The Inflammatory Microenvironment in Colorectal Neoplasia

Abstract

Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. Inflammatory activity within the stroma of invasive colorectal tumours is known to be a key predictor of disease activity with type, density and location of immune cells impacting on patient prognosis. To date, there has been no report of inflammatory phenotype within pre-malignant human colonic adenomas. Assessing the stromal microenvironment and particularly, inflammatory activity within colorectal neoplastic lesions is central to understanding early colorectal carcinogenesis. Inflammatory cell infiltrate was assessed by immunohistochemistry in paired colonic adenoma and adjacent normal colonic mucosa samples, and adenomas exhibiting increasing degrees of epithelial cell dysplasia. Macrophage phenotype was assessed using double stain immunohistochemistry incorporating expression of an intracellular enzyme of function. A targeted array of inflammatory cytokine and receptor genes, validated by RT-PCR, was used to assess inflammatory gene expression. Inflammatory cell infiltrates are a key feature of sporadic adenomatous colonic polyps with increased macrophage, neutrophil and T cell (specifically helper and activated subsets) infiltration in adenomatous colonic polyps, that increases in association with characteristics of high malignant potential, namely, increasing degree of cell dysplasia and adenoma size. Macrophages within adenomas express iNOS, suggestive of a pro-inflammatory phenotype. Several inflammatory cytokine genes (CXCL1, CXCL2, CXCL3, CCL20, IL8, CCL23, CCL19, CCL21, CCL5) are dysregulated in adenomas. This study has provided evidence of increased inflammation within pre-malignant colonic adenomas. This may allow potential mechanistic pathways in the initiation and promotion of early colorectal carcinogenesis to be identified.

Custom Design of a GeXP Multiplexed Assay Used to Assess Expression Profiles of Inflammatory Gene Targets in Normal Colon, Polyp, and Tumor Tissue

“…12 The gene targets were identified using an inflammatory cytokine-targeted macroarray (Oligo-GEArray gene filter, Human Inflammatory Cytokines and Receptors, OHS-011, Superarray Bioscience, Frederick, MD), followed by single-plex SYBR real-time PCR assays using RNA extracted from human normal colon, polyp, and tumor biopsy samples 12 (see Supplemental Tables S1-S3 at http://jmd.amjpathol.org/). The gene target accession numbers were loaded into the Genome Lab GeXP database, together with reference genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-2-microglobulin (B2M)] to design suitable gene-specific primers for reverse transcription and PCR amplification (see Table 2) using GenomeLab eXpress Designer GeXP Software (Beckman Coulter).…”

“…The gene-specific primers were designed to generate PCR amplicons that differ in size by 4 to 7 bp, ranging in size from 137 bp to 325 bp ( Table 2). The 14 gene targets were incorporated, together with three reference genes that included GAPDH and B2M (both used previously as reference genes for normalization and calculation of fold changes in single-plex real-time PCR assay of the gene targets) 12 (see Supplemental Tables S2 and S3 at http://jmd.amjpathol.org) and an external synthetic reference control transcript Kan (supplied with the GeXP assay kit, Beckman Coulter) used to spike each reaction. Two reference genes were selected for normalization as recommended for relative quantitative gene expression analysis.…”

“…GeXP eXpress Analysis software was used to perform peak binning and normalize peak area values against the incorporated reference genes (B2M and GAPDH). Both B2M and GAPDH have been used in previous gene expression analysis studies because these transcripts previously were shown to have no significant variation of expression in a macroarray analysis of total RNA extracted from normal, polyp, or tumor colon specimens 12 (see Supplemental Table S1 at http://jmd.amjpathol.org). Attenuation of signals beyond the linear range was achieved by reducing reverseprimer concentration according to the manufacturer's instructions.…”

“…The selected targets were identified in our laboratory by a gene array study of inflammatory cytokine expression in human normal, polyp, and tumor tissue corroborated by singleplex, quantitative, real-time PCR. 12 A number of the gene targets selected are associated with inflammation in the This work was supported by the Scottish Government.…”

Epigenetics: Unravelling the cancer code

“…In colorectal cancer, for example, he discovered there is an inflammatory environment in and around premalignant lesions. Within this region he found nine differentially expressed genes linked with inflammation, including those responsible for IL-8 and the CXCL-family of cytokines 5 .…”