The mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using 2D substrates, much less is known about it in a physiologically more relevant 3D context. Here it is demonstrated that breast cancer tumor spheroids, growing in 3D polyethylene glycol‐heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa build up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy reveals that tumor spheroid stiffness increases with hydrogel stiffness. Also, constituent cell stiffness increases in a Rho associated kinase (ROCK)‐ and F‐actin‐dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase, and elevated levels of the cyclin‐dependent kinase inhibitor p21. Drug‐mediated ROCK inhibition not only reverses cell stiffening upon culture in stiff hydrogels but also increases tumor spheroid growth. Taken together, a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment is revealed here. Thus, the findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments.
The labeling of biomolecules has become standard practice in molecular biosciences. Modifications are used for detection, sorting and isolation of small molecules, complexes and entire cells. We have recently reported a method for introducing internal chemical and structural modifications into kbp-sized DNA target substrates that are frequently used in single-molecule experiments. It makes use of nicking enzymes that create single-stranded DNA gaps, which can be subsequently filled with labeled oligonucleotides. Here we provide a detailed protocol and further expand this method. We show that modifications can be introduced at distant loci within one molecule in a simple one-pot reaction. In addition, we achieve labeling on both strands at a specific locus, as demonstrated by Förster resonance energy transfer (FRET) experiments. The protocol requires an initial cloning of the target substrate (3-5 d), whereas the labeling itself takes 4-6 h. More elaborate purification and verification of label incorporation requires 2 h for each method.
Mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using twodimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK-and F-actin-dependent manner.Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.
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