Shaghaghi, B. | Search bioRxiv

bioRxiv2024

Deciphering stiffness-driven changes in colorectal cancer by proteomics

Tumor stiffening plays a pivotal role in cancer progression. Increased tumor stiffness, resulting from interactions between cancer cells and their surrounding microenvironment, alters the tumors mechanical properties and significantly impacts cancer growth and metastasis, the primary cause of cancer-related deaths. Despite the importance of tumor stiffness, systematic studies exploring its effect on proteomic profiles are limited. In this study, focused on colorectal cancer, we show that matrix stiffness significantly alters the expression of secreted proteins, while intracellular protein levels remain largely unaffected. Functional assays reveal that the changes in the secretome, driven by matrix stiffness, enhance cell migration, angiogenesis, and matrix remodeling, which collectively contribute to a more aggressive cancer phenotype. Our findings emphasize the critical role of matrix stiffness in driving colorectal cancer progression through changes in the secretome, offering valuable insights for the development of biomechanical cancer therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/618701v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@fb6b58org.highwire.dtl.DTLVardef@446a31org.highwire.dtl.DTLVardef@197e22forg.highwire.dtl.DTLVardef@82a896_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Synthesis and mechanical characterization of polyacrylamide (PAAm) hydrogels with different stiffnesses for large-batch cell culture applications

The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a step-by-step protocol for synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large circular unattached gels customizable in shape and size. We outline methods for their use in cell culture and downstream applications involving secretome or cell analysis, and protein visualization by fluorescence microscopy. This protocol is based on the recent work of Shi & Janmey who describe a novel and straightforward method for the production of large PAAm hydrogels for bulk cell culture and mechanobiology studies.1 Their procedure results in one large gel that is not attached to a supporting surface and therefore can be transferred and/or stamped to generate PAAm gels of custom shapes and sizes. The aim of this step-by-step procedure is therefore not to improve the reported protocol, but to create a clearly outlined and repeatable protocol that enables a smooth implementation in any lab for a diverse audience. In addition, our protocol describes besides harvesting of cells also the collection of secretome for downstream biochemical analyses, as well as immunofluorescence labeling using antibodies that can readily be multiplexed for optimization of labeling conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/613503v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@cefcc3org.highwire.dtl.DTLVardef@1348c93org.highwire.dtl.DTLVardef@f935acorg.highwire.dtl.DTLVardef@132dbd0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗