Stromal cells regulate mechanics of tumour spheroid.

The remarkable contractility and force generation ability exhibited by cancer cells empower them to overcome the resistance and steric hindrance presented by a three-dimensional, interconnected matrix. Cancer cells disseminate by actively remodelling and deforming their extracellular matrix (ECM). The process of tumour growth and its ECM remodelling have been extensively studied, but the effect of the cellular tumour microenvironment (TME) has been ignored in most studies that investigated tumour-cell-mediated ECM deformations and realignment. This study reports the integration of stromal cells in spheroid contractility assays that impacts the ECM remodelling and invasion abilities of cancer spheroids. To investigate this, we developed a novel multilayer in vitro assay that incorporates stromal cells and quantifies the contractile deformations that tumour spheroids exert on the ECM. We observed a negative correlation between the spheroid invasion potential and the levels of collagen deformation. The presence of stromal cells significantly increased cancer cell invasiveness and altered the cancer cells' ability to deform and realign collagen gel, due to upregulation of proinflammatory cytokines. Interestingly, this was observed consistently in both metastatic and non-metastatic cancer cells. Our findings contribute to a better understanding of the vital role played by the cellular TME in regulating the invasive outgrowth of cancer cells and underscore the potential of utilising matrix deformation measurements as a biophysical marker for evaluating invasiveness and informing targeted therapeutic opportunities.

Corrigendum: Hippocampus of the APPNL-G-F mouse model of Alzheimer's disease exhibits region-specific tissue softening concomitant with elevated astrogliosis.

[This corrects the article DOI: 10.3389/fnagi.2023.1212212.].

Hippocampus of the APPNL-G-F mouse model of Alzheimer's disease exhibits region-specific tissue softening concomitant with elevated astrogliosis.

Widespread neurodegeneration, enlargement of cerebral ventricles, and atrophy of cortical and hippocampal brain structures are classic hallmarks of Alzheimer's disease (AD). Prominent macroscopic disturbances to the cytoarchitecture of the AD brain occur alongside changes in the mechanical properties of brain tissue, as reported in recent magnetic resonance elastography (MRE) measurements of human brain mechanics. Whilst MRE has many advantages, a significant shortcoming is its spatial resolution. Higher resolution "cellular scale" assessment of the mechanical alterations to brain regions involved in memory formation, such as the hippocampus, could provide fresh new insight into the etiology of AD. Characterization of brain tissue mechanics at the cellular length scale is the first stepping-stone to understanding how mechanosensitive neurons and glia are impacted by neurodegenerative disease-associated changes in their microenvironment. To provide insight into the microscale mechanics of aging brain tissue, we measured spatiotemporal changes in the mechanical properties of the hippocampus using high resolution atomic force microscopy (AFM) indentation tests on acute brain slices from young and aged wild-type mice and the APPNL-G-F mouse model. Several hippocampal regions in APPNL-G-F mice are significantly softer than age-matched wild-types, notably the dentate granule cell layer and the CA1 pyramidal cell layer. Interestingly, regional softening coincides with an increase in astrocyte reactivity, suggesting that amyloid pathology-mediated alterations to the mechanical properties of brain tissue may impact the function of mechanosensitive astrocytes. Our data also raise questions as to whether aberrant mechanotransduction signaling could impact the susceptibility of neurons to cellular stressors in their microenvironment.

Endothelium and Subendothelial Matrix Mechanics Modulate Cancer Cell Transendothelial Migration.

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.

Bioengineered Multicellular Liver Microtissues for Modeling Advanced Hepatic Fibrosis Driven Through Non-Alcoholic Fatty Liver Disease.

Despite considerable efforts in modeling liver disease in vitro, it remains difficult to recapitulate the pathogenesis of the advanced phases of non-alcoholic fatty liver disease (NAFLD) with inflammation and fibrosis. Here, a liver-on-a-chip platform with bioengineered multicellular liver microtissues is developed, composed of four major types of liver cells (hepatocytes, endothelial cells, Kupffer cells, and stellate cells) to implement a human hepatic fibrosis model driven by NAFLD: i) lipid accumulation in hepatocytes (steatosis), ii) neovascularization by endothelial cells, iii) inflammation by activated Kupffer cells (steatohepatitis), and iv) extracellular matrix deposition by activated stellate cells (fibrosis). In this model, the presence of stellate cells in the liver-on-a-chip model with fat supplementation showed elevated inflammatory responses and fibrosis marker up-regulation. Compared to transforming growth factor-beta-induced hepatic fibrosis models, this model includes the native pathological and chronological steps of NAFLD which shows i) higher fibrotic phenotypes, ii) increased expression of fibrosis markers, and iii) efficient drug transport and metabolism. Taken together, the proposed platform will enable a better understanding of the mechanisms underlying fibrosis progression in NAFLD as well as the identification of new drugs for the different stages of NAFLD.

In Vitro Human Liver Model of Nonalcoholic Steatohepatitis by Coculturing Hepatocytes, Endothelial Cells, and Kupffer Cells.

The liver has a complex and unique microenvironment with multiple cell-cell interactions and internal vascular networks. Although nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with multiple phases, no proper model could fully recapitulate the in vivo microenvironment to understand NAFLD progression. Here, an in vitro human liver model of NAFLD by coculturing human hepatocytes, umbilical vein endothelial cells (HUVECs), and Kupffer cells (KCs) into spheroids is presented. Analysis of indirect cross-talk using conditioned media between steatotic spheroids-composed of hepatocellular carcinoma-derived cells (HepG2) and HUVECs-and mouse KCs reveals that the latter can be activated showing increased cell area, elevated production of reactive oxygen species (ROS), and proinflammatory cytokines. Spheroids incorporating human KCs (HKCs) can also be induced into steatotic stage by supplementing fat. Steatotic spheroids with/without HKCs show different levels of steatotic stages through lipid accumulation and ROS production. Steatotic spheroids made from an immortalized hepatic progenitor cell line (HepaRG) compared to those made from HepG2 cells display similar trends of functionality, but elevated levels of proinflammatory cytokines, and improved reversibility of steatosis. The in vitro human liver system proposed makes strides in developing a model to mimic and monitor the progression of NAFLD.

A Microfabricated Sandwiching Assay for Nanoliter and High-Throughput Biomarker Screening.

Cells secrete substances that are essential to the understanding of numerous immunological phenomena and are extensively used in clinical diagnoses. Countless techniques for screening of biomarker secretion in living cells have generated valuable information on cell function and physiology, but low volume and real-time analysis is a bottleneck for a range of approaches. Here, a simple, highly sensitive assay using a high-throughput micropillar and microwell array chip (MIMIC) platform is presented for monitoring of biomarkers secreted by cancer cells. The sensing element is a micropillar array that uses the enzyme-linked immunosorbent assay (ELISA) mechanism to detect captured biomolecules. When integrated with a microwell array where few cells are localized, interleukin 8 (IL-8) secretion can be monitored with nanoliter volume using multiple micropillar arrays. The trend of cell secretions measured using MIMICs matches the results from conventional ELISA well while it requires orders of magnitude less cells and volumes. Moreover, the proposed MIMIC is examined to be used as a drug screening platform by delivering drugs using micropillar arrays in combination with a microfluidic system and then detecting biomolecules from cells as exposed to drugs.

Dielectrophoresis-assisted creation of cell aggregates under flow conditions using planar electrodes.

We present a microfluidic platform allowing dielectrophoresis-assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom-up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell-cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.

A Human Liver-on-a-Chip Platform for Modeling Nonalcoholic Fatty Liver Disease.

The liver possesses a unique microenvironment with a complex internal vascular system and cell-cell interactions. Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, and although much effort has been dedicated to building models to target NAFLD, most in vitro systems rely on simple models failing to recapitulate complex liver functions. Here, an in vitro system is presented to study NAFLD (steatosis) by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) into spheroids. Analysis of colocalization of HepG2-HUVECs along with the level of steatosis reveals that the NAFLD pathogenesis could be better modeled when 20% of HUVECs are presented in HepG2 spheroids. Spheroids with fat supplements progressed to the steatosis stage on day 2, which could be maintained for more than a week without being harmful for cells. Transferring spheroids onto a chip system with an array of interconnected hexagonal microwells proves helpful for monitoring functionality through increased albumin secretions with HepG2-HUVEC interactions and elevated production of reactive oxygen species for steatotic spheroids. The reversibility of steatosis is demonstrated by simply stopping fat-based diet or by antisteatotic drug administration, the latter showing a faster return of intracellular lipid levels to the basal level.