Alteration of mechanical stresses in the murine brain by age and hemorrhagic stroke.

Residual mechanical stresses, also known as solid stresses, emerge during rapid differential growth or remodeling of tissues, as observed in morphogenesis and tumor growth. While residual stresses typically dissipate in most healthy adult organs, as the growth rate decreases, high residual stresses have been reported in mature, healthy brains. However, the origins and consequences of residual mechanical stresses in the brain across health, aging, and disease remain poorly understood. Here, we utilized and validated a previously developed method to map residual mechanical stresses in the brains of mice across three age groups: 5-7 days, 8-12 weeks, and 22 months. We found that residual solid stress rapidly increases from 5-7 days to 8-12 weeks and remains high in mature 22 months mice brains. Three-dimensional mapping revealed unevenly distributed residual stresses from the anterior to posterior coronal brain sections. Since the brain is rich in negatively charged hyaluronic acid, we evaluated the contribution of charged extracellular matrix (ECM) constituents in maintaining solid stress levels. We found that lower ionic strength leads to elevated solid stresses, consistent with its unshielding effect and the subsequent expansion of charged ECM components. Lastly, we demonstrated that hemorrhagic stroke, accompanied by loss of cellular density, resulted in decreased residual stress in the murine brain. Our findings contribute to a better understanding of spatiotemporal alterations of residual solid stresses in healthy and diseased brains, a crucial step toward uncovering the biological and immunological consequences of this understudied mechanical phenotype in the brain.

Emergence of nanoscale viscoelasticity from single cancer cells to established tumors.

Tumors are complex materials whose physical properties dictate growth and treatment outcomes. Recent evidence suggests time-dependent physical properties, such as viscoelasticity, are crucial, distinct mechanical regulators of cancer progression and malignancy, yet the genesis and consequences of tumor viscoelasticity are poorly understood. Here, using Wide-bandwidth AFM-based ViscoElastic Spectroscopy (WAVES) coupled with mathematical modeling, we probe the origins of tumor viscoelasticity. From single carcinoma cells to increasingly sized carcinoma spheroids to established tumors, we describe a stepwise evolution of dynamic mechanical properties that create a nanorheological signature of established tumors: increased stiffness, decreased rate-dependent stiffening, and reduced energy dissipation. We dissect this evolution of viscoelasticity by scale, and show established tumors use fluid-solid interactions as the dominant mechanism of mechanical energy dissipation as opposed to fluid-independent intrinsic viscoelasticity. Additionally, we demonstrate the energy dissipation mechanism in spheroids and established tumors is negatively correlated with the cellular density, and this relationship strongly depends on an intact actin cytoskeleton. These findings define an emergent and targetable signature of the physical tumor microenvironment, with potential for deeper understanding of tumor pathophysiology and treatment strategies.

SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders.

Protein-protein interactions (PPIs) are critical for biological processes and predicting the sites of these interactions is useful for both computational and experimental applications. We present a Structure-agnostic Language Transformer and Peptide Prioritization (SaLT&PepPr) pipeline to predict interaction interfaces from a protein sequence alone for the subsequent generation of peptidic binding motifs. Our model fine-tunes the ESM-2 protein language model (pLM) with a per-position prediction task to identify PPI sites using data from the PDB, and prioritizes motifs which are most likely to be involved within inter-chain binding. By only using amino acid sequence as input, our model is competitive with structural homology-based methods, but exhibits reduced performance compared with deep learning models that input both structural and sequence features. Inspired by our previous results using co-crystals to engineer target-binding "guide" peptides, we curate PPI databases to identify partners for subsequent peptide derivation. Fusing guide peptides to an E3 ubiquitin ligase domain, we demonstrate degradation of endogenous ß-catenin, 4E-BP2, and TRIM8, and highlight the nanomolar binding affinity, low off-targeting propensity, and function-altering capability of our best-performing degraders in cancer cells. In total, our study suggests that prioritizing binders from natural interactions via pLMs can enable programmable protein targeting and modulation.

Intravital measurements of solid stresses in tumours reveal length-scale and microenvironmentally dependent force transmission.

In cancer, solid stresses impede the delivery of therapeutics to tumours and the trafficking and tumour infiltration of immune cells. Understanding such consequences and the origin of solid stresses requires their probing in vivo at the cellular scale. Here we report a method for performing volumetric and longitudinal measurements of solid stresses in vivo, and findings from its applicability to tumours. We used multimodal intravital microscopy of fluorescently labelled polyacrylamide beads injected in breast tumours in mice as well as mathematical modelling to compare solid stresses at the single-cell and tissue scales, in primary and metastatic tumours, in vitro and in mice, and in live mice and post-mortem tissue. We found that solid-stress transmission is scale dependent, with tumour cells experiencing lower stresses than their embedding tissue, and that tumour cells in lung metastases experience substantially higher solid stresses than those in the primary tumours. The dependence of solid stresses on length scale and the microenvironment may inform the development of therapeutics that sensitize cancer cells to such mechanical forces.

The peritumor microenvironment: physics and immunity.

Cancer initiation and progression drastically alter the microenvironment at the interface between healthy and malignant tissue. This site, termed the peritumor, bears unique physical and immune attributes that together further promote tumor progression through interconnected mechanical signaling and immune activity. In this review, we describe the distinct physical features of the peritumoral microenvironment and link their relationship to immune responses. The peritumor is a region rich in biomarkers and therapeutic targets and thus is a key focus for future cancer research as well as clinical outlooks, particularly to understand and overcome novel mechanisms of immunotherapy resistance.

Low LINC01272 predicts poor prognosis of non-small cell lung cancer and its biological function in tumor cells by inhibiting miR-1303.

Non-small cell lung cancer (NSCLC) is a malignant tumor associated with poor prognosis. The clinical value of long non-coding RNAs (lncRNAs) in the pathomechanism of various types of human malignancy has attracted increasing attention. The present study aimed to investigate the expression of LINC01272 in NSCLC and to determine its prognostic value and biological role. Tumor and adjacent non-tumor tissues from 108 patients with NSCLC and NSCLC cell lines were used in this study. The expression levels of LINC01272 and microRNA (miR)-1303 in tissues of patients and NSCLC cell lines were evaluated by reverse transcription quantitative PCR. The relationship between LINC01272 and the overall survival of patients with NSCLC was analyzed by Kaplan-Meier survival curve and log-rank test. Cox regression analysis confirmed the prognostic value of LINC01272 in patients with NSCLC. Cell Counting Kit-8 assay was used to evaluate the proliferation of NSCLC cells. The migration and invasion of NSCLC cells were determined using Transwell assays. The interaction between LINC01272 and miR-1303 in NSCLC was confirmed by dual-luciferase reporter assay. LINC01272 downregulation in NSCLC tissues was associated with worse overall survival in patients based on bioinformatics analysis. Furthermore, LINC01272 expression, which was decreased in NSCLC tumor tissues and NSCLC cells, was considered as an independent prognostic biomarker in NSCLC. In addition, LINC01272 overexpression inhibited NSCLC cell proliferation, migration and invasion. miR-1303 expression, which was increased in tumor tissues, was sponged by LINC01272 and negatively correlated with LINC01272 expression. miR-1303 expression reversed the inhibitory effects of LINC01272 on NSCLC cell function. In summary, the findings from this study suggested that LINC01272 expression, which was decreased in NSCLC tumor tissues and NSCLC cells, may be used as an independent prognostic biomarker for patients with NSCLC and that its overexpression may suppress NSCLC cell proliferation, migration and invasion by inhibiting miR-1303.

Solid stress impairs lymphocyte infiltration into lymph-node metastases.

Strong and durable anticancer immune responses are associated with the generation of activated cancer-specific T cells in the draining lymph nodes. However, cancer cells can colonize lymph nodes and drive tumour progression. Here, we show that lymphocytes fail to penetrate metastatic lesions in lymph nodes. In tissue from patients with breast, colon, and head and neck cancers, as well as in mice with spontaneously developing breast-cancer lymph-node metastases, we found that lymphocyte exclusion from nodal lesions is associated with the presence of solid stress caused by lesion growth, that solid stress induces reductions in the number of functional high endothelial venules in the nodes, and that relieving solid stress in the mice increased the presence of lymphocytes in lymph-node lesions by about 15-fold. Solid-stress-mediated impairment of lymphocyte infiltration into lymph-node metastases suggests a therapeutic route for overcoming T-cell exclusion during immunotherapy.

In vivo compression and imaging in mouse brain to measure the effects of solid stress.

We recently developed an in vivo compression device that simulates the solid mechanical forces exerted by a growing tumor on the surrounding brain tissue and delineates the physical versus biological effects of a tumor. This device, to our knowledge the first of its kind, can recapitulate the compressive forces on the cerebellar cortex from primary (e.g., glioblastoma) and metastatic (e.g., breast cancer) tumors, as well as on the cerebellum from tumors such as medulloblastoma and ependymoma. We adapted standard transparent cranial windows normally used for intravital imaging studies in mice to include a turnable screw for controlled compression (acute or chronic) and decompression of the cerebral cortex. The device enables longitudinal imaging of the compressed brain tissue over several weeks or months as the screw is progressively extended against the brain tissue to recapitulate tumor growth-induced solid stress. The cranial window can be simply installed on the mouse skull according to previously established methods, and the screw mechanism can be readily manufactured in-house. The total time for construction and implantation of the in vivo compressive cranial window is <1 h (per mouse). This technique can also be used to study a variety of other diseases or disorders that present with abnormal solid masses in the brain, including cysts and benign growths.

Degradable poly(ethylene glycol) (PEG)-based hydrogels for spatiotemporal control of siRNA/nanoparticle delivery.

Despite great therapeutic potential and development of a repertoire of delivery approaches addressing degradation and cellular uptake limitations, small interfering RNA (siRNA) exhibits poorly controlled tissue-specific localization. To overcome this hurdle, siRNA was complexed to nanoparticles (siRNA/NP) embedded within poly(ethylene glycol)-poly(lactic acid)-dimethacrylate (PEG-PLA-DM) hydrogels with the hypothesis that hydrolytic degradation of ester bonds within the PLA crosslinks would provide tunable, sustained siRNA/NP release. Hydrogels formed from macromers with increasing PLA repeats (e.g., 0 or non-degradable to 5 PLA repeats flanking PEG cores) and mixtures of nondegradable PEG-DM (0 PLA) and degradable PEG-PLA5-DM macromers were investigated. Hydrogels formed only with fully degradable crosslinks degraded rapidly over 6-14 days with limited control over siRNA/NP release. However, hydrogels formed with mixtures of nondegradable and 20%, 50%, and 100% degradable macromers resulted in siRNA/NP release over 3 to 28 days. Subsequently, gene silencing mediated by released siRNA/NP from 20% and 50% degradable hydrogels was sustained for ~28 days. Furthermore, in vivo imaging showed that hydrogel degradation controlled siRNA/NP localization, with sustained siRNA/NP release from 0%, 20% and 50% degradable hydrogels over 28, 21, and 15 days. A model, which accounts for hydrogel degradation rate and siRNA/NP diffusion, was developed to enable rational design of siRNA/NP delivery depots. Overall, this study shows that siRNA/NP release can be sustained via encapsulation in hydrogels with tunable degradation kinetics and modeled for a priori design of delivery depots.