Figure 1.
Lymph nodes of two different patient groups suffering (pN1) or not suffering (pN0) from lymph node metastasis were investigated. From each patient regional lymph nodes (adjacent to the tumor) were compared to distant lymph nodes (reference as baseline). Key to the experiment is that all investigated lymph nodes, including those of pN1 patients, were made sure to be still free of metastasis as well as micro-metastases.
Table 1.
Top regulated genes in pN0 and pN1 patients' regional lymph nodes.
Figure 2.
RT-PCR results for validation of microarray results.
Displayed the signal-log-ratios of the regional lymph nodes in comparison to the tumor distant lymph nodes (reference) for a selected set of top regulated genes.
Figure 3.
Functional gene set enrichment.
Displayed are biological and molecular functions and sub-functions associated with the genes regulated in regional lymph nodes in comparison to the distant lymph nodes (reference) of pN1 or pN0 patients. The single transparent circles depict the main functions, the colored (red: activated, blue: inactivated) circles within depict the significantly enriched sub-functions (e.g. within pN1 cell death circle: ‘apoptosis of leukemia cell lines’ – red, or ‘cell survival’ – blue). Blue sub-functions have a strong prediction to be inactivated, red sub-functions to be activated. The deeper the color the more reliable (z-score) is the activation state's prediction. The size of the circles increases with the significance (p-value) of the gene list's association with the sub-functions. The analysis was performed with Ingenuity Pathway Analysis software. It can be clearly seen that metastasis-free regional lymph nodes of pN1 patients exhibit a completely different regulation-pattern that those of pN0 patients.
Figure 4.
The network analysis was performed using Ingenuity Pathway Analysis software. a) Mechanistic network of β-catenin downstream regulators displaying predicted activation state and the regulatory relation (activation or inhibition) between the single regulators. β-catenin and a number of downstream regulators are predicted to be inactivated. b) The analysis shows a tight connection between the genes regulated in the pN1 samples (light grey nodes) and those regulated in the pN0 samples (dark grey nodes) linked by ERK1/2 and DKK1 embedded within the network.
Figure 5.
Representative immunohistochemical staining images of the investigated lymph nodes.
Lymph nodes were stained using a polyclonal antibody against DKK1 (1∶100, abcam, #ab61034) and counterstained with hematoxylin. a) Representative DKK1 negative lymph node. Scale bar: 200 µm. b) Representative DKK1 positive lymph node. The subcapsular sinus is marked with (I) and the intermediary sinus and trabeculae with (II). Scale bar: 200 µm. c) DKK1 positive vascular endothelial cells. Scale bar: 100 µm.