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1.  Layered Electrophoretic Transfer – A Method for Pre- Analytic Processing of Histological Sections 
Proteomics  2011;11(5):883-889.
Current technologies for measuring protein expression across a tissue section are based on mass spectrometry or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico- chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed Layered Electrophoretic Transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: 1) A reduced complexity of the tissue proteome for subsequent analysis; 2) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or mass spectrometry for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.
doi:10.1002/pmic.201000476
PMCID: PMC3729035  PMID: 21280224
2D protein separation; layered electrophoretic transfer; tissue protein mapping; proteomics
2.  Validation of esophageal squamous cell carcinoma candidate genes from high-throughput transcriptomic studies 
In a recent study, a unique gene expression signature was observed when comparing esophageal squamous cell carcinoma (ESCC) epithelial cells to normal esophageal epithelial cells using laser capture microdissection (LCM) and cDNA microarray technology. To validate the expression of several intriguing genes from that study (KRT17, cornulin, CD44, and EpCAM), we employed two new technologies, expression microdissection (xMD) for high-throughput microdissection facilitating protein analysis and RNAscope for the evaluation of low abundant transcripts in situ. For protein measurements, xMD technology was utilized to specifically procure sufficient tumor and normal epithelium from frozen human tissue for immunoblot analysis of KRT17 (CK17) and cornulin. A novel in situ hybridization method (RNAscope) was used to determine the transcript level of two relatively low expressed genes, CD44 and EpCAM in both individual formalin-fixed paraffin-embedded (FFPE) tissue sections and in an ESCC tissue microarray (TMA). The results successfully confirmed the initial expression pattern observed for all four genes, potentially implicating them in the pathogenesis of ESCC. Additionally, the study provides important methodological information on the overall process of candidate gene validation.
PMCID: PMC3744019  PMID: 23977449
Expression microdissection; esophageal squamous cell carcinoma; RNAscope; immunoblot
3.  Measuring collective cell movement and extracellular matrix interactions using magnetic resonance imaging 
Scientific Reports  2013;3:1879.
Collective cell behaviors in migration and force generation were studied at the mesoscopic-level using cells grown in a 3D extracellular matrix (ECM) simulating tissues. Magnetic resonance imaging (MRI) was applied to investigate dynamic cell mechanics at this level. MDCK, NBT2, and MEF cells were embedded in 3D ECM, forming clusters that then migrated and generated forces affecting the ECM. The cells demonstrated MRI contrast due to iron accumulation in the clusters. Timelapse-MRI enabled the measurement of dynamic stress fields generated by the cells, as well as simultaneous monitoring of the cell distribution and ECM deformation/remodeling. We found cell clusters embedded in the 3D ECM can exert translational forces to pull and push, as well as torque, their surroundings. We also observed that the sum of forces generated by multiple cell clusters may result in macroscopic deformation. In summary, MRI can be used to image cell-ECM interactions mesoscopically.
doi:10.1038/srep01879
PMCID: PMC3662010  PMID: 23698816
4.  A dynamic magnetic shift method to increase nanoparticle concentration in cancer metastases: a feasibility study using simulations on autopsy specimens 
A nanoparticle delivery system termed dynamic magnetic shift (DMS) has the potential to more effectively treat metastatic cancer by equilibrating therapeutic magnetic nanoparticles throughout tumors. To evaluate the feasibility of DMS, histological liver sections from autopsy cases of women who died from breast neoplasms were studied to measure vessel number, size, and spatial distribution in both metastatic tumors and normal tissue. Consistent with prior studies, normal tissue had a higher vascular density with a vessel-to-nuclei ratio of 0.48 ± 0.14 (n = 1000), whereas tumor tissue had a ratio of 0.13 ± 0.07 (n = 1000). For tumors, distances from cells to their nearest blood vessel were larger (average 43.8 μm, maximum 287 μm, n ≈ 5500) than normal cells (average 5.3 μm, maximum 67.8 μm, n ≈ 5500), implying that systemically delivered nanoparticles diffusing from vessels into surrounding tissue would preferentially dose healthy instead of cancerous cells. Numerical simulations of magnetically driven particle transport based on the autopsy data indicate that DMS would correct the problem by increasing nanoparticle levels in hypovascular regions of metastases to that of normal tissue, elevating the time-averaged concentration delivered to the tumor for magnetic actuation versus diffusion alone by 1.86-fold, and increasing the maximum concentration over time by 1.89-fold. Thus, DMS may prove useful in facilitating therapeutic nanoparticles to reach poorly vascularized regions of metastatic tumors that are not accessed by diffusion alone.
doi:10.2147/IJN.S23724
PMCID: PMC3224717  PMID: 22131836
cancer; metastases; vasculature; drug delivery; magnetic; nanoparticles
5.  Overexpression of WRAP53 Is Associated with Development and Progression of Esophageal Squamous Cell Carcinoma 
PLoS ONE  2014;9(3):e91670.
Background
Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer whose underlying molecular mechanisms are poorly understood. The natural antisense transcript (NAT) WRAP53 regulates p53 expression and WRAP53 protein is a component of telomerase. NATs play key roles in carcinogenesis, and although WRAP53 is known to increase cancer cell survival, its role in ESCC clinicopathology is unknown. The aim of this study was to investigate WRAP53 expression in ESCC and to correlate it with clinicopathological characteristics.
Methods
WRAP53 mRNA and protein expression was measured by quantitative PCR (qRT-PCR) and western blotting, respectively, in 4 ESSC cells lines and in 45 paired ESCC and non-neoplastic esophageal mucosa tissues. To correlate WRAP53 protein expression with clinicopathological characteristics, immunohistochemistry (IHC) was performed on 134 ESCC and 85 non-neoplastic esophageal mucosa tissues.
Results
Expression of WRAP53 was detected in all ESCC cell lines and was upregulated in the ESCC tissues compared with the corresponding non-neoplastic tissues (P<0.01). More cells expressed WRAP53 protein in the ESCC tissues than in the non-neoplastic tissues (P<0.01). Overexpression of WRAP53 was significantly correlated with tumor infiltration depth (P = 0.000), clinical stage (P = 0.001), and lymph node metastasis (P = 0.025). Wrap53 expression was not correlated with age, gender, or tumor differentiation.
Conclusion
This report indicates increased expression of WRAP53 in ESCC and that WRAP53 overexpression is correlated with tumor progression. WRAP53 may play a significant role in ESCC; accordingly, WRAP53 could be a useful biomarker for ESCC.
doi:10.1371/journal.pone.0091670
PMCID: PMC3953598  PMID: 24626331
6.  A Novel Quantitative Approach for Eliminating Sample-To-Sample Variation Using a Hue Saturation Value Analysis Program 
PLoS ONE  2014;9(3):e89627.
Objectives
As computing technology and image analysis techniques have advanced, the practice of histology has grown from a purely qualitative method to one that is highly quantified. Current image analysis software is imprecise and prone to wide variation due to common artifacts and histological limitations. In order to minimize the impact of these artifacts, a more robust method for quantitative image analysis is required.
Methods and Results
Here we present a novel image analysis software, based on the hue saturation value color space, to be applied to a wide variety of histological stains and tissue types. By using hue, saturation, and value variables instead of the more common red, green, and blue variables, our software offers some distinct advantages over other commercially available programs. We tested the program by analyzing several common histological stains, performed on tissue sections that ranged from 4 µm to 10 µm in thickness, using both a red green blue color space and a hue saturation value color space.
Conclusion
We demonstrated that our new software is a simple method for quantitative analysis of histological sections, which is highly robust to variations in section thickness, sectioning artifacts, and stain quality, eliminating sample-to-sample variation.
doi:10.1371/journal.pone.0089627
PMCID: PMC3940696  PMID: 24595280
7.  Background Coloration of Squamous Epithelium in Esophago-Pharyngeal Squamous Cell Carcinoma: What Causes the Color Change? 
PLoS ONE  2014;9(1):e85553.
Objectives
This study aims to clarify the cause of background coloration in the epithelia between each dilated intra papillary capillary loop in esophago-pharyngeal squamous cell carcinoma.
Design
This is a single center retrospective study including 124 patients with 160 lesions who underwent esophagogastroduodenoscopy in Nagasaki University Hospital from September 2007 to March 2012; a detailed comparison between endoscopic images and pathology was performed. Immunohistological assessment using anti-human hemoglobin antibody (anti-Hb Ab) was performed to verify the presence of hemoglobin (Hb) component in the cancer cells. Real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) on Hb-β mRNA were performed to assess the production of Hb component within the cancer cells.
Results
A strong positivity for anti-Hb Ab was observed in the squamous cell carcinoma area, whereas non-cancerous mucosa showed no immunopositivity for Hb. The concordance rate between anti-Hb Ab immunoreactivity and the presence of BC was as high as 80.9%. The amount of Hb-β mRNA expression was three times higher in cancer tissues compared with the surrounding non-cancerous mucosa. ISH images showed that the expression exclusively occurred in cancer cells, indicating that Hb is probably produced within cancer cells.
Conclusions
The background coloration observed is partly due to an extravascular component of Hb. RT-PCR and ISH analyses indicate that Hb is produced within cancer cells.
doi:10.1371/journal.pone.0085553
PMCID: PMC3904868  PMID: 24489662
8.  Squamous Cell Carcinoma – Similarities and Differences among Anatomical Sites 
Squamous cell carcinoma (SCC) is an epithelial malignancy involving many anatomical sites and is the most common cancer capable of metastatic spread. Development of early diagnosis methods and novel therapeutics are important for prevention and mortality reduction. In this effort, numerous molecular alterations have been described in SCCs. SCCs share many phenotypic and molecular characteristics, but they have not been extensively compared. This article reviews SCC as a disease, including: epidemiology, pathology, risk factors, molecular characteristics, prognostic markers, targeted therapy, and a new approach to studying SCCs. Through this comparison, several themes are apparent. For example, HPV infection is a common risk factor among the four major SCCs (NMSC, HNSC, ESCC, and NSCLC) and molecular abnormalities in cell-cycle regulation and signal transduction predominate. These data reveal that the molecular insights, new markers, and drug targets discovered in individual SCCs may shed light on this type of cancer as a whole.
PMCID: PMC3175764  PMID: 21938273
Squamous cell carcinoma (SCC); Non-melanoma skin cancer (NMSC); Head and neck squamous cell carcinomas (HNSCC); esophageal squamous cell carcinoma (ESCC); Non-small cell lung cancer (NSCLC); epidemiology; risk factors; molecular characteristics; prognostic markers; targeted therapy
9.  MicroRNA analysis of microdissected normal squamous esophageal epithelium and tumor cells 
Previous studies have identified several dysregulated microRNAs in esophageal squamous cell carcinoma (ESCC); however, to date there are no ex vivo analyses comparing expression levels of these regulatory molecules in esophageal squamous cell tumors versus patient-matched normal epithelium. We describe here a technical strategy to evaluate microRNAs in normal esophageal basal cells (NB), normal esophageal differentiated cells (ND), and tumor cells (T). Laser capture microdissection was used to procure target populations from five cases and 18 ESCC-associated microRNAs were measured by RT-qPCR. Five microRNAs (miR-25, miR-106b, miR-21, miR-203, and miR-145) demonstrated consistent differential expression in at least one of the three comparisons: T vs. NB, T vs. ND, or NB vs. ND. The potential regulatory role of the microRNAs in ESCC was further evaluated by correlating their expression with a matched mRNA dataset, which included the same five cases and cell populations. In conclusion, the present work demonstrates the feasibility of studying microRNA levels in precisely dissected cell populations from clinical samples, and sheds light on the molecular mechanisms associated with ESCC.
PMCID: PMC3142940  PMID: 21796275
Esophageal squamous cell carcinoma; laser capture microdissection; microRNA; basal layer; differentiated layer; miR-25; miR-106b; miR-21; miR-203; miR-145
11.  3-D Visualization and Quantitation of Microvessels in Transparent Human Colorectal Carcinoma 
PLoS ONE  2013;8(11):e81857.
Microscopic analysis of tumor vasculature plays an important role in understanding the progression and malignancy of colorectal carcinoma. However, due to the geometry of blood vessels and their connections, standard microtome-based histology is limited in providing the spatial information of the vascular network with a 3-dimensional (3-D) continuum. To facilitate 3-D tissue analysis, we prepared transparent human colorectal biopsies by optical clearing for in-depth confocal microscopy with CD34 immunohistochemistry. Full-depth colons were obtained from colectomies performed for colorectal carcinoma. Specimens were prepared away from (control) and at the tumor site. Taking advantage of the transparent specimens, we acquired anatomic information up to 200 μm in depth for qualitative and quantitative analyses of the vasculature. Examples are given to illustrate: (1) the association between the tumor microstructure and vasculature in space, including the perivascular cuffs of tumor outgrowth, and (2) the difference between the 2-D and 3-D quantitation of microvessels. We also demonstrate that the optically cleared mucosa can be retrieved after 3-D microscopy to perform the standard microtome-based histology (H&E staining and immunohistochemistry) for systematic integration of the two tissue imaging methods. Overall, we established a new tumor histological approach to integrate 3-D imaging, illustration, and quantitation of human colonic microvessels in normal and cancerous specimens. This approach has significant promise to work with the standard histology to better characterize the tumor microenvironment in colorectal carcinoma.
doi:10.1371/journal.pone.0081857
PMCID: PMC3843693  PMID: 24324559
12.  An NIH intramural percubator as a model of academic-industry partnerships: from the beginning of life through the valley of death 
In 2009 the NIH publicly announced five strategic goals for the institutes that included the critical need to translate research discoveries into public benefit at an accelerated pace, with a commitment to find novel ways to engage academic investigators in the process. The emphasis on moving scientific advancements from the laboratory to the clinic is an opportune time to discuss how the NIH intramural program in Bethesda, the largest biomedical research center in the world, can participate in this endeavor. Proposed here for consideration is a percolator-incubator program, a 'percubator' designed to enable NIH intramural investigators to develop new medical interventions as quickly and efficiently as possible.
doi:10.1186/1479-5876-9-54
PMCID: PMC3118127  PMID: 21548992
13.  MicroRNA analysis of microdissected normal squamous esophageal epithelium and tumor cells 
Previous studies have identified several dysregulated microRNAs in esophageal squamous cell carcinoma (ESCC); however, to date there are no ex vivo analyses comparing expression levels of these regulatory molecules in esophageal squamous cell tumors versus patient-matched normal epithelium. We describe here a technical strategy to evaluate microRNAs in normal esophageal basal cells (NB), normal esophageal differentiated cells (ND), and tumor cells (T). Laser capture microdissection was used to procure target populations from five cases and 18 ESCC-associated microRNAs were measured by RT-qPCR. Five microRNAs (miR-25, miR-106b, miR-21, miR-203, and miR-145) demonstrated consistent differential expression in at least one of the three comparisons: T vs. NB, T vs. ND, or NB vs. ND. The potential regulatory role of the microRNAs in ESCC was further evaluated by correlating their expression with a matched mRNA dataset, which included the same five cases and cell populations. In conclusion, the present work demonstrates the feasibility of studying microRNA levels in precisely dissected cell populations from clinical samples, and sheds light on the molecular mechanisms associated with ESCC.
PMCID: PMC3142940  PMID: 21796275
Esophageal squamous cell carcinoma; laser capture microdissection; microRNA; basal layer; differentiated layer; miR-25; miR-106b; miR-21; miR-203; miR-145
14.  SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling 
Introduction:
Laser capture microdissection (LCM) facilitates procurement of defined cell populations for study in the context of histopathology. The morphologic assessment step in the LCM procedure is time consuming and tedious, thus restricting the utility of the technology for large applications.
Results:
Here, we describe the use of Spatially Invariant Vector Quantization (SIVQ) for histological analysis and LCM. Using SIVQ, we selected vectors as morphologic predicates that were representative of normal epithelial or cancer cells and then searched for phenotypically similar cells across entire tissue sections. The selected cells were subsequently auto-microdissected and the recovered RNA was analyzed by expression microarray. Gene expression profiles from SIVQ–LCM and standard LCM–derived samples demonstrated highly congruous signatures, confirming the equivalence of the differing microdissection methods.
Conclusion:
SIVQ–LCM improves the work-flow of microdissection in two significant ways. First, the process is transformative in that it shifts the pathologist's role from technical execution of the entire microdissection to a limited-contact supervisory role, enabling large-scale extraction of tissue by expediting subsequent semi-autonomous identification of target cell populations. Second, this work-flow model provides an opportunity to systematically identify highly constrained cell populations and morphologically consistent regions within tissue sections. Integrating SIVQ with LCM in a single environment provides advanced capabilities for efficient and high-throughput histological-based molecular studies.
doi:10.4103/2153-3539.78500
PMCID: PMC3073068  PMID: 21572509
Laser capture microdissection; microarray; Spatially Invariant Vector Quantization
15.  Image microarrays (IMA): Digital pathology's missing tool 
Introduction:
The increasing availability of whole slide imaging (WSI) data sets (digital slides) from glass slides offers new opportunities for the development of computer-aided diagnostic (CAD) algorithms. With the all-digital pathology workflow that these data sets will enable in the near future, literally millions of digital slides will be generated and stored. Consequently, the field in general and pathologists, specifically, will need tools to help extract actionable information from this new and vast collective repository.
Methods:
To address this limitation, we designed and implemented a tool (dCORE) to enable the systematic capture of image tiles with constrained size and resolution that contain desired histopathologic features.
Results:
In this communication, we describe a user-friendly tool that will enable pathologists to mine digital slides archives to create image microarrays (IMAs). IMAs are to digital slides as tissue microarrays (TMAs) are to cell blocks. Thus, a single digital slide could be transformed into an array of hundreds to thousands of high quality digital images, with each containing key diagnostic morphologies and appropriate controls. Current manual digital image cut-and-paste methods that allow for the creation of a grid of images (such as an IMA) of matching resolutions are tedious.
Conclusion:
The ability to create IMAs representing hundreds to thousands of vetted morphologic features has numerous applications in education, proficiency testing, consensus case review, and research. Lastly, in a manner analogous to the way conventional TMA technology has significantly accelerated in situ studies of tissue specimens use of IMAs has similar potential to significantly accelerate CAD algorithm development.
doi:10.4103/2153-3539.86829
PMCID: PMC3237063  PMID: 22200030
IMA; SIVQ; TMA; WSI
16.  Decrease in CD8+ lymphocyte number and altered cytokine profile in human prostate cancer 
The tumor microenvironment is comprised of multiple cell types arranged in a three-dimensional structure. Interactions amongst the various cell components play an important role in neoplasia, including the inflammatory reaction that occurs as part of the host response. In this study, the regional lymphocyte subpopulations and cytokine profiles associated with prostate cancer were examined using a quantitative imaging approach and expression microarray analysis. Lymphocytes were measured in four different epithelial phenotypes in prostate cancer specimens: carcinoma; prostatic intraepithelial neoplasia (PIN); benign prostate hyperplasia (BPH); and normal epithelium. The data indicate that CD8 positive, cytotoxic T lymphocytes are significantly decreased in regions adjacent to hyperplasia and carcinoma as compared to normal epithelium and PIN. In contrast the relative number of CD4 positive and CD20 positive lymphocytes did not change markedly. Parallel mRNA expression array analysis of the normal and tumor microenvironments identified a distinct cytokine profile in cancer, with 24 dysregulated genes in tumor epithelium and nine altered in tumor-associated stroma. Overall, these data indicate that the spatial distribution of CD8 positive, cytotoxic T lymphocytes is dysregulated in human prostate glands that contain cancer, and cytokine profiles are altered at the mRNA level.
PMCID: PMC3180108  PMID: 21969236
Prostate cancer; lymphocytes; cytokines; histomathematics; histopathology
18.  2D-PCR: a method of mapping DNA in tissue sections† 
Lab on a chip  2009;9(24):3526-3534.
A novel approach was developed for mapping the location of target DNA in tissue sections. The method combines a high-density, multi-well plate with an innovative single-tube procedure to directly extract, amplify, and detect the DNA in parallel while maintaining the two-dimensional (2D) architecture of the tissue. A 2D map of the gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was created from a tissue section and shown to correlate with the spatial area of the sample. It is anticipated that this approach may be easily adapted to assess the status of multiple genes within tissue sections, yielding a molecular map that directly correlates with the histology of the sample. This will provide investigators with a new tool to interrogate the molecular heterogeneity of tissue specimens.
doi:10.1039/b910807f
PMCID: PMC2910845  PMID: 20024032
19.  Current molecular diagnostics of breast cancer and the potential incorporation of microRNA 
Although comprehensive molecular diagnostics and personalized medicine have sparked excitement among researchers and clinicians, they have yet to be fully incorporated into today’s standard of care. This is despite the discovery of disease-related oncogenes, tumor-suppressor genes and protein biomarkers, as well as other biological anomalies related to cancer. Each year, new tests are released that could potentially supplement or surpass standard methods of diagnosis, including serum, protein and gene expression analyses. All of these novel approaches have shown great promise, but initial enthusiasm has diminished as difficulties in reproducibility, expense, standardization and proof of significance beyond current protocols have emerged. This review will focus on current and novel molecular diagnostic tools applied to breast cancer with special attention to the exciting new field of microRNA analysis
doi:10.1586/erm.09.25
PMCID: PMC2758104  PMID: 19580430
breast cancer; microarray; microRNA; molecular diagnostics; predictive value; prognostic value
20.  Influence of hypoxia induced by minimally invasive prostatectomy on gene expression: implications for biomarker analysis 
Handling and processing of clinical specimens during and after surgical resection may significantly skew the molecular data obtained from analysis of those samples. Minimally invasive prostatectomy was used as a model to specifically study effects of surgical ischemia on gene expression in human clinical samples. Normal prostatic urethra cup biopsies were procured from 12 patients at three time points during laparoscopic radical prostatectomy. Homogeneous cells (stroma and epithelium) were microdissected. Transcript analysis of 3 oxygen-dependent, 3 oxygen-independent, and 3 control class genes was performed using quantitative RT-PCR. Data were analyzed by relative quantitation and two-sided t-test. Patient demographic and time covariates were fit by a linear mixed model. VEGF, an oxygen-dependent gene, showed significant expression alterations across three time points in epithelium (p=0.008), but not in stroma (p=0.66). Expression levels of VHL, STAT5B, and CYPA showed significant changes at the p<0.05 level in the stroma only. Effects of age, PSA, prostate size, Gleason score, surgery type, total surgery time, total ischemia time, and estimated blood loss on VEGF expression over time were not significant at the p<0.01 level. Therefore, surgical manipulation and tissue processing methods need to be taken into account when assessing prostatic biomarkers; however, resection does not dramatically alter mRNA profiles in prostate specimens.
PMCID: PMC2892411  PMID: 20589162
Laparoscopic surgery; prostatectomy; warm ischemia; hypoxia; tissue microdissection; gene expression analysis
21.  Oncogenic Features of PHF8 Histone Demethylase in Esophageal Squamous Cell Carcinoma 
PLoS ONE  2013;8(10):e77353.
Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide. It has been reported that histone demethylases are involved in the carcinogenesis of certain types of tumors. Here, we studied the role of one of the histone lysine demethylases, plant homeodomain finger protein 8 (PHF8), in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). Using short hairpin RNA via lentiviral infection, we established stable ESCC cell lines with constitutive downregulation of PHF8 expression. Knockdown of PHF8 in ESCC cells resulted in inhibition of cell proliferation and an increase of apoptosis. Moreover, there were reductions of both anchorage-dependent and -independent colony formation. In vitro migration and invasion assays showed that knockdown of PHF8 led to a reduction in the number of migratory and invasive cells. Furthermore, downregulation of PHF8 attenuated the tumorigenicity of ESCC cells in vivo. Taken together, our study revealed the oncogenic features of PHF8 in ESCC, suggesting that PHF8 may be a potential diagnostic marker and therapeutic target for ESCC.
doi:10.1371/journal.pone.0077353
PMCID: PMC3795633  PMID: 24146981
22.  Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples 
Nature protocols  2009;4(6):902-922.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy, and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires approximately 16 hours.
doi:10.1038/nprot.2009.61
PMCID: PMC2760821  PMID: 19478806
quantitative measurements; microdissected tissues; qRT-PCR; validation; gene expression analysis; protocol; normalization strategy
23.  Squamous cell carcinoma – similarities and differences among anatomical sites 
Squamous cell carcinoma (SCC) is an epithelial malignancy involving many anatomical sites and is the most common cancer capable of metastatic spread. Development of early diagnosis methods and novel therapeutics are important for prevention and mortality reduction. In this effort, numerous molecular alterations have been described in SCCs. SCCs share many phenotypic and molecular characteristics, but they have not been extensively compared. This article reviews SCC as a disease, including: epidemiology, pathology, risk factors, molecular characteristics, prognostic markers, targeted therapy, and a new approach to studying SCCs. Through this comparison, several themes are apparent. For example, HPV infection is a common risk factor among the four major SCCs (NMSC, HNSC, ESCC, and NSCLC) and molecular abnormalities in cell-cycle regulation and signal transduction predominate. These data reveal that the molecular insights, new markers, and drug targets discovered in individual SCCs may shed light on this type of cancer as a whole.
PMCID: PMC3175764  PMID: 21938273
Squamous cell carcinoma (SCC); non-melanoma skin cancer (NMSC); head and neck squamous cell carcinomas (HNSCC); esophageal squamous cell carcinoma (ESCC); non-small cell lung cancer (NSCLC); epidemiology; risk factors; molecular characteristics; prognostic markers; targeted therapy
24.  A Survey of the Barriers Associated with Academic-based Cancer Research Commercialization 
PLoS ONE  2013;8(8):e72268.
Commercialization within the academic setting is associated with many challenges and barriers. Previous studies investigating these challenges/barriers have, in general, broadly focused on multiple disciplines and, oftentimes, several institutions simultaneously. The goal of the study presented here was to analyze a range of barriers that may be broadly associated with commercializing academic-based cancer research. This goal was addressed via a study of the barriers associated with cancer research commercialization at the University of Kentucky (UK). To this end, a research instrument in the form of an electronic survey was developed. General demographic information was collected on study participants and two research questions were addressed: 1) What are the general barriers inhibiting cancer research commercialization at UK? and 2) Would mitigation of the barriers potentially enhance faculty engagement in commercialization activities? Descriptive and statistical analysis of the data reveal that multiple barriers likely inhibit cancer research commercialization at UK with expense, time, infrastructure, and lack of industry partnerships being among the most commonly cited factors. The potential alleviation of these factors in addition to revised University policies/procedures, risk mitigation, more emphasis on commercialization by academia research field, and increased information on how to commercialize significantly correlated with the potential for increased commercialization activity. Furthermore, multivariate logistic regression modeling demonstrated that research commercialization would incrementally increase as barriers to the process are removed and that PhD-holding respondents and respondents in commercialization-supportive research fields would be more likely to commercialize their research upon barrier removal. Overall, as with other disciplines, these data suggest that for innovations derived from academic cancer-research to move more effectively and efficiently into the marketplace, university administrators and external agents, such as policymakers, need to address what are well-documented and defined issues.
doi:10.1371/journal.pone.0072268
PMCID: PMC3749229  PMID: 23991077
25.  Analysis of Transcription Factor mRNAs in Identified Oxytocin and Vasopressin Magnocellular Neurons Isolated by Laser Capture Microdissection 
PLoS ONE  2013;8(7):e69407.
The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV) vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun) are differentially expressed in the Oxt and Avp MCNs.
doi:10.1371/journal.pone.0069407
PMCID: PMC3722287  PMID: 23894472

Results 1-25 (39)