Project description:The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naive neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC). In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-a, IL-1-a/b), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages. This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations. Various types of myeloid cells have been shown to promote tumor progression by direct immune suppression and by production of angiogenic factors, matrix-degrading enzymes, or growth factors. In untreated tumors, neutrophils have been reported to produce angiogenic factors and matrix-degrading enzymes, support the acquisition of a metastatic phenotype, and suppress the anti-tumor immune response. Neutrophils, like all other leukocytes, move into tissues from the blood under the influence of specific chemokines (e.g. KC/CXCL-1, MIP-2a/CXCL-2 and GCP-2/CXCL-6), cytokines (e.g. TNFa and IFN-x), and cell adhesion molecules located on their own surface (e.g. CD11b) and on the surface of endothelial cells (e.g. selectins, ICAM-1 and PECAM-1). When they traffic into tumors, they are referred to as TAN. In mice, TAN can be defined by the specific surface markers CD11b and Ly6G with low expression of macrophage markers such as F4/80. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immune suppressive cells that are produced excessively in cancer. They comprise at least two subsets -granulocytic (Ly6G+, G-MDSC) and monocytic cells (Ly6C+, M-MDSC), potentially with different immunosuppressive properties. It has been previously shown that MDSC can enter tumors and differentiate to mature macrophages (TAM) or neutrophils (TAN). However, since no definitive markers have been established, it is unknown whether intratumoral N2 neutrophils (N2 TAN) are granulocytic MDSC from spleen that are attracted to the tumor or if they are typical blood-derived neutrophils that are then converted to an N2 phenotype by the tumor microenvironment, specifically by the high local concentrations of TGF-b. The purpose of this study was to use a transcriptomics approach to gain further information about TANs by comparing the RNA profile of these cells to naive bone-marrow neutrophils (NN) and to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC). We examined which pathways and gene-groups varied amongst these 3 populations of neutrophils and performed a detailed analysis on pathways related to the main functions of neutrophils, such as respiratory burst, granule proteins, phagocytosis, apoptosis, structural genes, antigen presentation and specific immune effects. Our data defines TAN as a unique population of neutrophils, quite distinct from both NN and G-MDSC.

Project description:Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2-neu) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+. We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER+ cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs may contribute to the more invasive properties and unfavorable prognosis of Her2+ breast cancer. These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer. We isolated CAFs from twenty primary breast cancer samples representing three main subtypes (ER+ (n=7), TNBC (n=7), Her2+ (n=6)) and performed gene expression profile analyses on RNA isolated from these early passage CAFs. Those samples were done in two batches with 4 samples repeated in both batches. One TNBC sample was found to be an outlier and not used in the analysis.

Project description:Elevated lipocalin-2 (Lcn2) has been observed in multiple human cancers, but its biological roles remain unclear. Our purpose in this study was to investigate whether Lcn2 is involved in hepatocellular carcinoma (HCC) development via its involvement in invasion and metastasis. Using unsupervised hierarchical clustering analysis of tissue samples, we identified preferential expression of Lcn2 (>2-fold change) in the tumor group (differentiated group) versus the non-tumor group (liver cirrhosis group). LCN2 immunoreactivity was positively correlated with TNM stage (r = 0.194, P = 0.020), but not with differentiation or recurrence of HCC. Over-expression of Lcn2 was observed in HLK-2, HKK-2, and HLK-5 HCC cells. Knock-down of Lcn2 by shRNA in HKK-2 cells was correlated with the down-regulation of E-cadherin, CK-8, CK-18, and desmoplakin I/II (DesI/II) and the up-regulation of N-cadherin, vimentin (VIM), and fibronectin (FN), which are markers of the epithelial-mesenchymal transition (EMT) associated with tumor progression. The characteristics of EMT were reversed by adenoviral transduction of Lcn2 into SH-J1 cells. EGF or TGF-β treatment resulted in downregulation of Lcn2 with a concomitant change in EMT. Stable Lcn2 expression in HCC cells reduced the expression of the transcription factor Twist1, resulting in the inhibition of cell proliferation, migration, and invasion in vitro, and suppressed tumor growth and metastatic ability in a mouse model. These findings suggest that Lcn2 can reverse the EMT in HCC through transcriptional suppression of Twist 1. Thus, Lcn2 is a candidate metastasis suppressor and a potential therapeutic target for HCC. Unsupervised hierarchical clustering separated the samples into two main groups: a non-tumor group (NT, n = 40) and a HCC group (HCC, n = 40), and into two subgroups: a liver cirrhosis group (LC, n = 20) and a differentiated HCC group (difHCC, n = 20). Lcn2 was one of the unique genes with a two-fold or greater difference in expression from the mean with P < 0.01 based on the t-test for hierarchical clustering analysis.

Project description:Background: It is a challenge to identify those patients who, after undergoing potentially curative treatments for hepatocellular carcinoma, are at greatest risk of recurrence. Such high-risk patients could receive novel interventional measures. An obstacle to the development of genome-based predictors of outcome in patients with hepatocellular carcinoma has been the lack of a means to carry out genomewide expression profiling of fixed, as opposed to frozen, tissues. Methods: We aimed to demonstrate the feasibility of gene-expression profiling of more than 6000 human genes in formalin-fixed paraffin-embedded tissues. We applied the method to tissues from 307 patients with hepatocellular carcinoma, from four series of patients, to discover and validate a gene-expression signature associated with survival. Results: The expression-profiling method for formalin-fixed, paraffin-embedded tissue was highly effective: samples from 90% of the patients yielded data of high quality, including samples that had been archived for more than 24 years. Gene-expression profiles of tumor tissue failed to yield a significant association with survival. In contrast, profiles of the surrounding nontumoral liver tissue were highly correlated with survival in a training set of 82 Japanese patients, and the signature was validated in tissues from an independent group of 225 patients from the United States and Europe (p = 0.04). Conclusions: We have demonstrated the feasibility of genomewide expression profiling of formalin-fixed, paraffin-embedded tissues and have shown that a reproducible gene-expression signature correlating with survival is present in liver tissue adjacent to the tumor in patients with hepatocellular carcinoma. Keywords: Hepatocellular carcinoma, Expression array, Illumina, Signatures, Outcome prediction Training cohort: 80 tumor and 82 non-tumor liver tissues surgically resected from patients with hepatocellular carcinoma (HCC); Validation cohort: 225 non-tumor liver tissues surgically resected from patients with HCC. Clinical data has been withheld from GEO due to privacy concerns.

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Resection of Non-Small Cell Lung Cancers Reverses Tumor-Induced Gene Expression Changes in the Peripheral Immune System

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