The tumor microenvironment determines tumor characteristics, and its recapitulation via in vitro tissue culture conditions is necessary for better understanding of molecular mechanisms of tumor behaviors and development of novel therapy. Rhabdomyosarcoma is a mesenchymal tumor that originates limb muscles. To assess the possible utility of the decellularized materials that contain native components of the microenvironment for the optimal humanized spheroid formation, we demonstrated co-culture of decellularized muscle extracts and rhabdomyosarcoma cells. The decellularized muscle extracts were prepared using a detergent, solubilized via pepsin digestion, and used for the spheroid culture of rhabdomyosarcoma cells in a low-attachment tissue culture plate. Here, we confirmed that these extracts contained proteins whose expression was unique to muscles. When rhabdomyosarcoma cells formed spheroids with decellularized muscle extracts, they exhibited heterogeneous morphology and a unique protein expression pattern. We conclude that in vitro cancer models established using decellularized tissue extracts from the originating organ have the potential to recapitulate features of in vivo tumor cells by providing the components of the tumor microenvironment. The precise molecular mechanisms for the interaction of cells and decellularized tissues are worth further investigation.

A complex of carboxylesterase and transferrin was obtained after cytosolic proteins in the mouse liver were separated by a combination of non-denaturing two-dimensional electrophoresis (2DE) and reversible staining. At the site where the complex was separated on the 2DE gel, the complex retained esterase activity because the relative intensity of the fluorescent esterase substrate was 1.6-fold higher than that within the 2DE gel in the absence of protein. Furthermore, the complex was extracted from the 2DE gel and bound to antibody-conjugated protein G plus A (protein A/G) agarose by non-denaturing electrophoresis after separation and detection. The esterase activity was retained after the complex was extracted and bound to antibody-conjugated protein A/G agarose. These results indicate that intact protein complexes were separated, detected, and extracted by non-denaturing electrophoresis.

Sixty-eight human tyrosine kinases were cloned into the Escherichia coli expression vector pET21a(+) and 48 of them were successfully expressed in the BL21(DE3) strain. Among them, 18 kinases were activated in the bacterial cells by autophosphorylation and they phosphorylated endogenous proteins. Since E. coli expression systems do not usually enable post-translational modifications, the in vivo phosphorylation should be due to the tyrosine kinase expressed. In this study, we attempted to use the bacterial expression system as a site for the kinase reaction to determine the substrate sequence preference of a certain kinase, namely, a phosphorylation motif. As a first example, phosphoproteomic analysis of E. coli expressing active bone marrow tyrosine kinase gene in chromosome X protein (BMX) was performed to extract the phosphorylation motifs. The motifs were compared with that obtained from Kinapple, the database of in vitro human kinome profiling, and common features rich in acidic amino acids were found. Furthermore, the phosphorylation state of expressed BMX was analyzed. Phos-tag SDS-PAGE in conjunction with alanine scans of the phosphorylation sites found by mass spectrometry revealed that Y224, Y234, and Y566 are predominantly phosphorylated. Moreover, phosphorylation at S453 and T572 was implicated in activation in E. coli, whereas these sites did not affect activity in 293 cells. These results suggest that, although the bacterial system does not fully reflect the physiological properties of the kinase, it is a simple experimental method that can infer the substrate preference of tyrosine kinases similar to that observed with substrates of human origin.