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1. Cells utilize strain hardening and crosslinking to establish their extracellular niche in fibrous tissue

   Jagiello, A.; Lim, M.; Botvinick, E. (March 2021, APS March Meeting 2021)

   Bulk measurements of ECM stiffness are commonly used in mechanobiology. However, peri-cellular stiffness can be quite heterogenous and divergent from the bulk properties. Here, we use optical tweezers active microrheology (AMR) to quantify how two different cell lines embedded in 1.0 and 1.5 mg/ml type 1 collagen (T1C) establish dissimilar patterns of peri-cellular stiffness. We found that dermal fibroblasts (DFs) increase local stiffness of 1.0 mg/ml T1C hydrogels, but surprisingly do not alter stiffness of 1.5 mg/ml T1C hydrogels. In contrast, MDA-MB-231 cells (MDAs) predominantly do not stiffen T1C hydrogels, as compared to cell-free controls. Results suggest that MDAs adapt to the bulk ECM stiffness, while DFs regulate local stiffness to levels they intrinsically “prefer”. Further, cells were subjected to treatments, that were previously shown to alter migration, proliferation and contractility of DFs and MDAs. Following treatment, both cell lines established different levels of stiffness magnitude and anisotropy, which were dependent on the cell line, T1C concentration and treatment. In summary, our findings demonstrate that AMR reveals otherwise masked mechanical properties such as spatial gradients and anisotropy, which are known to affect cell behavior at the macro-scale. 

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2. Cell mediated remodeling of stiffness matched collagen and fibrin scaffolds

   https://doi.org/10.1038/s41598-022-14953-w  

   Jagiełło, Alicja; Castillo, Ulysses; Botvinick, Elliot (December 2022, Scientific Reports)

   Abstract Cells are known to continuously remodel their local extracellular matrix (ECM) and in a reciprocal way, they can also respond to mechanical and biochemical properties of their fibrous environment. In this study, we measured how stiffness around dermal fibroblasts (DFs) and human fibrosarcoma HT1080 cells differs with concentration of rat tail type 1 collagen (T1C) and type of ECM. Peri-cellular stiffness was probed in four directions using multi-axes optical tweezers active microrheology (AMR). First, we found that neither cell type significantly altered local stiffness landscape at different concentrations of T1C. Next, rat tail T1C, bovine skin T1C and fibrin cell-free hydrogels were polymerized at concentrations formulated to match median stiffness value. Each of these hydrogels exhibited distinct fiber architecture. Stiffness landscape and fibronectin secretion, but not nuclear/cytoplasmic YAP ratio differed with ECM type. Further, cell response to Y27632 or BB94 treatments, inhibiting cell contractility and activity of matrix metalloproteinases, respectively, was also dependent on ECM type. Given differential effect of tested ECMs on peri-cellular stiffness landscape, treatment effect and cell properties, this study underscores the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. 

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3. Stiffness-matched extracellular matrices comprising collagen or fibrin differentially affect cell-mediated remodeling and peri-cellular stiffness

   https://doi.org/10.21203/rs.3.rs-1348112/v1  

   Jagiełło, A.; Castillo, U.; Botvinick, E. (February 2022, Research square)

   Cells are known to continuously remodel their local extracellular matrix (ECM) and in a reciprocal way, they can also respond to mechanical and biochemical properties of their fibrous environment. In this study, we measured how stiffness around dermal fibroblasts (DFs) and human fibrosarcoma HT1080 cells differs with concentration of rat tail type 1 collagen (T1C) and type of ECM. Peri-cellular stiffness was probed in four directions using multi-axes optical tweezers active microrheology (AMR). First, we found that neither cell type significantly altered local stiffness landscape at different concentrations of T1C. Next, rat tail T1C, bovine skin T1C and fibrin cell-free hydrogels were polymerized at concentrations formulated to match median stiffness value. Each of these hydrogels exhibited distinct fiber architecture. Stiffness landscape and fibronectin secretion, but not nuclear/cytoplasmic YAP ratio differed with ECM type. Further, cell response to Y27632 or BB94 treatments, inhibiting cell contractility and activity of matrix metalloproteinases, respectively, was also dependent on ECM type. Given differential effect of tested ECMs on peri-cellular stiffness landscape, treatment effect and cell properties, this study underscores the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. 

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4. Using light to establish and measure stiffness gradients in three-dimensional engineered tissues

   https://doi.org/10.1117/12.2595590  

   Jagiello, A; Hu, Q; Castillo, U (August 2021, spie: Optical Trapping and Optical Micromanipulation XVIII)

   Studies of cell-extracellular matrix (ECM) interactions within fibrous systems such as collagen or fibrin are challenging, particularly if peri-cellular stiffness cannot be monitored. Here we present our light-based method for non-invasive patterning of molecular crosslinking combined with multi-axes optical tweezers active microrheology to map ECM stiffness landscapes. This method allows us to generate prescribed stiffness gradients and associated anisotropies, which model stiffness of the natural peri-cellular ECM. Patterned crosslinking induces strain hardening and measured stiffness gradients are in agreement with predicted strain fields. Migratory cells respond to these gradients as assessed by change in F-actin distribution and morphological properties. 

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5. DDR2 signaling and mechanosensing orchestrate neuroblastoma cell fate through different transcriptome mechanisms

   https://doi.org/10.1002/2211-5463.13798  

   Vessella, Theadora; Xiang, Steven; Xiao, Cong; Stilwell, Madelyn; Fok, Jaidyn; Shohet, Jason; Rozen, Esteban; Zhou, H. Susan; Wen, Qi (March 2024, FEBS Open Bio)

   The extracellular matrix (ECM) regulates carcinogenesis by interacting with cancer cells via cell surface receptors. Discoidin Domain Receptor 2 (DDR2) is a collagen‐activated receptor implicated in cell survival, growth, and differentiation. Dysregulated DDR2 expression has been identified in various cancer types, making it as a promising therapeutic target. Additionally, cancer cells exhibit mechanosensing abilities, detecting changes in ECM stiffness, which is particularly important for carcinogenesis given the observed ECM stiffening in numerous cancer types. Despite these, whether collagen‐activated DDR2 signaling and ECM stiffness‐induced mechanosensing exert similar effects on cancer cell behavior and whether they operate through analogous mechanisms remain elusive. To address these questions, we performed bulk RNA sequencing (RNA‐seq) on human SH‐SY5Y neuroblastoma cells cultured on collagen‐coated substrates. Our results show that DDR2 downregulation induces significant changes in the cell transcriptome, with changes in expression of 15% of the genome, specifically affecting the genes associated with cell division and differentiation. We validated the RNA‐seq results by showing that DDR2 knockdown redirects the cell fate from proliferation to senescence. Like DDR2 knockdown, increasing substrate stiffness diminishes cell proliferation. Surprisingly, RNA‐seq indicates that substrate stiffness has no detectable effect on the transcriptome. Furthermore, DDR2 knockdown influences cellular responses to substrate stiffness changes, highlighting a crosstalk between these two ECM‐induced signaling pathways. Based on our results, we propose that the ECM could activate DDR2 signaling and mechanosensing in cancer cells to orchestrate their cell fate through distinct mechanisms, with or without involving gene expression, thus providing novel mechanistic insights into cancer progression. 

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