Abstract 1576: Molecular profiling of the desmoplastic reaction within the colorectal tumor microenvironment using the nCounter® platform
Authors: Nearchou Ines P. , Ao Tadakazu , Mochizuki Satsuki , Warren Sarah , Harashima Hirofumi , Caie Peter D. , Ueno Hideki 
Published: September 2020

Abstract
Background & Objectives: Desmoplastic reaction (DR) refers to the presence of fibrosis at the tumor's invasive margin within the tumor microenvironment (TME). DR has been associated with cancer aggressiveness across various studies in colorectal cancer (CRC). Previously, we have suggested a histological classification of DR into 3 prognostic categories; mature, intermediate or immature, based on the presence of keloid-like collagen and myxoid stroma at the extramural desmoplastic front, and we have shown that survival outcome differs significantly among the DR categories. Although DR is significantly associated with patient prognosis, its molecular background remains unclear. NanoString nCounter® analysis platform enables the multiplex gene expression analysis with up to 800 genes from a single mRNA sample. The main focus of our study is the evaluation of the molecular profiles that may drive the development of the DR types.
Methods: Four CRC patients of each DR category were included in this study (n = 12). Surgical specimens were split into two halves, one of which was frozen and the other was fixed in formalin. DR was assessed on slides of the formalin-fixed tissues whereas the fresh frozen tissues samples were used for the molecular profiling. Four µm thick sections from the formalin-fixed samples were stained with hematoxylin and eosin. The extramural tumor front within the slides was assessed for DR classification. Samples containing myxoid stroma with area greater than a microscopic field of a ×40 objective lens were classified as immature. Samples with absence of size-significant myxoid stroma and presence of keloid-like collagens were classified as intermediate. Samples were classified as mature if neither size-significant myxoid stroma nor keloid-like collagens were present. Ten µm thick sections from the fresh frozen samples were stained with hematoxylin. Two regions of interest per tissue sample were selected: a) fibrotic stromal regions and b) tumor areas adjacent to the fibrotic stroma. A total area of 5mm2 was microdissected from each region of interest. RNA was extracted from each sample and over 1400 gene expressions were measured for each sample using the nCounter® PanCancer Progression and Immune Profiling panels. Nanostring nSolver™ analysis software was used for the data analysis.
Results: Genes associated with cell cycle and function, epithelial to mesenchymal transition and metastasis showed significantly different expression among the stromal and tumor regions of the 3 DR categories. The distribution of immune cells within the TME was also shown to be significantly different between the DR types. Although differential gene expression was observed among all the DR classes, the immature class had the most distinct pattern of gene expression compared to the other two classes.
Conclusion: This is to our knowledge the first study reporting differences in gene expression between mature, intermediate and immature DR types.
Citation Format: Ines P. Nearchou, Tadakazu Ao, Satsuki Mochizuki, Sarah Warren, Hirofumi Harashima, Peter D. Caie, Hideki Ueno. Molecular profiling of the desmoplastic reaction within the colorectal tumor microenvironment using the nCounter® platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1576.