Paraffin-embedded Human Cancer Tissues Research Articles

Synthesis of SiO2-Coated Fe3O4 Nanoparticles Using Ultrasound and Its Application in DNA Extraction from Formalin-Fixed, Paraffin-Embedded Human Cancer Tissues

SiO2-coated Fe3O4 nanoparticles (Fe3O4@SiO2 NPs) were successfully synthesized using ultrasound in order to extract DNA from cancer tissues for application in diagnostics. The core 10.7-nm-diameter Fe3O4 nanoparticles were synthesized by co-precipitation of Fe3+ and Fe2+ as reaction substrates and NH4OH as precipitant, then coated with a thin layer of amorphous silica by a modified Stober method. Further SiO2 coating using alkaline hydrolysis of tetraethyl orthosilicate in ethanol and water mixture was accelerated in the presence of a 37-kHz ultrasound, resulting in the NPs having different sizes of 14.5 nm (version M1), 24.4 nm (version M2), and 34.9 nm (version M3) with saturation magnetization values of 50.2 emu/g, 18.6 emu/g, 10.3 emu/g, respectively. Among the three Fe3O4@SiO2 NPs versions, the M1 NPs allowed extraction of DNAs from 10 mg formalin-fixed and paraffin-embedded (FFPE) tissues of nasopharyngeal carcinoma patients with the highest recovery of about 100–500 ng/μl and good purity (A260/A280: 1.8–1.9). The extracted DNAs could be used as templates for downstream amplification of 252-bp sequencing specifically for the Braf cancer biomarker gene using polymerase chain reaction (PCR), as well as detection of the pathogenic Epstein–Barr virus (EBV) and the human papilloma-virus (HPV) using real-time PCR. DNA extraction recoveries of both EBV and HPV using Fe3O4@SiO2 NPs M1 were significantly better that those using commercialized Fe3O4@SiO2 microbeads, as indicated by lower threshold cycles of all fluorescent signals including fluorescein amidite (FAM) dye representative for EBV infection, hexachlorofluorescein (HEX) dye representative for β-globin (internal control), and SYBR Green dye representative for HPV infection in tested clinical samples from patients with nasopharyngeal carcinoma (NPC).

Abstract 2047: Expression of microRNAs in squamous cell carcinoma of human head and neck (HNSCC) and the esophagus (ESCC) and their regulation by hepatocyte growth factor

Abstract Introduction: MicroRNAs (miRNAs) are non-coding small RNAs that regulate cell proliferation and various cellular functions by interfering the translation of target mRNAs. Altered expression of miRNA is known to induce various human malignancies. Hepatoceyte growth factor (HGF) is a multifunctional growth factor that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is up-regulated in various tumors including squamous cell carcinoma of human head and neck (HNSCC) and the esophagus (ESCC), but how HGF affect the downstream gene expression have not yet been elucidated in detail. In this study, we examined the expression of miRNAs and their regulation by HGF in HNSCC and ESCC. Methods: Nine HNSCC, 8 ESCC and 2 normal squamous cell lines were used for cell culture. Total RNAs were extracted and used for microarray and following quantitative reverse transcription real-time PCR (qRT-PCR). Validation study was performed by qRT-PCR using total RNAs extracted from formalin-fixed paraffin-embedded human cancer tissues and paired normal tissues by laser-captured microdissection. For miRNA regulation study, HSC3 cells (HNSCC cell line) expressing MET was cultured with or without HGF stimulation, and their total RNAs were extracted and analyzed by miRNA microarray and following qRT-PCR. The expression of target mRNAs for miRNAs altered by HGF stimulation was also examined by qRT-PCR. Results: In HNSCC, ESCC, and normal squamous cell lines, miR-205 showed the highest expression in miRNA microarray analysis. High expression of miR-205 was observed in not only HNSCC and ESCC but also other organs’ SCC and adjacent normal squamous epithelia by qRT-PCR in validation study, although less expression was detected in cells and tissues other than squamous epithelia. MiR-21, which is an oncogenic miRNA in various malignancies, was also up-regulated in HNSCC and ESCC compared to paired normal squamous epithelia. By HGF stimulation, miR-200c and miR-27b were significantly down-regulated in HSC3 cells. Interestingly, the expression of ZEB1, a target mRNA for miR-200c, was up-regulated and E-cadherin, downstream molecule of ZEB1, was down-regulated in these cells after HGF stimulation. Conclusion: Our present results suggest that miRNA expression pattern and target mRNAs of HNSCC and ESCC are different from those of other organ's malignancies. MiR-205 might be a specific marker miRNA of both normal and malignant squamous epithelia, while miR-21 might be a putative oncogenic miRNA in HNSCC and ESCC. In addition, several miRNAs including miR-200c and miR-27b down-regulated by HGF stimulation might play an important role for epithelial-mesenchymal transition (EMT) followed by enhanced invasive characteristics of HNSCC and ESCC, through up-regulation of ZEB1 followed by down-regulation of E-cadherin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2047.

Immunohistochemical measurement of endothelial cell apoptosis and proliferation in formalin-fixed, paraffin-embedded human cancer tissue

There is a need for simple, reproducible methodology for measurement of endothelial cell (EC) apoptosis and proliferation in formalin-fixed, paraffin-embedded (FFPE) human tissue obtained in clinical trials of potential anti-angiogenic agents. Therefore, we developed colorimetric, dual-label immunohistochemical techniques for use on FFPE tissue, based on the use of single-stranded (ss) DNA and Ki-67 as markers of EC apoptosis and proliferation, respectively. Digital image analysis was used to obtain the total tumour microvessel density (MVD), from which the EC apoptosis index (AI) and proliferation index (PI) were derived manually as the number of positive ECs per vessel. Immunohistochemical measurement of EC apoptosis and proliferation was validated on human colorectal cancer liver metastases from a randomized, placebo-controlled trial of the selective cyclooxygenase-2 inhibitor rofecoxib. Proliferating and apoptotic ECs clustered in discrete areas of the tumour vasculature. ECAI (median [inter-quartile range, IQR] 0.0018 [0.0003-0.0064]) and ECPI (median [IQR] 0.0043 [0.002-0.014]) values were low and highly variable in tumours from both placebo- and rofecoxib-treated patients. Our novel dual-label immunohistochemical methods will be generally applicable in FFPE human cancer tissue and should prove invaluable for measuring the anti-angiogenic activity of experimental therapies in clinical trials. The low absolute level of and variability in EC apoptosis and proliferation detectable in human colorectal cancer liver metastases indicates that similar intervention studies using these end-points will need to ensure adequate size in order to be able to detect anti-angiogenic activity by immunohistochemical methods.