug gradients in the individual microchambers. Here, the microchamber array with composite ECM (MACECM) device aims to simulate multiple tumor cell niches composed of both breast epithelial cells (MCF-10A-GFP) and metastatic breast cancer cells (MDA-MB-231-RFP), which enables systematic studies of cell responses to a variety of biochemical conditions. The results obtained from the MACECM studies indicate that discoidin domain receptor 1 (DDR1) inhibitor 7rh and matrix metalloproteinase inhibitor batimastat, in association with epidermal growth factor (EGF) had no significant effects on the growth of MCF-10A-GFP cells, but had significant effects on DDR1 expression and the related migratory behavior of MDA-MB-231-RFP cells. The MACECM design not only enables the construction of a more realistic in vitro model for investigating cancer cell migration mechanisms but also has considerable potential for further development as a platform for next-generation high-throughput and therapeutic screening (e.g., anti-cancer drug evaluation) and personalized medicine.

DOI: https://doi.org/10.1039/d0lc00951b
IEEE Trans Biomed Eng. 2021 Jun 25. PP

Mechanical Modulation of Tumor Nodules under Flow.

Christina Conrad, Kaitlin Moore, William Polacheck, Imran Rizvi, Giuliano Scarcelli.

Perfusion models are valuable tools to mimic complex features of the tumor microenvironment and to study cell behavior. In ovarian cancer, mimicking disease pathology of ascites has been achieved by seeding tumor nodules on a basement membrane and subjecting them to long-term continuous flow. In this scenario it is particularly important to study the role of mechanical stress on cancer progression. Mechanical cues are already known to be important in key cancer processes such as survival, proliferation, and migration. However, probing cell mechanical properties within microfluidic platforms has not been achievable with current technologies since samples are not easily accessible within most microfluidic channels. Here, to analyze the mechanical properties of cells within a perfusion chamber, we use Brillouin confocal microscopy, an all-optical technique that requires no contact or perturbation to the sample. Our results indicate that ovarian cancer nodules under long-term continuous flow have a significantly lower longitudinal modulus compared to nodules maintained in a static condition. We further dissect the role of distinct mechanical perturbations (e.g. shear flow, osmolality) on tumor nodule properties. In summary, the unique combination of a long-term microfluidic culture and noninvasive mechanical analysis technique provides insights on the effects of physical forces in ovarian cancer pathology.

DOI: https://doi.org/10.1109/TBME.2021.3092641
Sci Rep. 2021 Jun 23. 11(1): 13193

A microfluidic device enabling drug resistance analysis of leukemia cells via coupled dielectrophoretic detection and impedimetric counting.

Yağmur Demircan Yalçın, Taylan Berkin Töral, Sertan Sukas, Ender Yıldırım, Özge Zorlu, Ufuk Gündüz, Haluk Külah.

We report the development of a lab-on-a-chip system, that facilitates coupled dielectrophoretic detection (DEP-D) and impedimetric counting (IM-C), for investigating drug resistance in K562 and CCRF-CEM leukemia cells without (immuno) labeling. Two IM-C units were placed upstream and downstream of the DEP-D unit for enumeration, respectively, before and after the cells were treated in DEP-D unit, where the difference in cell count gave the total number of trapped cells based on their DEP characteristics. Conductivity of the running buffer was matched the conductivity of cytoplasm of wild type K562 and CCRF-CEM cells. Results showed that DEP responses of drug resistant and wild type K562 cells were statistically discriminative (at p = 0.05 level) at 200 mS/m buffer conductivity and at 8.6 MHz working frequency of DEP-D unit. For CCRF-CEM cells, conductivity and frequency values were 160 mS/m and 6.2 MHz, respectively. Our approach enabled discrimination of resistant cells in a group by setting up a threshold provided by the conductivity of running buffer. Subsequent selection of drug resistant cells can be applied to investigate variations in gene expressions and occurrence of mutations related to drug resistance.

DOI: https://doi.org/10.1038/s41598-021-92647-5
Nat Cell Biol. 2021 Jun 21.

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration.

Carlos Pérez-González, Gerardo Ceada, Francesco Greco, Marija Matejčić, Manuel Gómez-González, Natalia Castro, Anghara Menendez, Sohan Kale, Denis Krndija, Andrew G Clark, Venkata Ram Gannavarapu, Adrián Álvarez-Varela, Pere Roca-Cusachs, Eduard Batlle, Danijela Matic Vignjevic, Marino Arroyo, Xavier Trepat.

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

DOI: https://doi.org/10.1038/s41556-021-00699-6
Biomaterials. 2021 Jun 10. pii: S0142-9612(21)00298-2. [Epub ahead of print]275 120942

Reversible blood-brain barrier opening utilizing the membrane active peptide melittin in vitro and in vivo.

Raleigh M Linville, Alexander Komin, Xiaoyan Lan, Jackson G DeStefano, Chengyan Chu, Guanshu Liu, Piotr Walczak, Kalina Hristova, Peter C Searson.

  The blood-brain barrier (BBB) tightly controls entry of molecules and cells into the brain, restricting the delivery of therapeutics. Blood-brain barrier opening (BBBO) utilizes reversible disruption of cell-cell junctions between brain microvascular endothelial cells to enable transient entry into the brain. Here, we demonstrate that melittin, a membrane active peptide present in bee venom, supports transient BBBO. From endothelial and neuronal viability studies, we first identify the accessible concentration range for BBBO. We then use a tissue-engineered model of the human BBB to optimize dosing and elucidate the mechanism of opening. Melittin and other membrane active variants transiently increase paracellular permeability via disruption of cell-cell junctions that result in transient focal leaks. To validate the results from the tissue-engineered model, we then demonstrate that transient BBBO can be reproduced in a mouse model. We identify a minimum clinically effective intra-arterial dose of 3 μM min melittin, which is reversible within one day and neurologically safe. Melittin-induced BBBO represents a novel technology for delivery of therapeutics into the brain.

Keywords:  Blood-brain barrier; Drug delivery; Melittin; Peptide; Tissue engineering

DOI:  https://doi.org/10.1016/j.biomaterials.2021.120942
Sci Rep. 2021 Jun 25. 11(1): 13334

Patient-derived scaffolds as a drug-testing platform for endocrine therapies in breast cancer.

Anna Gustafsson, Elena Garre, Maria Carmen Leiva, Simona Salerno, Anders Ståhlberg, Göran Landberg.

Three-dimensional cell culture platforms based on decellularised patient-based microenvironments provide in vivo-like growth conditions allowing cancer cells to interact with intact structures and components of the surrounding tissue. A patient-derived scaffold (PDS) model was therefore evaluated as a testing platform for the endocrine therapies (Z)-4-Hydroxytamoxifen (4OHT) and fulvestrant as well as the CDK4/6-inhibitor palbociclib, monitoring the treatment responses in breast cancer cell lines MCF7 and T47D adapted to the patient-based microenvironments. MCF7 cells growing in PDSs showed increased resistance to 4OHT and fulvestrant treatment (100- and 20-fold) compared to 2D cultures. Quantitative PCR analyses of endocrine treated cancer cells in PDSs revealed upregulation of pluripotency markers further supported by increased self-renewal capacity in sphere formation assays. When comparing different 3D growth platforms including PDS, matrigel, gelatin sponges and 3D-printed hydrogels, 3D based cultures showed slightly varying responses to fulvestrant and palbociclib whereas PDS and matrigel cultures showed more similar gene expression profiles for 4OHT treatment compared to the other platforms. The results support that the PDS technique maximized to provide a multitude of smaller functional PDS replicates from each primary breast cancer, is an up-scalable patient-derived drug-testing platform available for gene expression profiling and downstream functional assays.

DOI: https://doi.org/10.1038/s41598-021-92724-9