Project description:Analysis of purified immune and breast tumor cells from three major compartments where cancer and immune cells interact: primary tumor, tumor draining lymph nodes (tumor invaded or tumor free), and peripheral blood. The results suggests that node-positive patients’ immune regulation and functionality is down-regulated compared to node-negative patients. CD45+ Immune and ESA+ tumor cells were purified from breast cancer patients' primary tumor, tumor-draining lymph node, and peripheral blood (ficoll) and placed onto Agilent microarrays using the dye-swap method. A universal human reference was used as a reference for the patient samples.
Project description:Expression data from 4T1 subclones derived from mammary fat pad tumors (MFP), axillary lymph node tumors (AxLN), and axillary lymph node-derived lung metastases (AxLN-LuM). In parallel, expression data, in the same subclones, of tail vein-derived (TV) lung metastases. The mechanism of how lymph node metastases seed distant metastases is unknown. We used the 4T1 breast cancer cell line, which is an immune competent model of triple negative breast cancer and spontaneously metastasizes in balb/c mice. 4T1-GFP/fLuc cells were injected into MFP to form tumors and 4T1-mCherry/rLuc cells were injected into axillary lymph nodes to form tumors and then allowed to metastasize to lung. TV cells were allowed to metastasize in the lung. Cells were harvested at different time intervals after the injection. Tumors were extracted, dissociated, and then expanded in vitro to obtain MFP, AxLN, AxLN-LuM and TV-LuM subclones isolated after different time lags with respect to the injection.
Project description:To determine the influence of primary tumors on pre-metastatic lymph nodes, we have employed whole genome microarray expression profiling as a discovery platform to identify gene signatures of B cells from tumor-draining lymph nodes, compared with normal lymph nodes. We subcutaneously inoculated C57BL/6 mice with the 4T1 mammary carcinoma. Two weeks later, tumor-draining lymph nodes were dissociated and B cells (CD19+) were sorted. Lymph nodes B cells from normal mice without tumor bearing were set as controls.
Project description:To determine the influence of primary tumors on pre-metastatic lymph nodes, we have employed whole genome microarray expression profiling as a discovery platform to identify gene signatures of stromal cells from tumor-draining lymph nodes, compared with normal lymph nodes. We subcutaneously inoculated C57BL/6 mice with the 4T1 mammary carcinoma. Two weeks later, tumor-draining lymph nodes were dissociated and stromal cells (CD45-) were sorted. Lymph nodes stromal cells from normal mice without tumor bearing were set as controls.
Project description:To determine the different gene signatures between B lymphocytes from tumor draining lymph node (DLN) and normal lymph node (NLN), we have employed gene microarray as a discovery platform to identify gene signatures of tumor-educated B cells in DLN from tumor-bearing mice, taking NLN from normal mice as a control. We subcutaneously inoculated Balb/c mice with breast cancer cell line 4T1. Two weeks later, DLN was harvest and B cells were purified as descript in “treatment protocol”. From gene microarray, we found that B cells in DLN showed quite different transcript profiles from that in NLN.
Project description:Metastasis to lymph nodes is an early and prognostically important event in the progression of many human cancers, and is associated with expression of vascular endothelial growth factor-D (VEGF-D). Changes to lymph node vasculature occur during metastasis, and may establish a metastatic niche capable of attracting and supporting tumor cells. We used microarrays to characterise the molecular profiles of endothelial cells from lymph nodes draining metastatic (VEGF-D-overexpressing) and non-metastatic tumors, and to identify differentially-expressed genes that might have therapeutic or prognostic potential. Draining lymph nodes of metastatic (VEGF-D-overexpressing) or non-metastatic tumors were pooled from 1-5 mice and enzymatically digested. Lymph nodes draining metastatic tumors were included for the analysis only if macroscopically enlarged, indicating the presence of metastatic cells. After digestion, tumor cells and leukocytes were depleted via immunomagnetic selection, and the resulting lymph node stromal cells were cultured briefly. Podoplanin was then used as a positive immunomagnetic selection marker to enrich for lymphatic and other endothelial cells in the lymph node. RNA was isolated from biological duplicate lymph node endothelial cell (LN EC) preparations and analysed by microarray.
Project description:Analysis of purified immune and breast tumor cells from three major compartments where cancer and immune cells interact: primary tumor, tumor draining lymph nodes (tumor invaded or tumor free), and peripheral blood. The results suggests that node-positive patients’ immune regulation and functionality is down-regulated compared to node-negative patients.
Project description:Dendritic cells (DCs) express high levels of PD-L1 in the tumor microenvironment and draining lymph nodes. Here, we explore the roles of PD-L1 signaling during immunogenic chemotherapy. DC-conditional PD-L1 knockout mice were inoculated with MC38-OVA tumors and treated with doxorubicin. T cells were isolated from draining lymph node for single cell RNA sequencing.
Project description:Lymph node involvement is a major prognostic variable in breast cancer. Whether the molecular mechanisms that drive breast cancer cells to colonize lymph nodes are shared with their capacity to form distant metastases is yet to be established. In a transcriptomic survey aimed at identifying molecular factors associated with lymph node involvement of ductal breast cancer, we found that luminal differentiation, assessed by the expression of estrogen receptor (ER) and/or progesterone receptor (PR) and GATA3, was only infrequently lost in node-positive primary tumors and in matched lymph node metastases. The transcription factor GATA3 critically determines luminal lineage specification of mammary epithelium and is widely considered a tumor and metastasis suppressor in breast cancer. Strong expression of GATA3 and ER in a majority of primary node-positive ductal breast cancer was corroborated by quantitative RT-PCR and immunohistochemistry in the initial sample set, and by immunohistochemistry in an additional set from 167 patients diagnosed of node-negative and positive primary infiltrating ductal breast cancer, including 102 samples from loco-regional lymph node metastases matched to their primary tumors, as well as 37 distant metastases. These observations suggest that loss of luminal differentiation is not a major factor driving the ability of breast cancer cells to colonize regional lymph nodes. The transcriptomic study comprises 16 samples from Lymph node metastasis from infiltrating ductal breast carcinoma, 18 samples from Primary node-positive infiltrating ductal,7 samples from Primary node-negative infiltrating ductal and 3 samples from Unaffected lymph node were included. Their RNA was isolated and prepared for hybridization to human Affymetrix GeneChip arrays.
Project description:Metastasis is responsible for the majority of deaths in a variety of cancer types, including breast cancer. Although several factors or biomarkers have been identified to predict the outcome of patients with breast cancer, few studies have been conducted to identify metastasis-associated biomarkers. Quantitative iTRAQ proteomics analysis was used to detect differentially expressed proteins between lymph node metastases and their paired primary tumor tissues from 23 patients with metastatic breast cancer. Immunohistochemistry was performed to validate the expression of two upregulated (EpCAM, FADD) and two downregulated (NDRG1, αB-crystallin) proteins in 190 paraffin-embedded tissue samples. These four proteins were further analyzed for their correlation with clinicopathological features in 190 breast cancer patients. We identified 637 differentially regulated proteins (397 upregulated and 240 downregulated) in lymph node metastases compared with their paired primary tumor tissues. Furthermore, bioinformatics analysis using GEO profiling confirmed the difference in the expression of EpCAM between metastases and primary tumors tissues. Two upregulated (EpCAM, FADD) and two downregulated (NDRG1, αB-crystallin) proteins were associated with the progression of breast cancer. Obviously, EpCAM plays a role in the metastasis of breast cancer cells to the lymph node. We further identified αB-crystallin as an independent biomarker to predict lymph node metastasis and the outcome of breast cancer patients.