Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles of mouse sentinel lymph nodes (SLNs) in xenotransplanted and non tumor bearing CB17 SCID.

ABSTRACT: Cutaneous melanoma first metastasizes into sentinel lymph nodes that control the lymphatic drain from the area of the primary tumor. This observation is used clinically for melanoma patients with primary melanomas thicker than 1mm (tumor stage ≥T2a), for these patients sentinel lymph node biopsy has become an important and routinely performed diagnostic procedure. The importance of sentinel node analysis is reflected by a significant better prognosis of melanoma patients with tumor free sentinel nodes compared to patients with metastatic sentinel nodes. Although intensively studied, not much is known about mechanisms responsible for the development of melanoma metastasis.To analyze gene expression in mouse SLNs of M24met tumor bearing animals as compared to tumor free control animals, SLNs were taken at different time points and analyzed for the presence of human M24met to classify SLNs into control, negative, macro metastatic SLN. After categorized SLNs were subjected to microarray analysis. To analyze gene expression in mouse SLNs of human M24met tumor bearing animals as compared to tumor free control animals, SLNs were taken at different time points and analyzed for the presence of human M24met to classify SLNs into control, negative, macro metastatic SLN. Briefly, explanted SLNs were stored in RNA later (Ambion, Austin, TX) and DNA as well as RNA was extracted using AllPrep DNA/RNA Mini Kit and RNeasy Micro Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. To detect human cells in mouse SLNs we used a polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17. For analysis of gene expression RNA from control SLN from tumor free animals (control), RNA from tumor negative SLN (negative), and RNA from macro metastatic SLN (positive) from tumor bearing animals was used to analyze gene expression on Mouse Genome 430A 2.0 Arrays (Affymetrix, Santa Clara, CA)

ORGANISM(S): Mus musculus

SUBMITTER: Andreas Bracher

PROVIDER: E-GEOD-30688 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Sentinel lymph node, the first node draining the primary tumor, is a key component of tumor microenvironment promoting immune tolerance. In melanoma, sentinel node is one of the most important prognostic factors and the most frequent site of regional metastasis. To unravel the immunomodulatory pathways that are triggered by melanoma cells in the draining node that allow tumor spreading, transcriptional profiles associated to disease progression were analyzed in archival sentinel node biopsies (SNB). Gene expression profiles of melanoma-positive and negative SNB selected to maximize the differences in terms of disease stage and course, revealed subgroups correlating with regional node involvement and disease progression within positive biopsies. Transcriptional profiles revealed that genes showing differential expression between tumor-positive SNB with/without disease progression were mainly related to inflammatory response and that were mostly down regulated in patients with poor prognosis. TNFRSF8 encoding CD30 showing up regulation in SNB from patients with progressing disease displayed higher expression by immunohistochemical staining compared to SNB from non progressing patients. Subpopulations of CD30 positive CD4/CD8 double negative and CD4 Foxp3/PD-1 CD147 positive T cells were identified by flow cytometry analysis in metastatic nodes, suggesting a potential role of regulatory and tolerogenic T cells in melanoma progression. Cutaneous melanoma patients undergoing SNB biopsy in 2001-2004 with available 5M-bM-^@M-^Syear follow-up clinical data were selected. Data relative to 752 cases was extracted, 80% (n= 603) with a negative SNB and 20% (n=149) with SNB positivity for melanoma metastases. The latter underwent regional lymphadenectomy by CLND and 30% (n=44) resulted positive for melanoma metastases at non-sentinel regional lymph nodes while 70% (n=105) resulted negative. Analysis of follow-up data showed that disease recurrence at 5 yrs occurred in 9% SNB negative patients, 14% of the patients with positive SNB resulting negative at CLND, and in 57% of the patients with positive SNB resulting positive at CLND, consistent to the range of previously reported rates (Santinami M 2009, Balch CM 2009). In order to exploit gene expression profiles to unravel molecular modifications occurring in SNB from patients with progressing disease we selected two groups of cases representing the extremes of the survey, i.e. patients positive at CLND (stage IIIB-C) recurring within 5 years follow-up (SNB-PP), and patients negative at CLND (stage IIIA-B) and non-relapsing at 5 years (SNB-PN). In addition, a group of negative SNB patients without disease recurrence at 5 years was selected and analysed for comparison as tumor negative SNB (SNB-N).

Project description:Background Breast cancer patients who present in the early stage of disease are affected by metastasis to the axillary group of lymph nodes. The first among this group that is affected is called as sentinel lymph node, and metastasis to this lymph node is crucial for the staging of cancer and the quality of surgical intervention. Sentinel Lymph Node Biopsy (SLNB) that is currently used to assess lymph node metastasis is neither sensitive, nor specific, is time-consuming, thereby necessitating the identification of novel biomarkers that can flag sentinel lymph node metastasis. Methods Breast cancer patients were screened, and those with early stage were recruited in the study. Surgical resection of the breast was followed by identification of sentinel lymph nodes by methylene fluorescent technique. Histo-pathology of fresh frozen section biopsy was used as the gold standard to assign the clinical phenotypes of metastatic (SLNM+) and non-metastatic sentinel lymph nodes (SLNM-). Discovery phase of the experiment included isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique comprising of six comparative experiments coupled with mass spectrometric analysis on Orbitrap Fusion to identify differentially expressed proteins on Proteome Discoverer 2.4. Functional enrichment and pathway analyses of differentially regulated genes was carried out in DAVID functional annotation tool. Validation was done by ELISA and protein concentrations were used to estimate the ROC for computing diagnostic parameters. Results Based on MS/MS spectra there were 2396 unique protein groups and 81 differentially expressed proteins comparing SLNB + and SLNB -. Nineteen proteins up-regulated, and eight proteins that were down regulated in SLNB+ as compared to SLNB-. Bioinformatic analysis showed the implication of extra cellular matrix proteins and ECM-receptor interaction pathways to be implicated in lymph node metastasis. ELISA confirmed the up-regulation of caveolin 1, collagen α-1, desmin, fibrillin-1, and microfibrillar associated glycoprotein 4 in metastatic, as compared to non-metastatic lymph nodes. These proteins are known to be integral in tumorogenesis, cell proliferation, invasion, cell survival and anti-apoptosis. These proteins have 80%-100%, of sensitivity and specificity to differentiate the two clinical phenotypes. Conclusion Identified extra cellular matrix protein biomarkers have requisite diagnostic parameters to be developed as a translational tool to assess the status of sentinel lymph nodes during mastectomy procedure to guide surgical therapy of axillary lymph nodes in early breast oncology.

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Gene expression profiles of mouse sentinel lymph nodes (SLNs) in xenotransplanted and non tumor bearing CB17 SCID.

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