Gene expression signatures for murine stromal cells from tumor-draining lymph nodes
ABSTRACT: 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. Overall design: Primary tumor induced gene expression in stromal cells from tumor-draining lymph nodes was measured at 2 weeks after tumor inoculation subcutaneously. Lymph nodes stromal cells from normal mice without tumor bearing were set as controls.
INSTRUMENT(S): Agilent-074809 SurePrint G3 Mouse GE v2 8x60K Microarray [Probe Name version]
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. Overall design: Primary tumor induced gene expression in B cells from tumor-draining lymph nodes was measured at 2 weeks after tumor inoculation subcutaneously. Lymph node B 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. Overall design: Examination of different gene signatures between B cells from tumor draining lymph node and normal lymph node
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:Lymph node (LN) stromal cells, particularly fibroblastic reticular cells (FRCs), provide critical structural support and regulate immunity, tolerance and transport properties of LNs. In many tumors, LN metastasis is predictive of poor prognosis, however, stromal contribution to the evolving microenvironment of tumor draining LNs (TDLN) remains poorly understood. Here we present comparative transcriptional data of resting and TDLN FRCs after different time points of tumor drainage. FRCs were isolated from lymph nodes and FACS sorted based on the expression of CD45-, CD31- and PDPN+ Overall design: FRCs were pooled per animal of 2 brachial LNs and gene array analysis was performed on triplicates
Project description:Foxp3+ regulatory T cells (Tregs) are critical for the maintenance of peripheral tolerance to self-antigens, and to non-self commensal microbiota or food-born antigens. Accordingly, gut-draining mesenteric lymph nodes (mLN) are superior in peripheral de novo induction of Foxp3+ Tregs. Moreover, it has been recently shown that this can be partly attributed to lymph node stromal cells, including fibroblastic reticular cells (FRCs) dominating the T cell zone. To investigate molecular details of peripheral tolerance, immortalized FRCs (iFRCs) of mLN and skin-draining peripheral LNs (pLN) have been generated. Differential de novo Treg induction was observed not only in the presence of mLN- and pLN-iFRCs, but also when mLN- and pLN-iFRC-derived supernatant was added to the in vitro cultures. Transcriptional profiling of iFRCs from mLN and pLN revealed differential expression of genes encoding secreted molecules that could be responsible for the tolerogenic phenotype of mLN iFRCs. Taken together, our findings represent a novel way of intercellular communication between FRCs and T cells. Overall design: Identification of differentially expressed genes in immortalized fibroblastic reticular cells from mesenteric or skin-draining lymph nodes of specific-pathogen-free housed mice.
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in heaves-affected horses versus matched controls. Mediastinal (pulmonary-draining) lymph nodes were sterilely obtained from affected and control horses, dissected, and frozen at -80oC. RNA was extracted from these tissues for downstream applications. These experiments utilized a commercially available Agilent horse array that featured >43,000 probes on a 4x44k array format. Mediastinal lymph node RNA from seven heaves-affected horses was compared to matching RNA from healthy, normal control horses.
Project description:mRNA vaccines are emerging as a powerful vaccine platform as they are well-tolerated and scalable. Modified non-replicating mRNA encoding Influenza hemagglutinin and encapsulated in lipid nanoparticles (LNP) induced robust antibody and CD4 T cell responses after intramuscular or intradermal delivery in rhesus macaques. We investigated the local innate immune responses modulating such vaccine-induced immunity at the sites of immunization (skeletal muscle and skin) and their draining lymph nodes (LNs). Rapid mobilization of antigen presenting cells was found at the LNP/mRNA-injection sites and LNs. Dendritic cells efficiently internalized the LNPs, translated the mRNA cargo and upregulated co-stimulatory molecules. In addition, several type I interferon-inducible genes were expressed at the immunization sites and draining LNs. The innate immune activation was transient and resulted in priming of antigen-specific CD4+ T cells exclusively in the vaccine-draining LNs. Collectively, mRNA-based vaccines induce type I interferon-polarized innate immunity and antigen production by antigen presenting cells, which resulted potent vaccine-specific responses. Overall design: Two groups of rhesus macaques (n=4/group) receiving either intramuscular (I.M.) or intradermal (I.D.) injection of lipid nanoparticle (LNP)-encapsulated mRNA vaccine plus a saline control injection given at a different site on the same animal. Injection site biopsies and draining lymph nodes from I.M. and I.D. group were collected for gene expression analysis.
Project description:We employed single-cell RNA sequencing to understand stromal changes in murine melanomas and draining lymph nodes at single cell resolution at different points of tumour development.