The extracellular matrix (ECM) provides structural support to cells, thereby forming a functional tissue. In cancer, the growth of the tumor creates internal mechanical stress, which, together with the remodeling activity of tumor cells and fibroblasts, alters the ECM structure, leading to an increased stiffness of the pathological ECM. The enhanced ECM stiffness, in turn, stimulates tumor growth and activates tumor-promoting fibroblasts and tumor cell migration, leading to metastasis and increased therapy resistance. While the relationship between matrix stiffness and migration has been studied before, their connection to internal tumor stress remains unresolved. Here we used 3D ECM-embedded spheroids and hydrogel particle stress sensors to quantify and correlate internal tumor spheroid pressure, ECM stiffness, ECM remodeling, and tumor cell migration. We note that 4T1 breast cancer spheroids and SV80 fibroblast spheroids showed increased invasion—described by area, complexity, number of branches, and branch area—in a stiffer, cross-linked ECM. On the other hand, changing ECM stiffness only minimally changed the radial alignment of fibers but highly changed the amount of fibers. For both cell types, the pressure measured in spheroids gradually decreased as the distance into the ECM increased. For 4T1 spheroids, increased ECM stiffness resulted in a further reach of mechanical stress into the ECM, which, together with the invasive phenotype, was reduced by inhibition of ROCK-mediated contractility. By contrast, such correlation between ECM stiffness and stress-reach was not observed for SV80 spheroids. Our findings connect ECM stiffness with tumor invasion, ECM remodeling, and the reach of tumor-induced mechanical stress into the ECM. Such mechanical connections between tumor and ECM are expected to drive early steps in cancer metastasis.

Background: Pancreatic ductal adenocarcinoma acquired resistance to chemotherapy poses a major limitation to patient survival. Despite understanding some biological mechanisms of chemoresistance, much about those mechanisms remains to be uncovered. Mechanobiology, which studies the physical properties of cells, holds promise as a potential target for addressing the challenges of chemoresistance in PDAC. Therefore, we, here in an initial step, assessed the altered mechanobiology of PDAC cells with acquired chemoresistance to gemcitabine and paclitaxel. Methods: Five PDAC cell lines and six stably resistant subclones were assessed for force generation on elastic micropillar arrays. Those measurements of mechanical phenotype were complemented by single-cell motility and invasion in 3D collagen-based matrix assays. Further, the nuclear translocation of Yes-associated protein (YAP), as a measure of active mechanical status, was compared, and biomarkers of the epithelial-to-mesenchymal transition (EMT) were evaluated using RT-qPCR. Results: The PDAC cells with acquired chemoresistance exert higher traction forces than their parental/wild-type (WT) cells. In 2D, single-cell motility was altered for all the chemoresistant cells, with a cell-type specific pattern. In 3D, the spheroids of the chemoresistant PDAC cells were able to invade the matrix and remodel collagen more than their WT clones. However, YAP nuclear translocation and EMT were not significantly altered in relation to changes in other physical parameters. Conclusions: This is the first study to investigate and report on the altered mechanobiological features of PDAC cells that have acquired chemoresistance. A better understanding of mechanical features could help in identifying future targets to overcome chemoresistance in PDAC.

High-resolution and super-resolution techniques become more frequently used in thick, inhomogeneous samples. In particular for imaging life cells and tissue in which one wishes to observe a biological process at minimal interference and in the natural environment, sample inhomogeneities are unavoidable. Yet sample-inhomogeneities are paralleled by refractive index variations, for example between the cell organelles and the surrounding medium, that will result in the refraction of light, and therefore lead to sample-induced astigmatism. Astigmatism in turn will result in positional inaccuracies of observations that are at the heart of all super-resolution techniques. Here we introduce a simple model and define a figure-of-merit that allows one to quickly assess the importance of astigmatism for a given experimental setting. We found that astigmatism caused by the cell’s nucleus can easily lead to aberrations up to hundreds of nanometers, well beyond the accuracy of all super-resolution techniques. The astigmatism generated by small objects, like bacteria or vesicles, appear to be small enough to be of any significance in typical super-resolution experimentation.

The reciprocal interplay between cancer cells and their local environment, mediated by mechanical forces, necessitates a deeper experimental understanding. This requires precise quantitative measurements of cellular forces within the intricate three-dimensional context of the extracellular matrix. While methods such as traction-force microscopy and micropillar-array technology have effectively reported on cellular forces in two-dimensional cell culture, extending these techniques to three dimensions has proven exceedingly challenging. In the current study, we introduced a novel approach utilizing soft, elastic hydrogel microparticles, resembling the size of cells, to serve as specific and sensitive traction probes in three-dimensional cell culture of collagen-embedded tumoroids. Our methodology relies on high-resolution detection of microparticle deformations. These deformations are translated into spatially resolved traction fields, reaching a spatial resolution down to 1 μm and thereby detecting traction forces as low as 30 Pa. By integrating this high-resolution traction analysis with three-dimensional cell segmentation, we reconstructed the traction fields originating from individual cells. Our methodology enables us to explore the relationships between cellular characteristics, extracellular traction fields, and cellular responses. We observed that cellular stresses ranged from 10 to 100 Pa, integrating to cellular forces from 0.1 to 100 nN, which correlated with the localization of the cells actin skeleton, and the interaction area that cells developed towards the microparticles. Interestingly, the interaction of cells with inert microparticles appeared to be governed by contact mechanics resembling that two soft particles. The methodology presented here not only addresses the challenges of extending traditional stress-probe techniques to three dimensions, but also opens a strategy for the study of specific interactions between cells and the local tumoroid environment in a strive to fully understand cell-matrix reciprocity in tissue.

Background Pancreatic ductal adenocarcinoma (PDAC) acquired resistance to chemotherapy poses a major limitation to patient survival. Despite understanding of some biological mechanisms of chemoresistance, much of those mechanisms remain to be uncovered. Mechanobiology, which studies physical properties of cells, holds promise as a potential target for addressing challenges of chemoresistance in PDAC. Therefore, we here in an initial step, assessed the altered mechanobiology of PDAC cells with acquired chemoresistance to gemcitabine and paclitaxel. Methods Five PDAC cell lines and six stably-resistant subclones were assessed for force generation on elastic micropillar arrays. Those measurements of mechanical phenotype were complemented by single-cell motility and invasion in collagen matrix were investigated using 2D models and 3D extracellular matrix-mimetic, respectively. Further the nuclear translocation of Yes-associted protein (YAP), as a measure of active mechanical status, was compared, and biomarkers of the epithelial-to-mesenchymal transition (EMT) were evaluated using RT-PCR. Results PDAC cells with acquired chemoresistance exert higher traction forces than their parental/wild-type (WT) cells. In 2D, single-cell motility was altered for all chemoresistant cells, with a cell-type specific pattern. In 3D, spheroids of chemoresistant PDAC cells were able to invade the matrix, and remodel collagen more than their WT clones. However, YAP nuclear translocation and EMT were not significantly altered in relation to changes in other physical parameters. Conclusion This is the first study to investigate and report on the altered mechanobiological features for PDAC cells that have acquired chemoresistance. A better understanding of mechanical features could help in identifying future targets to overcome chemoresistance in PDAC.

The extracellular matrix (ECM) provides structural support to cells thereby forming a functional tissue. In cancer, the growth of the tumor creates an internal mechanical stress which, together with remodeling activity of tumor cells and fibroblasts, alters the ECM structure leading to an increased stiffness of the pathological ECM. The enhanced ECM stiffness in turn stimulates tumor growth, activates tumor-promoting fibroblasts and tumor cell migration, leading to metastasis and an increased therapy resistance. The connection between internal tumor stress, ECM stiffness, ECM remodeling, and cell migration are unresolved. Here we used 3D ECM-embedded spheroids and hydrogel-particle stress sensors, to quantify and correlate internal tumor-spheroid pressure, ECM stiffness, ECM remodeling, and tumor cell migration. 4T1 breast cancer spheroids and SV80 fibroblast spheroids showed increased invasion - described by area, complexity, number of branches and branch area - in a stiffer, cross-linked ECM. On the other hand, changing the ECM stiffness only minimally changed the radial alignment of fibers but highly changed the amount of fibers.. For both cell types, the pressure measured in spheroids gradually decreased as the distance into the ECM increased. For 4T1 spheroids, increased ECM stiffness resulted in a further reach of mechanical stress into the ECM which, together with the invasive phenotype, was reduced by inhibition of ROCK-mediated contractility. By contrast, such correlation between ECM stiffness and stress-reach was not observed for SV80 spheroids. Our findings connect ECM stiffness with tumor invasion, ECM remodeling, and the reach of tumor-induced mechanical stress into the ECM. Such mechanical connections between tumor and ECM are expected to drive early steps in cancer metastasis.