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The unique physical tumor microenvironment (TME) and aberrant immune metabolic status are two obstacles that must be overcome in cancer immunotherapy to improve clinical outcomes. Here, an in situ mechano-immunometabolic therapy involving the injection of a biomimetic hydrogel is presented with sequential release of the anti-fibrotic agent pirfenidone, which softens the stiff extracellular matrix, and small interfering RNA IDO1, which disrupts kynurenine-mediated immunosuppressive metabolic pathways, together with the multi-kinase inhibitor sorafenib, which induces immunogenic cell death. This combination synergistically augmented tumor immunogenicity and induced anti-tumor immunity. In mouse models of clear cell renal cell carcinoma, a single-dose peritumoral injection of a biomimetic hydrogel facilitated the perioperative TME toward a more immunostimulatory landscape, which prevented tumor relapse post-surgery and prolonged mouse survival. Additionally, the systemic anti-tumor surveillance effect induced by local treatment decreased lung metastasis by inhibiting epithelial-mesenchymal transition conversion. The versatile localized mechano-immunometabolic therapy can serve as a universal strategy for conferring efficient tumoricidal immunity in "cold" tumor postoperative interventions.

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Understanding the signals from the physical microenvironment is critical for deciphering the processes of neurogenesis and neurodevelopment. The discovery of how surrounding physical signals shape human developing neurons is hindered by the bottleneck of conventional cell culture and animal models. Notwithstanding neural organoids provide a promising platform for recapitulating human neurogenesis and neurodevelopment, building neuronal physical microenvironment that accurately mimics the native neurophysical features is largely ignored in current organoid technologies. Here, it is discussed how the physical microenvironment modulates critical events during the periods of neurogenesis and neurodevelopment, such as neural stem cell fates, neural tube closure, neuronal migration, axonal guidance, optic cup formation, and cortical folding. Although animal models are widely used to investigate the impacts of physical factors on neurodevelopment and neuropathy, the important roles of human stem cell-derived neural organoids in this field are particularly highlighted. Considering the great promise of human organoids, building neural organoid microenvironments with mechanical forces, electrophysiological microsystems, and light manipulation will help to fully understand the physical cues in neurodevelopmental processes. Neural organoids combined with cutting-edge techniques, such as advanced atomic force microscopes, microrobots, and structural color biomaterials might promote the development of neural organoid-based research and neuroscience.

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Treatment of large bone defects represents a great challenge in orthopedic and craniomaxillofacial surgery. Traditional strategies in bone tissue engineering have focused primarily on mimicking the extracellular matrix (ECM) of bone in terms of structure and composition. However, the synergistic effects of other cues from the microenvironment during bone regeneration are often neglected. The bone microenvironment is a sophisticated system that includes physiological (e.g., neighboring cells such as macrophages), chemical (e.g., oxygen, pH), and physical factors (e.g., mechanics, acoustics) that dynamically interact with each other. Microenvironment-targeted strategies are increasingly recognized as crucial for successful bone regeneration and offer promising solutions for advancing bone tissue engineering. This review provides a comprehensive overview of current microenvironment-targeted strategies and challenges for bone regeneration and further outlines prospective directions of the approaches in construction of bone organoids.

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Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools. Among which mammalian cell protein expression approach is the most powerful one, which can express complex proteins or genetic engineered biological drugs, such as PD-1. However, the high expense, which partially derives from its low protein yielding efficiency, limited the further application of such approach in large scale production of target proteins. To address this issue, we proposed a novel technique to promote the protein production efficiency of mammal cells without using conventional genetic engineered approaches. By placing 293T cells in a hydrogel 3D cell culture platform and adjusting the stress relaxation of the matrix hydrogel, cells formed multicellular spheroids by self-organization. In particular, the multicellular spheroids have a significantly enhanced ability to transiently express multiple proteins (SHH-N, PD-1 and PDL-1). We also examined in detail the mechanism underlying this phenomenon, and found that the reorganization of cytoskeleton during spheroids formation enhances the translation process of protein by recruiting ribosomes. Overall, this finding provides a novel approach for subsequent improvement of large-scale mammalian protein expression cell systems.

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Cancer cells in secondary tumors are found to form metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, the role of deleterious mechanical stresses in this change of metastatic potential is unclear. Here, by forcing cancer cells to flow through small capillary-sized constrictions, it is demonstrated that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. These results highlight a potential link between the microenvironmental physical stresses and the enhanced malignancy of metastasizing cancer cells which may be utilized as a therapeutic strategy in preventing the metastatic spread of cancer cells.

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The therapy of solid tumors is always hampered by the intrinsic tumor physical microenvironment (TPME) featured with compact and rigid extracellular matrix (ECM) microstructures. Herein, we introduce nattokinase (NKase), a thrombolytic healthcare drug, to comprehensively regulate the TPME for versatile enhancement of various therapy modalities. Intratumoral injection of NKase not only degrades the major ECM component fibronectin but also inhibits cancer-associated fibroblasts (CAFs) in generating fibrosis, resulting in decreased tumor stiffness, enhanced perfusion, and hypoxia alleviation. The NKase-mediated regulation of the TPME significantly promotes the tumoral accumulation of therapeutic agents, leading to efficient chemotherapy without inducing side effects. Additionally, the enhancement of tumor radiotherapy based on radiosensitizers was also achieved by the pretreatment of intratumorally injected NKase, which could be ascribed to the elevated oxygen saturation level in NKase-treated tumors. Moreover, a xenografted human breast MDB-MA-231 tumor model is established to evaluate the influence of NKase on chimeric antigen receptor (CAR)-T cell therapy, illustrating that the pretreatment of NKase could boost the infiltration of CAR-T cells into tumors and thus be a benefit for tumor inhibition. These findings demonstrate the great promise of the NKase-regulated TPME as a translational strategy for universal enhancement of therapeutic efficacy in solid tumors by various treatments.

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The clinical benefits of immunotherapy are proven in many cancers, but a significant number of patients do not respond well to immunotherapy. The tumor physical microenvironment (TpME) has recently been shown to affect the growth, metastasis and treatment of solid tumors. The tumor microenvironment (TME) has unique physical hallmarks: 1) unique tissue microarchitecture, 2) increased stiffness, 3) elevated solid stress, and 4) elevated interstitial fluid pressure (IFP), which contribute to tumor progression and immunotherapy resistance in a variety of ways. Radiotherapy, a traditional and powerful treatment, can remodel the matrix and blood flow associated with the tumor to improve the response rate of immune checkpoint inhibitors (ICIs) to a certain extent. Herein, we first review the recent research advances on the physical properties of the TME and then explain how TpME is involved in immunotherapy resistance. Finally, we discuss how radiotherapy can remodel TpME to overcome immunotherapy resistance.

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Nanoparticles (NPs) are confronted with limited and disappointing delivery efficiency in tumors clinically. The tumor extracellular matrix (ECM), whose physical traits have recently been recognized as new hallmarks of cancer, forms a main steric obstacle for NP diffusion, yet the role of tumor ECM physical traits in NP diffusion remains largely unexplored. Here, we characterized the physical properties of clinical gastric tumor samples and observed limited distribution of NPs in decellularized tumor tissues. We also performed molecular dynamics simulations and in vitro hydrogel experiments through single-particle tracking to investigate the diffusion mechanism of NPs and understand the influence of tumor ECM physical properties on NP diffusion both individually and collectively. Furthermore, we developed an estimation matrix model with evaluation scores of NP diffusion efficiency through comprehensive analyses of the data. Thus, beyond finding that loose and soft ECM with aligned structure contribute to efficient diffusion, we now have a systemic model to predict NP diffusion efficiency based on ECM physical traits and provide critical guidance for personalized tumor diagnosis and treatment.

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Chimeric antigen receptor (CAR) T cell therapy has achieved unprecedented clinical success against hematologic malignancies. However, the transition of CAR-T cell therapies for solid tumors is limited by heterogenous antigen expression, immunosuppressive microenvironment (TME), immune adaptation of tumor cells and impeded CAR-T-cell infiltration/transportation. Recent studies increasingly reveal that tumor physical microenvironment could affect various aspects of tumor biology and impose profound impacts on the antitumor efficacy of CAR-T therapy. In this review, we discuss the critical roles of four physical cues in solid tumors for regulating the immune responses of CAR-T cells, which include solid stress, interstitial fluid pressure, stiffness and microarchitecture. We highlight new strategies exploiting these features to enhance the therapeutic potency of CAR-T cells in solid tumors by correlating with the state-of-the-art technologies in this field. A perspective on the future directions for developing new CAR-T therapies for solid tumor treatment is also provided.

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The success of anticancer therapies is often limited by heterogeneity within and between tumors. While much attention has been devoted to understanding the intrinsic molecular diversity of tumor cells, the surrounding tissue microenvironment is also highly complex and coevolves with tumor cells to drive clinical outcomes. Here, we propose that diverse types of solid tumors share common physical motifs that change in time and space, serving as universal regulators of malignancy. We use breast cancer and glioblastoma as instructive examples and highlight how invasion in both diseases is driven by the appropriation of structural guidance cues, contact-dependent heterotypic interactions with stromal cells, and elevated interstitial fluid pressure and flow. We discuss how engineering strategies show increasing value for measuring and modeling these physical propertiesfor mechanistic studies. Moreover, engineered systems offer great promise for developing and testing novel therapies that improve patient prognosis by normalizing the physical tumor microenvironment.

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A low response rate is the major challenge for existing cancer immunotherapies. Physical cues in the tumor microenvironment (TME) have been recognized as new hallmarks of cancer, which may synergistically contribute to low immunotherapy response. Recent evidence indicates that the physical hallmarks of cancer hold great potential as new targets for improved immunotherapy.

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Macrophages play essential roles in innate immunity and their functions can be activated by different signals at pathological sites. Concerning changes in the rigidity of the microenvironment as a disease progresses, the influence of stiffened substrates on macrophage physiology remains elusive. In this study, to evaluate the effect of stiffened substrates on macrophages, we used J774A.1 cells as the macrophage model to investigate its mechanoinflammation responses using engineered polymeric substrates with various physiological rigidities (approximately 0.6 to 100 kPa). Under lipopolysaccharide (LPS) and adenosine triphosphate (ATP) stress, approximately 4-fold higher cytoplasmic reactive oxygen species (ROS) were triggered in cells on the softer substrate, compared with cells on the stiff substrates. The enhanced ROS response was found to be regulated mainly by NADPH oxidase. Moreover, mitochondrial ROS (mtROS), a crucial intracellular ROS source, are produced in response to substrate rigidity. The results showed higher mtROS production when cells were grown on a soft substrate with LPS/ATP stimuli, and the mechano-mtROS alteration was eliminated by Rho kinase inhibitor Y-27632. We suggest that substrate rigidity can coincide with LPS/ATP in regulating the ROS generation of macrophages. As a result of the pivotal role of ROS in regulating inflammation, increased NLRP-3 inflammasome formation and higher NO secretion (an approximately 300% increase) were observed with macrophages grown on soft substrates. Although no substantial genomic distinction was identified in our experiments, based on the phenotypic and functional results, softer substrates prime macrophages toward the proinflammatory (M1)-like phenotype. In summary, this study demonstrated the mechanosensitive inflammatory response of macrophages and the alteration of ROS, as secondary inflammation signals, may contribute to the functional status of macrophages. These findings not only provide an alternative interpretation of the functional transitions of macrophages influenced by substrate rigidity but may also support the manipulation of the inflammatory responses of macrophages via physical microenvironment modifications.

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BACKGROUND: Overconsumption of energy from food contributes to high rates of overweight and obesity in many populations. A promising set of interventions tested in pilot studies in worksite cafeterias, suggests energy intake may be reduced by increasing the proportion of healthier - i.e. lower energy - food options available, and decreasing portion sizes. The current study aims to assess the impact on energy purchased of i. increasing the proportion of lower energy options available; ii. combining this with reducing portion sizes, in a full trial. METHODS: A stepped-wedge randomised controlled trial in 19 worksite cafeterias, where the proportion of lower energy options available in targeted food categories (including main meals, snacks, and cold drinks) will be increased; and combined with reduced portion sizes. The primary outcome is total energy (kcal) purchased from targeted food categories using a pooled estimate across all sites. Follow-up analyses will test whether the impact on energy purchased varies according to the extent of intervention implementation. DISCUSSION: This study will provide the most reliable estimate to date of the effect sizes of two promising interventions for reducing energy purchased in worksite cafeterias. TRIAL REGISTRATION: The study was prospectively registered on ISRCTN (date: 24.05.19; TRN: ISRCTN87225572; doi: https://doi.org/10.1186/ISRCTN87225572).

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BACKGROUND: There is considerable uncertainty regarding the impact of tableware size on food consumption. Most existing studies have used small and unrepresentative samples and have not followed recommended procedures for randomised controlled trials, leading to increased risk of bias. In the first pre-registered study to date, we examined the impact on consumption of using larger versus smaller plates for self-served food. We also assessed impact on the underlying meal micro-structure, such as number of servings and eating rate, which has not previously been studied. METHODS: The setting was a purpose-built naturalistic eating behaviour laboratory. A general population sample of 134 adult participants (aged 18-61 years) was randomly allocated to one of two groups varying in the size of plate used for self-serving lunch: large or small. The primary outcome was amount of food energy (kcal) consumed during a meal. Additionally, we assessed impact on meal micro-structure, and examined potential modifying effects of executive function, socio-economic position, and sensitivity to perceptual cues. RESULTS: There was no clear evidence of a difference in consumption between the two groups: Cohen's d = 0.07 (95% CI [- 0.27, 0.41]), with participants in the large plate group consuming on average 19.2 (95% CI [- 76.5, 115.0]) more calories (3%) compared to the small plate group (large: mean (SD) = 644.1 (265.0) kcal, versus small: 624.9 (292.3) kcal). The difference between the groups was not modified by individual characteristics. There was no evidence of impact on meal micro-structure, with the exception of more food being left on the plate when larger plates were used. CONCLUSIONS: This study suggests that previous meta-analyses of a low-quality body of evidence may have considerably overestimated the effects of plate size on consumption. However, the possibility of a clinically significant effect - in either direction - cannot be excluded. Well-conducted trials of tableware size in real-world field settings are now needed to determine whether changing the size of tableware has potential to contribute to efforts to reduce consumption at population-level. TRIAL REGISTRATION: The study protocol ( https://osf.io/e3dfh/ ) and data analysis plan ( https://osf.io/sh5u7/ ) were pre-registered on the Open Science Framework.

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BACKGROUND: Reducing the portion sizes of foods available in restaurants and cafeterias is one promising approach to reducing energy intake, but there is little evidence of its impact from randomised studies in field settings. This study aims to i. examine the feasibility and acceptability, and ii. estimate the impact on energy purchased, of reducing portion sizes in worksite cafeterias. METHODS: Nine worksites in England were recruited to reduce by at least 10% the portion sizes of foods available in their cafeterias from targeted categories (main meals, sides, desserts, cakes). In a stepped wedge randomised controlled pilot trial, each site was randomised to a date of implementation, staggered fortnightly, following a baseline period of four weeks. Impact on energy purchased was analysed using generalised linear mixed modelling. We also assessed feasibility, acceptability, and fidelity of intervention implementation. RESULTS: Data from six of the nine randomised sites were analysed, with three sites excluded for not providing sufficient data and/or not implementing the intervention. The extent to which the intervention was implemented varied by site, with between 6 and 49% of products altered within targeted categories. Feedback following the intervention suggested it was broadly acceptable to customers and cafeteria staff. For the primary outcome of daily energy (kcal) purchased from intervention categories, there was no statistically significant change when data from all six sites were pooled: percentage change - 8.9% (95% CI: -16.7, - 0.4; p = 0.081). Each of these six sites showed reductions in energy purchased, ranging from - 15.6 to - 0.3%, which were borderline statistically significant at two sites (respective percentage changes (95% CIs): - 15.6% (- 26.7, - 2.8); - 14.0% (- 25.0, - 1.2)). Secondary outcome data are suggestive of a compensatory increase in energy purchased from food categories not targeted by the intervention, with no overall effect observed on energy purchased across all categories. CONCLUSIONS: The results of this pilot trial suggest that reducing portion sizes could be effective in reducing energy purchased and consumed from targeted food categories, and merits investigation in a larger trial. Future studies will need to address factors that prevented optimal implementation including site dropout and application across a limited range of products. TRIAL REGISTRATION: ( ISRCTN52923504 ). Registered on 20th September 2016.

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Tendon injuries are common musculoskeletal system disorders, but the tendons have poor regeneration ability. To address this issue, tendon tissue engineering provides potential strategies for future therapeutic treatment. Elements of the physical microenvironment, such as the mechanical force and surface topography, play a vital role in regulating stem cell fate, enhancing the differentiation efficiency of seed cells in tendon tissue engineering. Various inducible scaffolds have been widely explored for tendon regeneration, and scaffold-enhancing modifications have been extensively studied. In this review, we systematically summarize the effects of the physical microenvironment on stem cell differentiation and tendon regeneration; we also provide an overview of the inducible scaffolds for stem cell tenogenic differentiation. Finally, we suggest some potential scaffold-based therapies for tendon injuries, presenting an interesting perspective on tendon regenerative medicine.

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BACKGROUND: For working adults, about one-third of energy is consumed in the workplace making this an important context in which to reduce energy intake to tackle obesity. The aims of the current study were first, to identify barriers to the feasibility and acceptability of implementing calorie labelling in preparation for a larger trial, and second, to estimate the potential impact of calorie labelling on energy purchased in worksite cafeterias. METHODS: Six worksite cafeterias were randomised to the intervention starting at one of six fortnightly periods, using a stepped wedge design. The trial was conducted between August and December 2016, across 17 study weeks. The intervention comprised labelling all cafeteria products for which such information was available with their calorie content (e.g. "250 Calories") displayed in the same font style and size as for price. A post-intervention survey with cafeteria patrons and interviews with managers and caterers were used to assess the feasibility and acceptability of the intervention. Intervention impact was assessed using generalised linear mixed modelling. The primary outcome was the total energy (kcal) purchased from intervention items in each cafeteria each day. RESULTS: Recruitment and retention of worksite cafeterias proved feasible, with post-intervention feedback suggesting high levels of intervention acceptability. Several barriers to intervention implementation were identified, including chefs' discretion at implementing recipes and the manual recording of sales data. There was no overall effect of the intervention: -0.4% (95%CI -3.8 to 2.9, p = .803). One site showed a statistically significant effect of the intervention, with an estimated 6.6% reduction (95%CI -12.9 to - 0.3, p = .044) in energy purchased in the day following the introduction of calorie labelling, an effect that diminished over time. The remaining five sites did not show robust changes in energy purchased when calorie labelling was introduced. CONCLUSIONS: A calorie labelling intervention was acceptable to both cafeteria operators and customers. The predicted effect of labelling to reduce energy purchased was only evident at one out of six sites studied. Before progressing to a full trial, the calorie labelling intervention needs to be optimised, and a number of operational issues resolved. TRIAL REGISTRATION: ISRCTN52923504 ; Registered: 22/09/2016; retrospectively registered.

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BACKGROUND: An estimated one third of energy is consumed in the workplace. The workplace is therefore an important context in which to reduce energy consumption to tackle the high rates of overweight and obesity in the general population. Altering environmental cues for food selection and consumption-physical micro-environment or 'choice architecture' interventions-has the potential to reduce energy intake. The first aim of this pilot trial is to estimate the potential impact upon energy purchased of three such environmental cues (size of portions, packages and tableware; availability of healthier vs. less healthy options; and energy labelling) in workplace cafeterias. A second aim of this pilot trial is to examine the feasibility of recruiting eligible worksites, and identify barriers to the feasibility and acceptability of implementing the interventions in preparation for a larger trial. METHODS: Eighteen worksite cafeterias in England will be assigned to one of three intervention groups to assess the impact on energy purchased of altering (a) portion, package and tableware size (n = 6); (b) availability of healthier options (n = 6); and (c) energy (calorie) labelling (n = 6). Using a stepped wedge design, sites will implement allocated interventions at different time periods, as randomised. DISCUSSION: This pilot trial will examine the feasibility of recruiting eligible worksites, and the feasibility and acceptability of implementing the interventions in preparation for a larger trial. In addition, a series of linear mixed models will be used to estimate the impact of each intervention on total energy (calories) purchased per time frame of analysis (daily or weekly) controlling for the total sales/transactions adjusted for calendar time and with random effects for worksite. These analyses will allow an estimate of an effect size of each of the three proposed interventions, which will form the basis of the sample size calculations necessary for a larger trial. TRIAL REGISTRATION: ISRCTN52923504.

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Thorough understanding of the effects of shear stress on stem cells is critical for the rationale design of large-scale production of cell-based therapies. This is of growing importance as emerging tissue engineering and regenerative medicine applications drive the need for clinically relevant numbers of both pluripotent stem cells (PSCs) and cells derived from PSCs. Here, we describe the use of a custom parallel plate bioreactor system to impose fluid shear stress on a layer of PSCs adhered to protein-coated glass slides. This system can be useful both for basic science studies in mechanotransduction and as a surrogate model for bioreactors used in large-scale production.

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Conventional in vitro culture studies on flat surfaces do not reproduce tissue environments, which have inherent topographical mechanical signals. To understand the impact of these mechanical signals better, we use a cell imprinting technique to replicate cell features onto hard polymer culture surfaces as an alternative platform for investigating biomechanical effects on cells; the high-resolution replication of cells offers the micro- and nanotopography experienced in typical cell-cell interactions. We call this platform a Bioimprint. Cells of an endometrial adenocarcinoma cell line, Ishikawa, were cultured on a bioimprinted substrate, in which Ishikawa cells were replicated on polymethacrylate (pMA) and polystyrene (pST), and compared to cells cultured on flat surfaces. Characteristics of cells, incorporating morphology and cell responses, including expression of adhesion-associated molecules and cell proliferation, were studied. In this project, we fabricated two different topographies for the cells to grow on: a negative imprint that creates cell-shaped hollows and a positive imprint that recreates the raised surface topography of a cell layer. We used two different substrate materials, pMA and pST. We observed that cells on imprinted substrates of both polymers, compared to cells on flat surfaces, exhibited higher expression of ß1-integrin, focal adhesion kinase, and cytokeratin-18. Compared to cells on flat surfaces, cells were larger on imprinted pMA and more in number, whereas on pST-imprinted surfaces, cells were smaller and fewer than those on a flat pST surface. This method, which provided substrates in vitro with cell-like features, enabled the study of effects of topographies that are similar to those experienced by cells in vivo. The observations establish that such a physical environment has an effect on cancer cell behavior independent of the characteristics of the substrate. The results support the concept that the physical topography of a cell's environment may modulate crucial oncological signaling pathways; this suggests the possibility of cancer therapies that target pathways associated with the response to mechanical stimuli.

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