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.

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.

Mathematical model of oxygen, nutrient, and drug transport in tuberculosis granulomas.

Physiological abnormalities in pulmonary granulomas-pathological hallmarks of tuberculosis (TB)-compromise the transport of oxygen, nutrients, and drugs. In prior studies, we demonstrated mathematically and experimentally that hypoxia and necrosis emerge in the granuloma microenvironment (GME) as a direct result of limited oxygen availability. Building on our initial model of avascular oxygen diffusion, here we explore additional aspects of oxygen transport, including the roles of granuloma vasculature, transcapillary transport, plasma dilution, and interstitial convection, followed by cellular metabolism. Approximate analytical solutions are provided for oxygen and glucose concentration, interstitial fluid velocity, interstitial fluid pressure, and the thickness of the convective zone. These predictions are in agreement with prior experimental results from rabbit TB granulomas and from rat carcinoma models, which share similar transport limitations. Additional drug delivery predictions for anti-TB-agents (rifampicin and clofazimine) strikingly match recent spatially-resolved experimental results from a mouse model of TB. Finally, an approach to improve molecular transport in granulomas by modulating interstitial hydraulic conductivity is tested in silico.

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.

Do Tumor Mechanical Stresses Promote Cancer Immune Escape?

Immune evasion-a well-established cancer hallmark-is a major barrier to immunotherapy efficacy. While the molecular mechanisms and biological consequences underpinning immune evasion are largely known, the role of tissue mechanical stresses in these processes warrants further investigation. The tumor microenvironment (TME) features physical abnormalities (notably, increased fluid and solid pressures applied both inside and outside the TME) that drive cancer mechanopathologies. Strikingly, in response to these mechanical stresses, cancer cells upregulate canonical immune evasion mechanisms, including epithelial-mesenchymal transition (EMT) and autophagy. Consideration and characterization of the origins and consequences of tumor mechanical stresses in the TME may yield novel strategies to combat immunotherapy resistance. In this Perspective, we posit that tumor mechanical stresses-namely fluid shear and solid stresses-induce immune evasion by upregulating EMT and autophagy. In addition to exploring the basis for our hypothesis, we also identify explicit gaps in the field that need to be addressed in order to directly demonstrate the existence and importance of this biophysical relationship. Finally, we propose that reducing or neutralizing fluid shear stress and solid stress-induced cancer immune escape may improve immunotherapy outcomes.

The heterogeneous morphology of networked collagen in distal colon and rectum of mice quantified via nonlinear microscopy.

Visceral pain from the distal colon and rectum (colorectum) is a major complaint of patients with irritable bowel syndrome. Mechanotransduction of colorectal distension/stretch appears to play a critical role in visceral nociception, and further understanding requires improved knowledge of the micromechanical environments at different sub-layers of the colorectum. In this study, we conducted nonlinear imaging via second harmonic generation to quantify the thickness of each distinct through-thickness layer of the colorectum, as well as the principal orientations, corresponding dispersions in orientations, and the distributions of diameters of collagen fibers within each of these layers. From C57BL/6 mice of both sexes (8-16 weeks of age, 25-35 g), we dissected the distal 30 mm of the large bowel including the colorectum, divided these into three even segments, and harvested specimens (~8 × 8 mm2) from each segment. We stretched the specimens either by colorectal distension to 20 mmHg (reference) or 80 mmHg (deformed) or by biaxial stretch to 10 mN (reference) or 80 mN (deformed), and fixed them with 4% paraformaldehyde. We then conducted SHG imaging through the wall thickness and analyzed post-hoc using custom-built software to quantify the orientations of collagen fibers in all distinct layers. We also quantified the thickness of each layer of the colorectum, and the corresponding distributions of collagen density and diameters of fibers. We found collagen concentrated in the submucosal layer. The average diameter of collagen fibers was greatest in the submucosal layer, followed by the serosal and muscular layers. Collagen fibers aligned with muscle fibers in the two muscular layers, whereas their orientation varied greatly with location in the serosal layer. In colonic segments, thick collagen fibers in the submucosa presented two major orientations aligned approximately ±30° to the axial direction, and form a patterned network. Our results indicate the submucosa is likely the principal passive load-bearing structure of the colorectum. In addition, afferent endings in those collagen-rich regions present likely candidates of colorectal nociceptors to encode noxious distension/stretch.

The Macro- and Micro-Mechanics of the Colon and Rectum II: Theoretical and Computational Methods.

Abnormal colorectal biomechanics and mechanotransduction associate with an array of gastrointestinal diseases, including inflammatory bowel disease, irritable bowel syndrome, diverticula disease, anorectal disorders, ileus, and chronic constipation. Visceral pain, principally evoked from mechanical distension, has a unique biomechanical component that plays a critical role in mechanotransduction, the process of encoding mechanical stimuli to the colorectum by sensory afferents. To fully understand the underlying mechanisms of visceral mechanical neural encoding demands focused attention on the macro- and micro-mechanics of colon tissue. Motivated by biomechanical experiments on the colon and rectum, increasing efforts focus on developing constitutive frameworks to interpret and predict the anisotropic and nonlinear biomechanical behaviors of the multilayered colorectum. We will review the current literature on computational modeling of the colon and rectum as well as the mechanical neural encoding by stretch sensitive afferent endings, and then highlight our recent advances in these areas. Current models provide insight into organ- and tissue-level biomechanics as well as the stretch-sensitive afferent endings of colorectal tissues yet an important challenge in modeling theory remains. The research community has not connected the biomechanical models to those of mechanosensitive nerve endings to create a cohesive multiscale framework for predicting mechanotransduction from organ-level biomechanics.

The Macro- and Micro-Mechanics of the Colon and Rectum I: Experimental Evidence.

Many lower gastrointestinal diseases are associated with altered mechanical movement and deformation of the large intestine, i.e., the colon and rectum. The leading reason for patients' visits to gastrointestinal clinics is visceral pain, which is reliably evoked by mechanical distension rather than non-mechanical stimuli such as inflammation or heating. The macroscopic biomechanics of the large intestine were characterized by mechanical tests and the microscopic by imaging the load-bearing constituents, i.e., intestinal collagen and muscle fibers. Regions with high mechanical stresses in the large intestine (submucosa and muscularis propria) coincide with locations of submucosal and myenteric neural plexuses, indicating a functional interaction between intestinal structural biomechanics and enteric neurons. In this review, we systematically summarized experimental evidence on the macro- and micro-scale biomechanics of the colon and rectum in both health and disease. We reviewed the heterogeneous mechanical properties of the colon and rectum and surveyed the imaging methods applied to characterize collagen fibers in the intestinal wall. We also discussed the presence of extrinsic and intrinsic neural tissues within different layers of the colon and rectum. This review provides a foundation for further advancements in intestinal biomechanics by synergistically studying the interplay between tissue biomechanics and enteric neurons.

Load-bearing function of the colorectal submucosa and its relevance to visceral nociception elicited by mechanical stretch.

Mechanical distension beyond a particular threshold evokes visceral pain from distal colon and rectum (colorectum), and thus biomechanics plays a central role in visceral nociception. In this study we focused on the layered structure of the colorectum through the wall thickness and determined the biomechanical properties of layer-separated colorectal tissue. We harvested the distal 30 mm of mouse colorectum and dissected this tissue into inner and outer composite layers. The inner composite consists of the mucosa and submucosa, whereas the outer composite includes the muscular layers and serosa. We divided each composite axially into three 10-mm-long segments and conducted biaxial mechanical extension tests and opening-angle measurements for each tissue segment. In addition, we quantified the thickness of the rich collagen network in the submucosa by nonlinear imaging via second-harmonic generation (SHG). Our results reveal that the inner composite is slightly stiffer in the axial direction, whereas the outer composite is stiffer circumferentially. The stiffness of the inner composite in the axial direction is about twice that in the circumferential direction, consistent with the orientations of collagen fibers in the submucosa approximately ±30° to the axial direction. Submucosal thickness measured by SHG showed no difference from proximal to distal colorectum under the load-free condition, which likely contributes to the comparable tension stiffness of the inner composite along the colorectum. This, in turn, strongly indicates the submucosa as the load-bearing structure of the colorectum. This further implies nociceptive roles for the colorectal afferent endings in the submucosa, which likely encode tissue-injurious mechanical distension.NEW & NOTEWORTHY Visceral pain from distal colon and rectum (colorectum) is usually elicited from mechanical distension/stretch, rather than from heating, cutting, or pinching, which usually evoke pain from the skin. We conducted layer-separated biomechanical tests on mouse colorectum and identified an unexpected role of submucosa as the load-bearing structure of the colorectum. Outcomes of this study will focus attention on sensory nerve endings in the submucosa that likely encode tissue-injurious distension/stretch to cause visceral pain.

Differential biomechanical properties of mouse distal colon and rectum innervated by the splanchnic and pelvic afferents.

Visceral pain is one of the principal complaints of patients with irritable bowel syndrome, and this pain is reliably evoked by mechanical distension and stretch of distal colon and rectum (colorectum). This study focuses on the biomechanics of the colorectum that could play critical roles in mechanical neural encoding. We harvested the distal 30 mm of the colorectum from mice, divided evenly into three 10-mm-long segments (colonic, intermediate and rectal), and conducted biaxial mechanical stretch tests and opening-angle measurements for each tissue segment. In addition, we determined the collagen fiber orientations and contents across the thickness of the colorectal wall by nonlinear imaging via second harmonic generation (SHG). Our results reveal a progressive increase in tissue compliance and prestress from colonic to rectal segments, which supports prior electrophysiological findings of distinct mechanical neural encodings by afferents in the lumbar splanchnic nerves (LSN) and pelvic nerves (PN) that dominate colonic and rectal innervations, respectively. The colorectum is significantly more viscoelastic in the circumferential direction than in the axial direction. In addition, our SHG results reveal a rich collagen network in the submucosa and orients approximately ±30° to the axial direction, consistent with the biaxial test results presenting almost twice the stiffness in axial direction versus the circumferential direction. Results from current biomechanical study strongly indicate the prominent roles of local tissue biomechanics in determining the differential mechanical neural encoding functions in different regions of the colorectum. NEW & NOTEWORTHY Mechanical distension and stretch-not heat, cutting, or pinching-reliably evoke pain from distal colon and rectum. We report different local mechanics along the longitudinal length of the colorectum, which is consistent with the existing literature on distinct mechanotransduction of afferents innervating proximal and distal regions of the colorectum. This study draws attention to local mechanics as a potential determinant factor for mechanical neural encoding of the colorectum, which is crucial in visceral nociception.