Simulating the Impact of Tumor Mechanical Forces on Glymphatic Networks in the Brain Parenchyma.

Background: The brain glymphatic system is currently being explored in the context of many neurological disorders and diseases, including traumatic brain injury, Alzheimer's disease, and ischemic stroke. However, little is known about the impact of brain tumors on glymphatic function. Mechanical forces generated during tumor development and growth may be responsible for compromised glymphatic transport pathways, reducing waste clearance and cerebrospinal fluid (CSF) transport in the brain parenchyma. One such force is solid stress, i.e., growth-induced forces from cell hyperproliferation and excess matrix deposition. Because there are no prior studies assessing the impact of tumor-derived solid stress on glymphatic system structure and performance in the brain parenchyma, this study serves to fill an important gap in the field. Methods: We adapted a previously developed Electrical Analog Model using MATLAB Simulink for glymphatic transport coupled with Finite Element Analysis for tumor mechanical stresses and strains in COMSOL. This allowed simulation of the impact of tumor mechanical force generation on fluid transport within brain parenchymal glymphatic units - which include paravascular spaces, astrocytic networks, interstitial spaces, and capillary basement membranes. We conducted a parametric analysis to compare the contributions of tumor size, tumor proximity, and ratio of glymphatic subunits to the stress and strain experienced by the glymphatic unit and corresponding reduction in flow rate of CSF. Results: Mechanical stresses intensify with proximity to the tumor and increasing tumor size, highlighting the vulnerability of nearby glymphatic units to tumor-derived forces. Our stress and strain profiles reveal compressive deformation of these surrounding glymphatics and demonstrate that varying the relative contributions of astrocytes vs. interstitial spaces impact the resulting glymphatic structure significantly under tumor mechanical forces. Increased tumor size and proximity caused increased stress and strain across all glymphatic subunits, as does decreased astrocyte composition. Indeed, our model reveals an inverse correlation between extent of astrocyte contribution to the composition of the glymphatic unit and the resulting mechanical stress. This increased mechanical strain across the glymphatic unit decreases the venous efflux rate of CSF, dependent on the degree of strain and the specific glymphatic subunit of interest. For example, a 20% mechanical strain on capillary basement membranes does not significantly decrease venous efflux (2% decrease in flow rates), while the same magnitude of strain on astrocyte networks and interstitial spaces decreases efflux flow rates by 7% and 22%, respectively. Conclusion: Our simulations reveal that solid stress from brain tumors directly reduces glymphatic fluid transport, independently from biochemical effects from cancer cells. Understanding these pathophysiological implications is crucial for developing targeted interventions aimed at restoring effective waste clearance mechanisms in the brain.This study opens potential avenues for future experimental research in brain tumor-related glymphatic dysfunction.

Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells.

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Ratiometric near-infrared fluorescent probe for nitroreductase activity enables 3D imaging of hypoxic cells within intact tumor spheroids.

Fluorescent molecular probes that report nitroreductase activity have promise as imaging tools to elucidate the biology of hypoxic cells and report the past hypoxic history of biomedical tissue. This study describes the synthesis and validation of a "first-in-class" ratiometric, hydrophilic near-infrared fluorescent molecular probe for imaging hypoxia-induced nitroreductase activity in 2D cell culture monolayers and 3D multicellular tumor spheroids. The probe's molecular structure is charge-balanced and the change in ratiometric signal is based on Förster Resonance Energy Transfer (FRET) from a deep-red, pentamethine cyanine donor dye (Cy5, emits ∼660 nm) to a linked near-infrared, heptamethine cyanine acceptor dye (Cy7, emits ∼780 nm). Enzymatic reduction of a 4-nitrobenzyl group on the Cy7 component induces a large increase in Cy7/Cy5 fluorescence ratio. The deep penetration of near-infrared light enables 3D optical sectioning of intact tumor spheroids, and visualization of individual hypoxic cells (i.e., cells with raised Cy7/Cy5 ratio) as a new way to study tumor spheroids. Beyond preclinical imaging, the near-infrared fluorescent molecular probe has high potential for ratiometric imaging of hypoxic tissue in living subjects.

Novel 3-D macrophage spheroid model reveals reciprocal regulation of immunomechanical stress and mechano-immunological response.

Purpose: In many diseases, an overabundance of macrophages contributes to adverse outcomes. While numerous studies have compared macrophage phenotype after mechanical stimulation or with varying local stiffness, it is unclear if and how macrophages themselves contribute to mechanical forces in their microenvironment. Methods: Raw 264.7 murine macrophages were embedded in a confining agarose gel, where they proliferated to form spheroids over time. Gels were synthesized at various concentrations to tune the stiffness and treated with various growth supplements to promote macrophage polarization. The spheroids were then analyzed by immunofluorescent staining and qPCR for markers of proliferation, mechanosensory channels, and polarization. Finally, spheroid geometries were used to computationally model the strain generated in the agarose by macrophage spheroid growth. Results: Macrophages form spheroids and generate growth-induced mechanical forces (i.e., solid stress) within confining agarose gels, which can be maintained for at least 16 days in culture. Increasing agarose concentration restricts spheroid expansion, promotes discoid geometries, limits gel deformation, and induces an increase in iNOS expression. LPS stimulation increases spheroid growth, though this effect is reversed with the addition of IFN-γ. Ki67 expression decreases with increasing agarose concentration, in line with the growth measurements. Conclusions: Macrophages alone both respond to and generate solid stress. Understanding how macrophage generation of growth-induced solid stress responds to different environmental conditions will help to inform treatment strategies for the plethora of diseases that involve macrophage accumulation.

Single-cell mechanical analysis reveals viscoelastic similarities between normal and neoplastic brain cells.

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes (IHAs) and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real-time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in IHAs and GBM cells via immunofluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic behavior. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future study to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Mechanical and metabolic interplay in the brain metastatic microenvironment.

In this Perspective, we provide our insights and opinions about the contribution-and potential co-regulation-of mechanics and metabolism in incurable breast cancer brain metastasis. Altered metabolic activity can affect cancer metastasis as high glucose supply and demand in the brain microenvironment favors aerobic glycolysis. Similarly, the altered mechanical properties of disseminating cancer cells facilitate migration to and metastatic seeding of the brain, where local metabolites support their progression. Cancer cells in the brain and the brain tumor microenvironment often possess opposing mechanical and metabolic properties compared to extracranial cancer cells and their microenvironment, which inhibit the ease of extravasation and metastasis of these cells outside the central nervous system. We posit that the brain provides a metabolic microenvironment that mechanically reinforces the cellular structure of cancer cells and supports their metastatic growth while restricting their spread from the brain to external organs.

Engineering a 3D collective cancer invasion model with control over collagen fiber alignment.

Prior to cancer cell invasion, the structure of the extracellular matrix (ECM) surrounding the tumor is remodeled, such that circumferentially oriented matrix fibers become radially aligned. This predisposed radially aligned matrix structure serves as a critical regulator of cancer invasion. However, a biomimetic 3D model recapitulating a tumor's behavioral response to these ECM structures is not yet available. In this study, we have developed a phase-specific, force-guided method to establish a 3D dual topographical tumor model in which each tumor spheroid/organoid is surrounded by radially aligned collagen I fibers on one side and circumferentially oriented fibers on the opposite side. A coaxial rotating cylinder system was employed to construct the dual fiber topography and to pre-seed tumor spheroids/organoids within a single device. This system enables the application of different force mechanisms in the nucleation and elongation phases of collagen fiber polymerization to guide fiber alignment. In the nucleation phase, fiber alignment is enhanced by a horizontal laminar Couette flow driven by the inner cylinder rotation. In the elongation phase, fiber growth is guided by a vertical gravitational force to form a large aligned collagen matrix gel (35 × 25 × 0.5 mm) embedded with >1000 tumor spheroids. The fibers above each tumor spheroid are radially aligned along the direction of gravitational force in contrast to the circumferentially oriented fibers beneath each tumor spheroid/organoid, where the presence of the tumor interferes with the gravity-induced fiber alignment. After tumor invasion, there are more disseminated multicellular clusters on the radially aligned side, compared to the side of the tumor spheroid/organoid facing circumferentially oriented fibers. These results indicate that our 3D dual topographical model recapitulates the preference of tumors to invade and disseminate along radially aligned fibers. We anticipate that this 3D dual topographical model will have broad utility to those studying collective tumor invasion and that it has the potential to identify cancer invasion-targeted therapeutic agents.