Dataset Information

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma

ABSTRACT: We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to unmanipulated control animals and to animals that underwent an incomplete Solt-Farber protocol to activate hepatic progenitor cells. Regions of formalin-fixed, paraffin-embedded tissue were obtained by laser capture microdissection (LCM). Proteomic analysis yielded 11,070 unique peptides representing 2,227 proteins. Quantitation of the peptides showed increased abundance of known HCC markers (e.g., glutathione S-transferase-P, epoxide hydrolase, 6 others) and potential markers (e.g., aflatoxin aldehyde reductase, glucose 6-phosphate dehydrogenase, 10 others) in foci from placebo-treated and rapamycin-treated rats. Peptides derived from cytochrome P450 enzymes were generally reduced. Comparisons of the rapamycin samples to normal liver and to the progenitor cell model indicated that rapamycin attenuated a loss of differentiation relative to placebo. We conclude that early administration of rapamycin in the Solt-Farber model not only inhibits the growth of pre-neoplastic foci but also attenuates the loss of differentiated function. In addition, we have demonstrated that the combination of LCM and mass spectrometry-based proteomics is an effective approach to characterize focal liver lesions.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Hepatocyte, Liver

DISEASE(S): Hepatocellular Carcinoma

SUBMITTER: Arthur Salomon

LAB HEAD: Philip A. Gruppuso

PROVIDER: PXD005845 | Pride | 2018-10-26

REPOSITORIES: Pride

ACCESS DATA

Publications

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma.

Adebayo Michael Adeola O AO Ahsan Nagib N Zabala Valerie V Francois-Vaughan Heather H Post Stephanie S Brilliant Kate E KE Salomon Arthur R AR Sanders Jennifer A JA Gruppuso Philip A PA

Oncotarget 20170401 16

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks af ...[more]

PMID: 28199961

Similar Datasets

Project description:Glioblastoma multiforme (GBM), the most advanced form of a large subset of brain tumors collectively known as glioma, is the most aggressive and invasive type of brain tumor. Patients usually experience a median survival range of 9 to 12 months. GBMs are extremely difficult to manage because of the tumor cellsâ tendency to migrate and pervade into adjacent tissues. Surgical resection of GBMs generally only slows disease progression because any remaining tumor cells proceed to migrate through brain tissues and reform a new tumor mass. It is hypothesized that the invasive phenotype of these tumor cells may be attributable to unique gene expression. Stationary core and invasive rim tumor cells were collected separately by laser capture microdissection (LCM) from 19 biopsy samples. Identification of differentially expressed genes in the tumor core and invasive rim can give valuable insight to the genes and pathways potentially involved with the invasive phenotype. This information can then be used to generate possible biomarkers, diagnostic markers, or drug targets. Human glioblastoma tumor samples were obtained from patients who underwent primary therapeutic brain tumor resection. All specimens were collected and submitted to the study under institutional review board approved protocols. None of the patients had been subjected to chemotherapy or radiotherapy prior to resection, in order to avoid genetic signatures that are due to exposure to alkylating agents and/or ionizing radiation. All specimens were verified as GBMs by a neuropathologist. Tumors are embedded in OCT, then sectioned in a cryostat at -20C, to a thickness of 8-10 um and placed onto HistoGene slides. Sections to be microdissected are removed from the -80C fixed and stained with an abbreviated Hematoxylin and Eosin protocol. Two thousand tumor core and invasive cells are dissected onto separate caps using the PixCell II instrument using CapSureâ¢ Macro LCM Caps. The CapSureâ¢ Macro LCM Caps LCM 0211 should be used with the AutoPix instrument. Cells in the tumor core are identified by nuclear atypia and size and captured using the larger spot sizes. Tumor cells immediately adjacent to necrotic areas, cortical areas, cells with small regular nuclei, endothelial and blood cells should be avoided. Individual white matter invading GBM cells can be identified by means of their nuclear atypia and heteropyknotic staining, which is consistent with that of the cells within the tumor core. They should be microdissected using the 7.5 um laser spot size and the initial power settings recommended by the PixCell II manual.Â After microdissection all harvested material should immediately be lysed on the cap by applying the lysis buffer (XB) from the PicoPure RNA Isolation Kit according to the PicoPure protocol and stored -80C. Cell populations harvested on different caps can be pooled at the time of RNA isolation. RNA integrity is varified by identification of distinct 28S and 18S ribosomal bands with an Agilent Bioanalyzer using the RNA 6000 Nano LabChip kit. Total RNA was isolated from 2000 LCM cells (to ensure at least 500 ng total RNA) using the PicoPure RNA solation Kit, following manufacturers protocol. mRNA is reverse transcribed with the RiboAmp RNA Amplification kit. 500 ng total RNA is amplified with the RiboAmp RNA Amplification kit, following manufacturerâs instructions. The total yield that can be expected falls between 30 and 60 Âµg copy RNA. The size of the copy RNA should be verified by gel electrophoresis. Approximately 500ng of copy RNA can be separated on a 1% agarose gel in TAE buffer. A smear of amplified material should be seen between 200 and 3000bp. Six Âµg amplified RNA are labeled in a RT with SuperScriptIII in the presence of dUTP Cy5 utilizing 6 Âµg random hexamers as primers. Universal reference RNA is amplified one round in the same manner and labeled with Cy3 dUTP. Labeled cDNA is hybridized overnight onto cDNA microarray. Following hybridization, arrays are washed, scanned and quantitated with the Axon GenePix 4000 microarray reader (Axon Instruments). Gene expression results are analyzed using GeneSpring (Silicon Genetics) software. Intensity dependent normalization is applied, where the ratio is reduced to the residual Lowess fit of the intensity versus ratio curve. The measured intensity of each gene is divided by its reference channel (signal from the universal reference RNA) in each sample. When the reference channel is below 10, the data point is considered uninformative. The ratios (sample over reference) for the tumor core experiments and invasive rim experiments are averaged and compared. Genes that are more than two-fold differentially regulated in the majority of the matched core/invasive rim sets are selected.

Project description:Many human cancers present as multi-focal lesions. Understanding the clonal origin of multi-focal cancers is of both etiological and clinical importance. The molecular basis of multi-focal prostate cancer has previously been explored using only a limited number of isolated markers and, although independent origin is widely believed, the clonal origin of multi-focal prostate cancer is still debatable. We attempted to address clonal origin using a genome-wide copy-number analysis of individual cancer and high-grade prostatic intraepithelial neoplasia (HGPIN) lesions. Using Affymetrix array 6.0 copy-number analysis, we compared the genomic changes detected in 54 individual cancer and HGPIN lesions, isolated from 20 clinically localized prostate cancer cases. Identical genomic copy-number changes, shared by all same-case cancer foci, were detected in all 16 informative multi-tumor cases. In addition, both HGPIN lesions in the two multi-HGPIN cases available shared identical genomic changes. Commonly known genomic alterations, including losses at 6q15, 8p21.3-8p21.2, 10q23.2-10q23.31, 13q21.31-13q21.32, 16q22.3, 16q23.2-16q23.3 and 21q22.2-21q22.3 regions and gain of 8q24.3 were the most frequently detected changes in this multi-focal prostate cancer study, occurring in all same-case foci in at least one case. Microarray data were confirmed by fluorescence in situ hybridization in selected foci. Our high-resolution genome-wide copy-number data suggest that many multi-focal cases derive from a single prostate cancer precursor clone and that this precursor may give rise to separate HGPIN foci, which through clonal expansion may progress to multi-focal invasive prostate cancer. These findings, which demonstrate the monoclonal origin of multi-focal prostate cancer, should significantly enhance our understanding of prostate carcinogenesis and potentially improve clinical management of the disease. Copy number analysis of Affymetrix SNP 6.0 array was performed for a total of 48 cancer and HGPIN lesions from 18 prostate cancer cases. All samples have case-matched normal controls. PL = high grade PIN from left side, PR = high grade PIN from right side, PM = high grade PIN from middle of the tissue, TL = tumour from left side, TR = tumour from right side.

Dataset Information

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma

Publications

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure