Potential effect of matrix stiffness on the enrichment of tumor initiating cells under three-dimensional culture conditions.

Cancer stem cell (CSC) or tumor initiating cell (TIC) plays an important role in tumor progression and metastasis. Biophysical forces in tumor microenvironment have an important effect on tumor formation and development. In this study, the potential effect of matrix stiffness on the biological characteristics of human head and neck squamous cell carcinoma (HNSCC) TICs, especially the enrichment of HNSCC TICs, was investigated under three-dimensional (3D) culture conditions by means of alginate gel (ALG) beads with different matrix stiffnesses. ALG beads with soft (21 kPa), moderate (70 kPa) and hard (105 kPa) stiffness were generated by changing alginate concentration. It was found that significant HNSCC TIC enrichment was achieved in the ALG beads with moderate matrix stiffness (70 kPa). The gene expression of stemness markers Oct3/4 and Nanog, TIC markers CD44 and ABCG2 was enhanced in cells under this moderate (70 kPa) stiffness. HNSCC TIC proportion was also highly enriched under moderate matrix stiffness, accompanying with higher tumorigenicity, metastatic ability and drug resistance. And it was also found that the possible molecular mechanism underlying the regulated TIC properties by matrix stiffness under 3D culture conditions was significantly different from 2D culture condition. Therefore, the results achieved in this study indicated that 3D biophysical microenvironment had an important effect on TIC characteristics and alginate-based biomimetic scaffolds could be utilized as a proper platform to investigate the interaction between tumor cells and 3D microenvironment.

Role of three-dimensional matrix stiffness in regulating the chemoresistance of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) was the most common primary liver cancer, and its resistance to anti-tumor drugs often caused the death of patients suffering with HCC. Matrix stiffness was reported to be closely related to tumor chemoresistance; however, the relationship between HCC drug resistance and three-dimensional (3D) matrix stiffness is still unclear at present. In this study, alginate gel (ALG) beads with controllable matrix stiffness were used to mimic tumor tissue rigidity, and the role of 3D matrix stiffness in regulating the chemoresistance of HCC cells was investigated by using these ALG beads. It was found that HCC cells in ALG beads with 105 kPa stiffness had highest resistance to paclitaxel, 5-FU, and cisplatin. Although the mechanism was still uncovered, ABC transporters and endoplasmic reticulum stress-related molecules were highly expressed in ALG bead-encapsulated HCC cells compared with two-dimensional-cultured cells, which suggested a very complex mechanism underlying HCC drug resistance in 3D culture conditions. In addition, to mimic the specific stiffness of HCC tumor tissue, or other tumor tissues in vivo, response surface methodology (RSM) was used to build up a prediction mathematical model so that ALG beads with desired matrix stiffness could be prepared by simply changing three factors: molecular weight, G content, and alginate concentration.

Effect of surface wettability and charge on protein adsorption onto implantable alginate-chitosan-alginate microcapsule surfaces.

Alginate-chitosan-alginate (ACA) microcapsules have been developed as a device for the transplantation of living cells. However, protein adsorption onto the surface of microcapsules immediately upon their implantation decides their ultimate biocompatibility. In this work, the chemical composition of the ACA membranes was determined using X-ray photoelectron spectroscopy (XPS). The surface wettability and charge were determined by contact angle and zeta potential measurements, respectively. Then, the effects of surface wettability and charge on bovine fibrinogen (Fgn) and gamma globulin (IgG) adsorption onto ACA microcapsules were evaluated. The results showed that ACA microcapsules had a hydrophilic membrane. So, the surface wettability of ACA microcapsules had little effect on protein adsorption. There was a negative zeta potential of ACA microcapsules which varies with the viscosity or G content of alginate used, indicating a varying degree of net negatively charged groups on the surface of ACA microcapsules. The amount of adsorbed protein increased with increasing of positive charge. Furthermore, the interaction between proteins and ACA microcapsules is dominated by electrostatic repulsion at pH 7.4 and that is of electrostatic attraction at pH 6.0. This work could help to explain the bioincompatibility of ACA microcapsules and will play an important role in the optimization of the microcapsule design.

[Cloning of the RgpAcd gene of Porphyromonas gingivalis and its expression in E. coli].

OBJECTIVE: To clone the catalytic domain gene sequence of RgpAcd of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. METHODS: The desired DNA fragment RgpAcd was obtained by PCR and was separately sequenced and identified by inserting into inter-vector pMD18-T vector. The correctly fragment was linked with and cloned into a prokaryotic expression vector pET-15b. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 (DE3) and expression of fusion protein was induced by IPTG. RESULTS: A 1 476 bp specific fragment was obtained and DNA sequencing showed that the fragment was consistent with those of the published. After induction with IPTG, a fusion protein of 5 x 10(4) was visualized on SDS-PAGE gel. CONCLUSION: The protein of RgpAcd will be obtained for further study and its protein was correctly expressed in E. coli BL21 cells.