How cytoskeletal crosstalk makes cells move: Bridging cell-free and cell studies.

Cell migration is a fundamental process for life and is highly dependent on the dynamical and mechanical properties of the cytoskeleton. Intensive physical and biochemical crosstalk among actin, microtubules, and intermediate filaments ensures their coordination to facilitate and enable migration. In this review, we discuss the different mechanical aspects that govern cell migration and provide, for each mechanical aspect, a novel perspective by juxtaposing two complementary approaches to the biophysical study of cytoskeletal crosstalk: live-cell studies (often referred to as top-down studies) and cell-free studies (often referred to as bottom-up studies). We summarize the main findings from both experimental approaches, and we provide our perspective on bridging the two perspectives to address the open questions of how cytoskeletal crosstalk governs cell migration and makes cells move.

Tumor decellularization reveals proteomic and mechanical characteristics of the extracellular matrix of primary liver cancer.

Tumor initiation and progression are critically dependent on interaction of cancer cells with their cellular and extracellular microenvironment. Alterations in the composition, integrity, and mechanical properties of the extracellular matrix (ECM) dictate tumor processes including cell proliferation, migration, and invasion. Also in primary liver cancer, consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the dysregulation of the extracellular environment by liver fibrosis and tumor desmoplasia is pertinent. Yet, the exact changes occurring in liver cancer ECM remain uncharacterized and underlying tumor-promoting mechanisms remain largely unknown. Herein, an integrative molecular and mechanical approach is used to extensively characterize the ECM of HCC and CCA tumors by utilizing an optimized decellularization technique. We identified a myriad of proteins in both tumor and adjacent liver tissue, uncovering distinct malignancy-related ECM signatures. The resolution of this approach unveiled additional ECM-related proteins compared to large liver cancer transcriptomic datasets. The differences in ECM protein composition resulted in divergent mechanical properties on a macro- and micro-scale that are tumor-type specific. Furthermore, the decellularized tumor ECM was employed to create a tumor-specific hydrogel that supports patient-derived tumor organoids, which provides a new avenue for personalized medicine applications. Taken together, this study contributes to a better understanding of alterations to composition, stiffness, and collagen alignment of the tumor ECM that occur during liver cancer development.

Modelling metastatic colonization of cholangiocarcinoma organoids in decellularized lung and lymph nodes.

Cholangiocarcinoma (CCA) is a type of liver cancer with an aggressive phenotype and dismal outcome in patients. The metastasis of CCA cancer cells to distant organs, commonly lung and lymph nodes, drastically reduces overall survival. However, mechanistic insight how CCA invades these metastatic sites is still lacking. This is partly because currently available models fail to mimic the complexity of tissue-specific environments for metastatic CCA. To create an in vitro model in which interactions between epithelial tumor cells and their surrounding extracellular matrix (ECM) can be studied in a metastatic setting, we combined patient-derived CCA organoids (CCAOs) (n=3) with decellularized human lung (n=3) and decellularized human lymph node (n=13). Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin. Proteomic analyses showed a tissue-specific ECM protein signature reflecting tissue functioning aspects. The macro and micro-scale mechanical properties, as determined by rheology and micro-indentation, revealed the local heterogeneity of the ECM. When growing CCAOs in decellularized lung and lymph nodes genes related to metastatic processes, including epithelial-to-mesenchymal transition and cancer stem cell plasticity, were significantly influenced by the ECM in an organ-specific manner. Furthermore, CCAOs exhibit significant differences in migration and proliferation dynamics dependent on the original patient tumor and donor of the target organ. In conclusion, CCA metastatic outgrowth is dictated both by the tumor itself as well as by the ECM of the target organ. Convergence of CCAOs with the ECM of its metastatic organs provide a new platform for mechanistic study of cancer metastasis.

Deconstruction of activity-dependent covalent modification of heme in human neutrophil myeloperoxidase by multistage mass spectrometry (MS(4)).

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.

Modification of amyloid-beta(1-40) by a protease inhibitor creates risk of error in mass spectrometric quantitation of amyloid-beta(1-42).

Matrix-assisted laser desorption mass spectrometry successfully analyzes mixed populations of amyloid-beta (Abeta) peptides, providing a profile in which changes caused by drug action are directly observed. A spectrum of Abeta immunocaptured from guinea pig brain included a novel component with monoisotopic [M+H]+ at 4511.22, close to the monoisotopic value of [M+H]+ for Abeta(1-42) of 4512.27 and overlapping and interfering with the authentic Abeta(1-42) peak. Hypothesis and experiment led to the conclusion that modification of Abeta(1-40) by the protease inhibitor aminoethylbenzenesulfonyl fluoride generates a product with monoisotopic [M+H]+ at 4511.19, and that this accounts for the interfering peak.