An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework’s potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.

[1]  Sashwati Roy,et al.  Laser capture microdissection: Big data from small samples. , 2015, Histology and histopathology.

[2]  W. Sommergruber,et al.  IGFBP7, a novel tumor stroma marker, with growth-promoting effects in colon cancer through a paracrine tumor–stroma interaction , 2014, Oncogene.

[3]  P. Butler,et al.  Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas , 2016, Journal of histotechnology.

[4]  G. V. van Muijen,et al.  Type I collagen expression contributes to angiogenesis and the development of deeply invasive cutaneous melanoma , 2007, International journal of cancer.

[5]  J. DeVoss,et al.  Analysis of neuronal gene expression with laser capture microdissection , 2002, Journal of neuroscience research.

[6]  B. Di Camillo,et al.  Measuring differential gene expression with RNA-seq: challenges and strategies for data analysis. , 2015, Briefings in functional genomics.

[7]  Yong Hou,et al.  Current Challenges in the Bioinformatics of Single Cell Genomics , 2013, Front. Oncol..

[8]  S. Makhzami,et al.  Efficient gene expression profiling of laser‐microdissected melanoma metastases , 2012, Pigment cell & melanoma research.

[9]  Ahmad M Khalil,et al.  Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens. , 2014, Lung cancer.

[10]  T. Deller,et al.  Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression , 2004, Journal of Neuroscience Methods.

[11]  Yixing Han,et al.  Advanced Applications of RNA Sequencing and Challenges , 2015, Bioinformatics and biology insights.

[12]  J. Warrington,et al.  Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis. , 2003, The Journal of molecular diagnostics : JMD.

[13]  L. Liotta,et al.  IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication. , 2000, Kidney international.

[14]  Y. Takeshima,et al.  Heterogeneous genetic alterations in ovarian mucinous tumors: application and usefulness of laser capture microdissection. , 2001, Human pathology.

[15]  Amaya Garcia Munoz,et al.  Targeting promiscuous heterodimerization overcomes innate resistance to ERBB2 dimerization inhibitors in breast cancer , 2019, Breast Cancer Research.

[16]  John M. Daly,et al.  ErbB2 Potentiates Breast Tumor Proliferation through Modulation of p27Kip1-Cdk2 Complex Formation: Receptor Overexpression Does Not Determine Growth Dependency , 2000, Molecular and Cellular Biology.

[17]  A. Munnich,et al.  Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions. , 2008, RNA.

[18]  Xue Gao,et al.  Improvement of tissue preparation for laser capture microdissection: application for cell type-specific miRNA expression profiling in colorectal tumors , 2010, BMC Genomics.

[19]  Jinhai Yu,et al.  MiR-129-5p suppresses gastric cancer cell invasion and proliferation by inhibiting COL1A1. , 2018, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[20]  Rajyalakshmi Luthra,et al.  Implementation of next generation sequencing in clinical molecular diagnostic laboratories: advantages, challenges and potential , 2016 .

[21]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[22]  Z. Molnár,et al.  High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection , 2009, BMC Molecular Biology.

[23]  Wei Li,et al.  RSeQC: quality control of RNA-seq experiments , 2012, Bioinform..

[24]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[25]  D. Gao,et al.  Identification of COL1A1 as an invasion-related gene in malignant astrocytoma , 2018, International journal of oncology.

[26]  A. Conesa,et al.  Differential expression in RNA-seq: a matter of depth. , 2011, Genome research.

[27]  D. Carbone,et al.  Expression of angiomodulin (tumor‐derived adhesion factor/mac25) in invading tumor cells correlates with poor prognosis in human colorectal cancer , 2001, International journal of cancer.

[28]  J. Carpten,et al.  Translating RNA sequencing into clinical diagnostics: opportunities and challenges , 2016, Nature Reviews Genetics.

[29]  K. Tomczak,et al.  The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge , 2015, Contemporary oncology.

[30]  Alexander van Oudenaarden,et al.  Spatially resolved transcriptomics and beyond , 2014, Nature Reviews Genetics.

[31]  Falko Fend,et al.  Laser capture microdissection in pathology , 2000, Methods in enzymology.

[32]  Bao Zhang,et al.  Integrated analysis of microRNA regulatory network in nasopharyngeal carcinoma with deep sequencing , 2016, Journal of Experimental & Clinical Cancer Research.

[33]  B. Kaang,et al.  Cell type-specific gene expression profiling in brain tissue: comparison between TRAP, LCM and RNA-seq , 2015, BMB reports.

[34]  Meijuan Huang,et al.  Development and characterization of a novel method to analyze global gene expression profiles in endothelial cells derived from primary tissues , 2008, American journal of hematology.

[35]  Charles Giardina,et al.  Distinct Transcriptional Changes and Epithelial–Stromal Interactions Are Altered in Early-Stage Colon Cancer Development , 2016, Molecular Cancer Research.

[36]  J. Eberwine,et al.  Single-neuron isolation for RNA analysis using pipette capture and laser capture microdissection. , 2015, Cold Spring Harbor protocols.

[37]  T. B. Taylor,et al.  High-quality RNA from cells isolated by laser capture microdissection. , 2002, BioTechniques.

[38]  Fatih Ozsolak,et al.  RNA sequencing: advances, challenges and opportunities , 2011, Nature Reviews Genetics.

[39]  Hui-Rong Qian,et al.  Optimization and validation of small quantity RNA profiling for identifying TNF responses in cultured human vascular endothelial cells. , 2006, Journal of pharmacological and toxicological methods.

[40]  R. Sandberg,et al.  Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling , 2016, Nature Communications.

[41]  T. Mikkelsen,et al.  Insulin-like growth factor binding protein 7 mediates glioma cell growth and migration. , 2008, Neoplasia.

[42]  Camille Stephan-Otto Attolini,et al.  Stromal gene expression defines poor-prognosis subtypes in colorectal cancer , 2015, Nature Genetics.

[43]  S. Makhzami,et al.  Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection , 2010, BMC Cell Biology.

[44]  Ziding Feng,et al.  Improving the Quality of Biomarker Discovery Research: The Right Samples and Enough of Them , 2015, Cancer Epidemiology, Biomarkers & Prevention.

[45]  Hai-Jian Sun,et al.  Recent advances and current issues in single-cell sequencing of tumors. , 2015, Cancer letters.

[46]  D. Rosenberg,et al.  Targeted Transcriptional Profiling of Microdissected Biopsy Specimens Representing Early Colonic Neoplasia , 2016, Journal of cellular biochemistry.