RESULTS: Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated.
CONCLUSIONS: XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.
METHODS: Immunoblotting, immunofluorescence, quantitative real-time PCR and flow cytometry assays investigated the expression of E-cadherin and CXCR3 isoforms. Intrasplenic inoculation of human prostate cancer (PCa) cells with spontaneous metastasis to the liver analyzed E-cadherin and CXCR3-B expression during cancer progression in vivo.
RESULTS: We found reciprocal regulation of E-cadherin and CXCR3 isoforms. E-cadherin surface expression promoted CXCR3-B presentation on the cell membrane, and to a lesser extent increased its mRNA and total protein levels. In turn, forced expression of CXCR3-A reduced E-cadherin expression level, whereas CXCR3-B increased E-cadherin in PCa. Meanwhile, a positive correlation of E-cadherin and CXCR3-B expression was found both in experimental PCa liver micro-metastases and patients' tissue.
CONCLUSIONS: CXCR3-B and E-cadherin positively correlated in vitro and in vivo in PCa cells and liver metastases, whereas CXCR3-A negatively regulated E-cadherin expression. These results suggest that CXCR3 isoforms may play important roles in cancer progression and dissemination via diametrically regulating tumor's phenotype.
METHODS: We analysed expression of NFIA and NFIB in mRNA expression data of high-grade astrocytoma and with immunofluorescence co-staining. Furthermore, we induced NFI expression in patient-derived subcutaneous glioblastoma xenografts via in vivo electroporation.
RESULTS: The expression of NFIA and NFIB is reduced in glioblastoma as compared to lower grade astrocytomas. At a cellular level, their expression is associated with differentiated and mature astrocyte-like tumour cells. In vivo analyses consistently demonstrate that expression of either NFIA or NFIB is sufficient to promote tumour cell differentiation in glioblastoma xenografts.
CONCLUSION: Our findings indicate that both NFIA and NFIB may have an endogenous pro-differentiative function in astrocytomas, similar to their role in normal astrocyte differentiation. Overall, our study establishes a basis for further investigation of targeting NFI-mediated differentiation as a potential differentiation therapy.
METHODS: Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13-18 days).
RESULTS: The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation.
CONCLUSION: In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs.
OBJECTIVES: This study was performed to identify mechanisms of afatinib resistance and to explore potential afatinib-based combination treatments with other targeted inhibitors in oral squamous cell carcinoma.
METHODS: We determined the anti-proliferative effects of afatinib on a panel of oral squamous cell carcinoma cell lines using a crystal violet-growth inhibition assay, click-iT 5-ethynyl-2'-deoxyuridine staining, and cell-cycle analysis. Biochemical assays were performed to study the underlying mechanism of drug treatment as a single agent or in combination with the MEK inhibitor trametinib. We further evaluated and compared the anti-tumor effects of single agent and combined treatment by using oral squamous cell carcinoma xenograft models.
RESULTS: In this study, we showed that afatinib inhibited oral squamous cell carcinoma cell proliferation via cell-cycle arrest at the G0/G1 phase, and inhibited tumor growth in xenograft mouse models. Interestingly, we demonstrated reactivation of the mitogen-activated protein kinase (ERK1/2) pathway in vitro, which possibly reduced the effects of ErbB inhibition. Concomitant treatment of oral squamous cell carcinoma cells with afatinib and trametinib synergized the anti-tumor effects in oral squamous cell carcinoma-bearing mouse models.
CONCLUSIONS: Our findings provide insight into the molecular mechanism of resistance to afatinib and support further clinical evaluation into the combination of afatinib and MEK inhibition in the treatment of oral squamous cell carcinoma.