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1.  Gas6/Axl is the sensor of Arginine-Auxotrophic Response in Targeted Chemotherapy with Arginine-Depleting Agents 
Oncogene  2015;35(13):1632-1642.
Many human malignancies lack de novo biosynthesis of arginine (Arg) because the key enzyme argininosuccinate synthetase 1 (ASS1) is silenced. These tumors acquire ectopic Arg for survival, and depleting this source by Arg-depleting recombinant enzyme ADI-PEG20 results in cell death. Mechanisms underlying Arg auxotrophy in these tumors and how they respond to Arg-auxotrophic stress are poorly understood. Here, we report that an immediate early event of Arg-auxotrophic response involves ROS-mediated secretion of Gas6 which interacts with its receptor Axl and activates the downstream Ras/PI3K/Akt growth signal leading to accumulation of c-Myc by protein stabilization. Arg-auxotrophic challenge also transcriptionally upregulates c-Myc expression which feedbacks to enhance Axl expression. c-Myc is a positive regulator of ASS1, but elevated ASS1 feedbacks to suppress c-Myc and Axl. Our results revealed multiple inter-regulatory pathways in Arg-auxotrophic response consisting of Axl, c-Myc, ASS1 that regulate Arg homeostasis and ADI-PEG20 sensitivity. These pathways provide potential targets for improving the efficacy of treating Arg-auxotrophic tumors using Arg deprivation strategies.
PMCID: PMC4835044  PMID: 26096933
Gas6; Axl; Arginine; c-Myc; ROS; ADI-PEG20
2.  Systematic siRNA Screen Unmasks NSCLC Growth Dependence by Palmitoyltransferase DHHC5 
Molecular cancer research : MCR  2015;13(4):784-794.
Protein S-palmitoylation is a widespread and dynamic post-translational modification that regulates protein-membrane interactions, protein-protein interactions, and protein stability. A large family of palmitoyl acyl transferases, termed the DHHC family due to the presence of a common catalytic motif, catalyzes S-palmitoylation; the role of these enzymes in cancer is largely unexplored. In this study, an RNAi-based screen targeting all 23 members of the DHHC family was conducted to examine the effects on the growth in non-small cell cancer (NSCLC). Interestingly, siRNAs directed against DHHC5 broadly inhibited the growth of multiple NSCLC lines but not normal human bronchial epithelial cell (HBEC) lines. Silencing of DHHC5 by lentivirus-mediated expression of DHHC5 shRNAs dramatically reduced in vitro cell proliferation, colony formation and cell invasion in a subset of cell lines that were examined in further detail. The phenotypes were restored by transfection of a wild-type DHHC5 plasmid but not by a plasmid expressing a catalytically inactive DHHC5. Tumor xenograft formation was severely inhibited by DHHC5 knockdown and rescued by DHHC5 expression, using both a conventional and tetracycline-inducible shRNA. These data indicate that DHHC5 has oncogenic capacity and contributes to tumor formation in NSCLC; thus representing a potential novel therapeutic target.
PMCID: PMC4398612  PMID: 25573953
lung cancer; post-translational lipid modifications; S-palmitoylation; xenografts; drug target
3.  Immunoguided Microdissection Techniques 
Over the past 15 years, laser-based microdissection has improved the precision by which scientists can procure cells of interest from a heterogeneous tissue section. However, for studies that require a large amount of material (e.g., proteomics) or for cells that are scattered and difficult to identify by standard histological stains, an immunostain-based, automated approach becomes essential. In this chapter, we discuss the use of immunohistochemistry (IHC) and immunofluorescence (IF) to guide the microdissection process via manual and software-driven auto-dissection methods. Although technical challenges still exist with these innovative approaches, we present here methods and protocols to successfully perform immuno-based microdissection on commercially available laser dissection systems.
PMCID: PMC4794997  PMID: 21761293
Automation; Immunohistochemistry; Microdissection
4.  Expression microdissection adapted to commercial laser dissection instruments 
Nature protocols  2011;6(4):457-467.
Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis.
PMCID: PMC4795005  PMID: 21412274
5.  Multiplex Quantitative Measurement of mRNAs from Fixed Tissue Microarray Sections 
The development of prognostic and diagnostic biomarkers, such as those from gene expression studies, requires independent validation in clinical specimens. Immunohistochemistry on tissue microarrays (TMAs) of formalin-fixed paraffin-embedded (FFPE) tissue is often used to increase statistical power, and it is used more often than in situ hybridization (ISH), which can be technically limiting. Here we introduce a method for performing quantitative gene expression analysis across a TMA using an adaptation of 2D-RT-qPCR, a recently developed technology for measuring transcript levels in a histological section while maintaining two-dimensional positional information of the tissue sample. As a demonstration of utility, a TMA with tumor and normal human prostate samples was used to validate expression profiles from previous array-based gene discovery studies of prostate cancer. The results show that 2D-RT-qPCR expands the utility of TMAs to include sensitive and accurate gene expression measurements.
PMCID: PMC4022353  PMID: 24809843
6.  Oncogenic mutation profiling in new lung cancer and mesothelioma cell lines 
OncoTargets and therapy  2015;8:195-209.
Thoracic tumor, especially lung cancer, ranks as the top cancer mortality in most parts of the world. Lung adenocarcinoma is the predominant subtype and there is increasing knowledge on therapeutic molecular targets, namely EGFR, ALK, KRAS, and ROS1, among lung cancers. Lung cancer cell lines established with known clinical characteristics and molecular profiling of oncogenic targets like ALK or KRAS could be useful tools for understanding the biology of known molecular targets as well as for drug testing and screening.
Materials and methods
Five new cancer cell lines were established from pleural fluid or biopsy tissues obtained from Chinese patients with primary lung adenocarcinomas or malignant pleural mesothelioma. They were characterized by immunohistochemistry, growth kinetics, tests for tumorigenicity, EGFR and KRAS gene mutations, ALK gene rearrangement and OncoSeq mutation profiling.
These newly established lung adenocarcinoma and mesothelioma cell lines were maintained for over 100 passages and demonstrated morphological and immunohistochemical features as well as growth kinetics of tumor cell lines. One of these new cell lines bears EML4-ALK rearrangement variant 2, two lung cancer cell lines bear different KRAS mutations at codon 12, and known single nucleotide polymorphism variants were identified in these cell lines.
Four new lung adenocarcinoma and one mesothelioma cell lines were established from patients with different clinical characteristics and oncogenic mutation profiles. These characterized cell lines and their mutation profiles will provide resources for exploration of lung cancer and mesothelioma biology with regard to the presence of known oncogenic mutations.
PMCID: PMC4303463  PMID: 25653542
lung adenocarcinomas; oncogenic mutations; EGFR; ALK; KRAS
Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant pre-clinical model. Canine melanomas rarely arise in sun-exposed sites. Most occur in the oral cavity, with a subset having intraepithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS and c-kit mutations uncommonly, compared to human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a pre-clinical model.
PMCID: PMC4066658  PMID: 24128326
melanoma; animal model; comparative study; clinical trial design; image analysis; digital telepathology; signal transduction
8.  A Re-evaluation of CD22 Expression by Human Lung Cancer 
Cancer research  2014;74(1):263-271.
CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B cell receptor and its co-receptor CD19. Recently it was reported that most human lung cancer cells and cell lines express CD22 making it an important new lung cancer therapeutic target (Can Res 72:5556, 2012). The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by qRT-PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200–60,000- fold lower than those observed in the human CD22+ Burkitt’s lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by CD22 antibodies or our highly potent anti-CD22 immunotoxin. By contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from over 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells and that these cells can not be killed by anti-CD22 immunotoxins.
PMCID: PMC3903042  PMID: 24395821
lung cancer; CD22 expression
9.  Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus - associated hepatocellular carcinoma 
The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC.
We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR) and confocal immunofluorescence.
Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK.
Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-014-0230-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4142136  PMID: 25141867
Hepatocellular carcinoma; Hepatitis B virus; Gene expression profiling; Laser capture microdissection; Confocal microscopy
10.  ZEB1 sensitizes lung adenocarcinoma to metastasis suppression by PI3K antagonism 
The Journal of Clinical Investigation  2014;124(6):2696-2708.
Epithelial tumor cells that have undergone epithelial-to-mesenchymal transition (EMT) are typically prone to metastasis and drug resistance and contribute to a poor clinical outcome. The transcription factor ZEB1 is a known driver of EMT, and mediators of ZEB1 represent potential therapeutic targets for metastasis suppression. Here, we have shown that phosphatidylinositol 3-kinase–targeted (PI3K-targeted) therapy suppresses metastasis in a mouse model of Kras/Tp53-mutant lung adenocarcinoma that develops metastatic disease due to high expression of ZEB1. In lung adenocarcinoma cells from Kras/Tp53-mutant animals and human lung cancer cell lines, ZEB1 activated PI3K by derepressing miR-200 targets, including amphiregulin (AREG), betacellulin (BTC), and the transcription factor GATA6, which stimulated an EGFR/ERBB2 autocrine loop. Additionally, ZEB1-dependent derepression of the miR-200 and miR-183 target friend of GATA 2 (FOG2) enhanced GATA3-induced expression of the p110α catalytic subunit of PI3K. Knockdown of FOG2, p110α, and RHEB ameliorated invasive and metastatic propensities of tumor cells. Surprisingly, FOG2 was not required for mesenchymal differentiation, suggesting that mesenchymal differentiation and invasion are distinct and separable processes. Together, these results indicate that ZEB1 sensitizes lung adenocarcinoma cells to metastasis suppression by PI3K-targeted therapy and suggest that treatments to selectively modify the metastatic behavior of mesenchymal tumor cells are feasible and may be of clinical value.
PMCID: PMC4038569  PMID: 24762440
11.  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.
PMCID: PMC3722287  PMID: 23894472
12.  The General Transcription Factor TAF7 Is Essential for Embryonic Development but Not Essential for the Survival or Differentiation of Mature T Cells 
Molecular and Cellular Biology  2012;32(10):1984-1997.
TAF7, a component of the TFIID complex that nucleates the assembly of transcription preinitiation complexes, also independently interacts with and regulates the enzymatic activities of other transcription factors, including P-TEFb, TFIIH, and CIITA, ensuring an orderly progression in transcription initiation. Since not all TAFs are required in terminally differentiated cells, we examined the essentiality of TAF7 in cells at different developmental stages in vivo. Germ line disruption of the TAF7 gene is embryonic lethal between 3.5 and 5.5 days postcoitus. Mouse embryonic fibroblasts with TAF7 deleted cease transcription globally and stop proliferating. In contrast, whereas TAF7 is essential for the differentiation and proliferation of immature thymocytes, it is not required for subsequent, proliferation-independent differentiation of lineage committed thymocytes or for their egress into the periphery. TAF7 deletion in peripheral CD4 T cells affects only a small number of transcripts. However, T cells with TAF7 deleted are not able to undergo activation and expansion in response to antigenic stimuli. These findings suggest that TAF7 is essential for proliferation but not for proliferation-independent differentiation.
PMCID: PMC3347399  PMID: 22411629
13.  Paracrine SLPI Secretion Upregulates MMP-9 Transcription and Secretion in Ovarian Cancer Cells 
Gynecologic oncology  2011;122(3):656-662.
Secretory leukocyte protease inhibitor (SLPI) is amplified in serous ovarian cancer. We have dissected its function, showing it is a survival factor for ovarian cancer and promotes tumorigenesis and paclitaxel-resistance. We hypothesized that the protease inhibitory function was responsible for modulating SLPI’s invasive capacity.
Stable HEYA8 ovarian cancer transfectants expressing vector, wild type SLPI, and protease inhibitor null (F-)SLPI were examined in vitro and in xenografts. Invasion, enzyme activity, and MMP production and function assays were applied. SLPI and MMP immunoexpression were graded on tissue microarray and clinical samples. Statistical comparisons used unpaired T test and ANOVA, where appropriate.
SLPI and F-SLPI cells caused greater parenchymal and peritoneal dissemination over control cells in xenografts and invasion assays (p<0.001). MMP-9 protease activity was increased in SLPI and F-SLPI cells over control. SLPI, but not F-SLPI, inhibited plasmin activity, necessary for MMP-9 activation and release, and inhibited activation of MMP-9. However, paradoxically, both induced quantitative MMP-9 transcription (p<0.05) and protein (p<0.008), yielding an increased net MMP-9 activity in the face of plasmin inhibition. SLPI and MMP-9 expression were strongly correlated in serous ovarian cancers (r2=0.986) and a set of ovarian cancers (p<0.02). SLPI expression was greater in serous than endometrioid ovarian cancers (p=0.04).
SLPI stimulates ovarian cancer invasion, modulated in part by its serine protease inhibitory activity attenuating MMP-9 release. However, SLPI induction of MMP-9, independent of protease inhibition activity, is greater yielding a net pro-invasive behavior. These findings further support SLPI as a molecular target for ovarian cancer.
PMCID: PMC3152651  PMID: 21676452
ovarian cancer; SLPI; MMP-9; metalloproteinase
14.  Clonally related Histiocytic/dendritic cell sarcoma and chronic lymphocytic leukemia/small lymphocytic lymphoma: A study of 7 cases 
Histiocytic and interdigitating dendritic cell sarcomas are rare tumors originating from bone marrow derived myeloid stem cells. Recent studies have shown evidence of cross-lineage transdifferentiation of B-cells in follicular lymphoma to histiocytic and dendritic cell sarcomas. In this study, we report the morphologic, molecular and cytogenetic analysis of 7 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma associated with histiocytic and dendritic cell sarcomas. All seven patients were elderly males (median age, 71 years). The B-cell neoplasms preceded the development of the histiocytic and dendritic cell sarcomas in 6 of 7 patients, and one patient had both tumors diagnosed at the same time. The tumors included 4 interdigitating dendritic cell sarcomas; 1 Langerhans cell sarcoma, 1 histiocytic sarcoma, and 1 immature neoplasm with evidence of histiocytic origin. Laser-capture microdissection and PCR analysis showed identical clonal immunoglobulin gene rearrangements in the two phenotypically distinct components in all cases. There was a preferential usage of IGHV4-39 by the V-D-J gene rearrangement. By FISH analysis two cases showed deletion 17p in both components, while 4 cases had normal cytogenetic findings by FISH in the CLL/SLL cells, but acquired cytogenetic abnormalities in the corresponding histiocytic and dendritic tumors. Chromosome 17p abnormalities were the most common cytogenetic abnormality detected in the sarcomas, seen in 5 of 6 cases studied. Compared with the CLL/SLL cells, the histiocytic/dendritic cells were largely negative for PAX5, but showed strong expression of PU.1 and variable and weak expression of CEBPβ. Our study provides evidence for transdifferentiation of CLL/SLL B-cells to tumors of dendritic and less often histiocytic lineage, and suggests that secondary genetic events may play a role in this phenomenon.
PMCID: PMC3175277  PMID: 21666687
CLL; SLL; histiocytic sarcoma; interdigitating dendritic cell sarcoma; clonal; transformation; transdifferentiation
15.  Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma 
BMC Research Notes  2012;5:73.
Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB), normal differentiated squamous epithelium (ND), and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets.
As a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA) were verified to be dysregulated in the same pattern at both the mRNA and protein levels.
These data reveal insight into genes and molecular pathways mediating ESCC development and provide information potentially useful in designing novel therapeutic interventions for this tumor type.
PMCID: PMC3283499  PMID: 22280838
16.  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.
PMCID: PMC3224717  PMID: 22131836
cancer; metastases; vasculature; drug delivery; magnetic; nanoparticles
17.  Opposing regulation of the Il17 locus through direct, reciprocal actions of STAT3 and STAT5 
Nature immunology  2011;12(3):247-254.
IL-2, a cytokine linked to human autoimmune diseases, limits IL-17 production. We show that deletion of STAT3 in T cells abrogates IL-17 production and attenuates autoimmunity associated with IL-2 deficiency. While STAT3 induces IL-17 and RORγt and inhibits FOXP3, IL-2 inhibited IL-17 independently of FOXP3 and RORγt. We found that STAT3 and STAT5 bound to multiple common sites across the Il17 genetic locus. The induction of STAT5 binding by IL-2 was associated with a reduction in STAT3 binding at these sites and the inhibition of associated active epigenetic marks. Titrating the relative activation of STAT3 and STAT5 modulated Th17 cell specification and thus, the balance rather than the absolute magnitude of these signals determine the propensity of cells to make a key inflammatory cytokine.
PMCID: PMC3182404  PMID: 21278738
18.  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
19.  Human Intestinal Tissue and Cultured Colonic Cells Contain Globotriaosylceramide Synthase mRNA and the Alternate Shiga Toxin Receptor Globotetraosylceramide ▿  
Infection and Immunity  2010;78(11):4488-4499.
Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) bacteria are not enteroinvasive but can cause hemorrhagic colitis. In some STEC-infected individuals, a life-threatening sequela of infection called the hemolytic uremic syndrome may develop that can lead to kidney failure. This syndrome is linked to the production of Stx by the infecting organism. For Stx to reach the kidney, the toxin must first penetrate the colonic epithelial barrier. However, the Stx receptor, globotriaosylceramide (Gb3), has been thought to be absent from human intestinal epithelial cells. Thus, the mechanisms by which the toxin associates with and traverses through the intestine en route to the kidneys have been puzzling aspects of STEC pathogenesis. In this study, we initially determined that both types of Stx made by STEC, Stx1 and Stx2, do in fact bind to colonic epithelia in fresh tissue sections and to a colonic epithelial cell line (HCT-8). We also discovered that globotetraosylceramide (Gb4), a lower-affinity toxin receptor derived from Gb3, is readily detectable on the surfaces of human colonic tissue sections and HCT-8 cells. Furthermore, we found that Gb3 is present on a fraction of HCT-8 cells, where it presumably functions to bind and internalize Stx1 and Stx2. In addition, we established by quantitative real-time PCR (qRT-PCR) that both fresh colonic epithelial sections and HCT-8 cells express Gb3 synthase mRNA. Taken together, our data suggest that Gb3 may be present in small quantities in human colonic epithelia, where it may compete for Stx binding with the more abundantly expressed glycosphingolipid Gb4.
PMCID: PMC2976364  PMID: 20732996
20.  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
21.  SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling 
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.
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.
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.
PMCID: PMC3073068  PMID: 21572509
Laser capture microdissection; microarray; Spatially Invariant Vector Quantization
23.  Image microarrays (IMA): Digital pathology's missing tool 
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.
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.
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.
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.
PMCID: PMC3237063  PMID: 22200030
24.  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
25.  Increased matrix metalloproteinase activation in esophageal squamous cell carcinoma 
Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC.
Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment.
Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.
PMCID: PMC2958908  PMID: 20920372

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