Abstract 209: Laser capture microdissection of metastatic colon adenocarcinoma samples enables RNA sequencing analysis of tumor and stromal compartments
Authors: Littlewood Stephanie , Krumbach Rebekka , Corr Gemma , Collins Laura , Carbone Francesco , Gambardella Adriana , Hickman Emma 
Published: August 2018

Abstract
Gene expression data from whole tumor samples represent averaged expression of all the tumor cells, stromal cells and normal adjacent cells present in a given sample. Identification of colon tumor-specific genes may be hampered by contamination from surrounding normal adjacent tissue or stroma. In order to better understand gene expression derived specifically from colon tumor cells we performed laser-capture microdissection to obtain separate samples of tumor and stromal/normal adjacent tissue. We generated data from 12 samples of well-differentiated colon adenocarcinoma metastasized to the liver, lung or ovary. Snap-frozen samples were sectioned, stained with an RNA-preserving haematoxylin and eosin staining procedure and tumor cell and stromal cell containing areas were defined with the support of a pathologist. Tumor and non-tumor samples (containing both stroma and normal adjacent tissue) were captured separately and total RNA isolated from these alongside whole (non-dissected) sections. RNA-sequencing was performed with Kapa HyperPrep Riboerase library preparation and Illumina sequencing at UCL Genomics, London, UK and FPKM values were calculated. For tumors metastasized to liver, analysis of liver-specific genes (including ALB, HP and ORM1) showed that in 6/9 samples these genes were present in the whole section, but absent in the microdissected tumor cells. Some samples (3/9) showed very low expression of liver-specific markers in either the whole section or the dissected non-tumor compartment, indicating low levels of normal adjacent tissue contamination, confirmed by pathology analysis. Similarly in tumors metastasized to the lung, we found absence of lung-specific genes including surfactants in the microdissected tumor cells (2/2 samples), despite high expression in whole sections and the non-tumor compartment. We were able to demonstrate localization of known and novel colon tumor genes in the tumor cell compartment. For example, a cancer testis antigen expressed in 13% of colon adenocarcinoma samples in the TCGA dataset was localized specifically to the tumor compartment in 3/12 microdissected samples and was absent from the non-tumor fraction. We were also able to identify genes derived from immune infiltrates and cancer-associated fibroblasts in the non-tumor compartment. This unique dataset can be used to identify genes expressed in metastatic colon adenocarcinoma without interference from stromal cell, immune infiltrate or normal adjacent tissue.
Citation Format: Stephanie Littlewood, Rebekka Krumbach, Gemma Corr, Laura Collins, Francesco Carbone, Adriana Gambardella, Emma Hickman. Laser capture microdissection of metastatic colon adenocarcinoma samples enables RNA sequencing analysis of tumor and stromal compartments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 209.