Osteosarcoma

The main usage of CentiScaPe is to rank the nodes of a network depending on their topological and experimental relevance. The numerical results are saved as node, edge or network attributes in the Cytoscape attributes browser, depending on the kind of parameters, so all the Cytoscape features for managing attributes are supported; after the computation the centralities are treated as normal Cytoscape attributes. CentiScaPe can be used in undirected networks
^{8 (link)}, in directed networks and in weighted networks. Centralities for directed networks (see
Supplementary materials: CentralitiesTutorial) are useful in the case of metabolic networks in which the direction describes the interaction between the substrates and reactants and the products of the chemical reactions and also in signal transduction networks, in which the direction depends on the flux of information. Considering the direction in the computation of centralities can lead to different results and interpretations than the undirected version.
As an example, in
Figure 1 we show the computation of the directed and undirected Stress applied to a network of Oncogenes (see
Supplementary materials: Oncogenes.txt and Oncogenes_edge_directions.txt). Results of both the computations are shown and discussed.
The image, obtained using Cytoscape’s graphical tool, represents the different Stress values by using the colour and the size of the nodes. The size describes the value obtained by using the directed Stress: the bigger the node the higher the value; the colour describes the value obtained by using the undirected Stress: red is used for the highest value, blue for the lowest value. For example a large blue node requires particular attention because it is showing a node with a high centrality value if the network is considered as directed but with a very low value if the network is considered as undirected.
By analyzing the Oncogenes network we saw that the large red node, i.e. AKT1, shows how its Stress values are high using both algorithms. It plays a central role in different cell processes like metabolism, proliferation, cell survival, growth and angiogenesis. This role may highlight its high Stress value but, on the other hand, the high values suggest us to deeply investigate its characteristics; it is also involved in two different kind of cancer: breast and colorectal (see
http://www.uniprot.org/uniprot/P31749). This evidence suggest that the node could be involved in cancer related processes but this assertion needs validation from several lab experiments.
Another interesting result is shown by the blue medium sized node RAF1. It shows how, using undirected Stress we obtained a low value, but by using the new algorithm we obtained an interesting Stress value. RAF1 was identified as a proto-oncogene with different and fundamental cellular functions (see
http://www.omim.org/entry/164760). The results could be interpreted by saying that the directed network gives us a better understanding about how the gene, and its product, are involved in the development of cancer and could highlight that the use of the direction enhance our ability in describing a complex biological process.
The opposite situation is found in the third highlighted node, the small green node, RB1 in the right bottom corner. In this case the value computed with undirected Stress is not very high, but the value computed with the directed Stress is very low. RB1 is a gene involved in coding a protein involved in the retinoblastoma and other type of cancer like bladder cancer and osteogenic sarcoma. It was the first tumor suppressor gene found (see
http://www.ncbi.nlm.nih.gov/gene/5925). As already said for RAF1, if the directed analysis is considered more reliable than the undirected one, then RB1 seems to be marginally involved in the Oncogenes network otherwise the undirected network is a better description. As already stated an experimental validation should be carried out in order to improve the results from the topological analysis and to better understand the role of each highlighted node.
All the results shown and described must be considered as a possible direction for further lab experiments. The main goal of this kind of analysis is to give us a comprehensive view that could be useful in describing the role of each node involved in a specific biological process and to drive future insights and investigations.
Second important features of the new version of CentiScaPe is the possibility of computing centralities for weighted networks, that are networks in which the edges are provided with an attribute that can be interpreted as a distance between the two connected nodes.
In the network in
Figure 2 we have a distance (
dist) attribute for each edge. The values are
dist(
A,
B) = 2,
dist(
B,
C) = 3 and
dist(
A,
C) = 7. Since A and C are connected by a single edge, in an unweighted computation, the distance from A to C is equal to one. But if the attributes of the edges are considered as distances, the shortest path between A and C is the one passing through B (= 2 + 3 = 5) since it is shorter, or
lighter, than the one connecting A directly to C (= 7). The computation of weighted shortest paths will result in completely different values from the case wherein the weight is not considered. The user should consider that the weight is used in the sense that close nodes are more important than distant nodes. Therefore depending on the meaning of the attributes, one can use the real value or its reciprocal. For example if the attribute represents the speed of a reaction instead of a distance, the reciprocal should be used. This is because the higher the value of the speed, the nearer the nodes are: an increasing speed determines a decreasing reciprocal and the distance decrease by consequence.
An example of usage of weighted networks centralities analysis, can be found in Holly
et al.^{9 (link)} where an euclidean distance is given to each edge depending on the difference between the phosphorylation level of the proteins connected by that edge.
All the graphical features of the previous version of CentiScaPe, as the plot-by-node, the plot-by-centrality and the boolean-based result panel have been maintained in the new version. A complete guide can be found in Scardoni
et al.^{8 (link)} or in the CentiScaPe userguide available from the website.

ESTIMATE was designed to count scores for reflecting the infiltration levels of immune cells and stromal cells within the tumor microenvironment on the foundation of the specific genes expression level of immune and stromal cells using the R package “ESTIMATE” (10 (link)). First, we used ESTIMATE algorithm based on the expression level of RNA-seq to count the Tumor Purity, ESTIMATE Score, Immune Score, and Stromal Score of 101 osteosarcoma samples in three clusters of TARGET database using the R package “estimate” to validate the effectuality of ssGSEA grouping and to picture clustering heatmap. The vioplots of Tumor Purity, ESTIMATE Score, Immune Score, and Stromal Score in three clusters were presented employing the R package “ggpubr”. Next, to investigate the difference of immune cell subtypes, the R package “CIBERSORT” was applied to count the proportion of 22 immune cells of all osteosarcoma samples on the foundation of expression file (11 (link)), and the difference of three clusters was validated again. Besides, we also used K-M analysis and the expression of HLA family and PD-L1 to validate the difference between three clusters applying the R package “survival” and “ggpubr” respectively.

Human bone marrow mesenchymal stem cell were obtained according to procedures approved by the Ethics Committee at Maoming People's Hospital. hBMSCs were isolated from healthy volunteers and patients with OP. The hBMSCs were cultured under 10% exosome-free FBS for 72 h. The cells, dead cells and debris were removed using several low-speed centrifugations. hBMSCs of passage 5 were used for in our experiments. Cells were cultured in a humidified incubator with 5% CO₂ at 37 °C and passaged with trypsin/EDTA after reaching the confluence. The human osteosarcoma cell line, MG63 cell, was purchased from ATCC company. MG63 cells were cultured in a humidified incubator with 5% CO₂ at 37 °C with RPMI 1604 complete medium.