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1.  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.
doi:10.1172/JCI72171
PMCID: PMC4038569  PMID: 24762440
2.  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
3.  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.
doi:10.1128/MCB.06305-11
PMCID: PMC3347399  PMID: 22411629
4.  Paracrine SLPI Secretion Upregulates MMP-9 Transcription and Secretion in Ovarian Cancer Cells 
Gynecologic oncology  2011;122(3):656-662.
Objectives
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1016/j.ygyno.2011.04.052
PMCID: PMC3152651  PMID: 21676452
ovarian cancer; SLPI; MMP-9; metalloproteinase
5.  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.
doi:10.1038/modpathol.2011.102
PMCID: PMC3175277  PMID: 21666687
CLL; SLL; histiocytic sarcoma; interdigitating dendritic cell sarcoma; clonal; transformation; transdifferentiation
6.  Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma 
BMC Research Notes  2012;5:73.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1756-0500-5-73
PMCID: PMC3283499  PMID: 22280838
7.  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
8.  Opposing regulation of the Il17 locus through direct, reciprocal actions of STAT3 and STAT5 
Nature immunology  2011;12(3):247-254.
Summary
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.
doi:10.1038/ni.1995
PMCID: PMC3182404  PMID: 21278738
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
10.  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.
doi:10.1128/IAI.00620-10
PMCID: PMC2976364  PMID: 20732996
11.  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
12.  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
14.  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
15.  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
16.  Increased matrix metalloproteinase activation in esophageal squamous cell carcinoma 
Background
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.
Methods
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).
Results
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.
Conclusions
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.
doi:10.1186/1479-5876-8-91
PMCID: PMC2958908  PMID: 20920372
17.  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
18.  Approaching Solid Tumor Heterogeneity on a Cellular Basis by Tissue Proteomics Using Laser Capture Microdissection and Biological Mass Spectrometry 
Journal of proteome research  2009;8(5):2310-2318.
The purpose of this study was to examine solid tumor heterogeneity on a cellular basis using tissue proteomics that relies on a functional relationship between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS). With the use of LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lymph node containing breast carcinoma metastasis. Six specimens ∼50 000 cell each (three from tumor proper and three from tumor stroma) were collected by LCM. Specimens were processed directly on LCM caps, using sonication in buffered methanol to lyse captured cells, solubilize, and digest extracted proteins. Prepared samples were analyzed by LC/MS/MS resulting in more than 500 unique protein identifications. Decoy database searching revealed a false-positive rate between 5 and 10%. Subcellular localization analysis for stromal cells revealed plasma membrane 14%, cytoplasm 39%, nucleus 11%, extracellular space 27%, and unknown 9%; and tumor cell results were 5%, 58%, 26%, 4%, and 7%, respectively. Western blot analysis confirmed specific linkage of validated proteins to underlying pathology and their potential role in solid tumor heterogeneity. With continued research and optimization of this method including analysis of additional clinical specimens, this approach may lead to an improved understanding of tumor heterogeneity, and serve as a platform for solid tumor biomarker discovery.
doi:10.1021/pr8009403
PMCID: PMC2858576  PMID: 19284784
laser capture microdissection (LCM); mass spectrometry (MS); solid tumor heterogeneity
19.  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
20.  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

Results 1-20 (20)