DOI: 10.5061/dryad.m905qfv4w
title: Osteosarcoma-enriched transcripts paradoxically generate osteosarcoma-suppressing extracellular proteins

description: Osteosarcoma (OS) is the common primary bone cancer that affects mostly
 children and young adults. To augment the standard-of-care chemotherapy,
 we examined the possibility of protein-based therapy using mesenchymal
 stem cells (MSCs)-derived proteomes and osteosarcoma-elevated proteins.
 While a conditioned medium (CM), collected from MSCs, did not present
 tumor-suppressing ability, the activation of PKA converted MSCs into
 induced tumor-suppressing cells (iTSCs). In a mouse model, the direct and
 hydrogel-assisted administration of CM inhibited tumor-induced bone
 destruction, and its effect was additive with Cisplatin. CM was enriched
 with proteins such as Calreticulin, which acted as an extracellular tumor
 suppressor by interacting with CD47. Notably, the level of Calr
 transcripts was elevated in OS tissues, together with other
 tumor-suppressing proteins, including histone H4, and PCOLCE. PCOLCE acted
 as an extracellular tumor-suppressing protein by interacting with amyloid
 precursor protein (APP), a prognostic OS marker with poor survival. The
 results supported the possibility of employing a paradoxical strategy of
 utilizing OS transcriptomes for the treatment of OS.
description: Here are the procedures for collecting and analyzing <em>in
 vitro </em>and <em>in vivo</em> data and conducting
 bioinformatics analysis. <strong><em> In
 vitro</em> assays</strong> 
 MTT-based metabolic activity (Figs. 1A, 1D, 2A, 2B, 2D, 2E, 2F,
 4G, 4H, 5H, 6B, 7E, and Figure 1-figure supplement 1A, 1C, Figure 5-figure
 supplement 4): The activity was evaluated using three osteosarcoma cell
 lines. The optical density was determined at 562 nm using a multi-well
 spectrophotometer (EL808, BioTek, VT, USA). Data were analyzed in
 Excel. Transwell invasion (Figs. 1C, 1F, 6D, 7C, and
 Figure 1-figure supplement 1A, 1D): Images were taken with an inverted
 optical microscope (magnification, 100x, Nikon, Tokyo, Japan). The average
 number of stained cells was determined with Image J (National Institutes
 of Health, Bethesda, MD, USA). Data were analyzed in Excel.
 Two-dimensional motility (Figs. 1B, 1E, 6E, 7D): Images were
 taken with an inverted optical microscope. The two-dimensional motility
 scratch areas were quantified by Image J software. Data were analyzed in
 Excel. Western blot analysis (Figs. 2C, 4A, 5D, 5E,
 5G, 6C, 7A, 7B, 7F, and Figure 5-figure supplement 2B, Figure 5-figure
 supplement 3, Figure 5-figure supplement 5): A luminescent image analyzer
 (LAS-3000, Fuji Film, Tokyo, Japan) was used to determine signal
 intensities for Western blot images. The relevant bands are labeled in
 PowerPoint. ELISA assay (Figs. 2B, 2C, 2D, 2E, 2F):
 According to the procedure provided by the manufacturer, protein levels in
 CW008-treated CM were determined using the ELISA kits (MyBioSource, San
 Diego, CA, USA). The absorbance of each well was measured at 450 nm using
 a multi-well spectrophotometer (EL808). Data were analyzed in
 Excel. Alizarin Red assay (Figure 5-figure supplement
 2): Alizarin Red staining was used to visualize calcium deposits. The
 optical density was measured at 562 nm using a multi-well
 spectrophotometer (EL808). Data were analyzed in Excel.
 <strong><em>In vivo</em> assays
 (mouse model)</strong> X-ray images
 (Fig. 5A): X-ray imaging was performed using a Faxitron radiographic
 system (Faxitron X-ray Co., Tucson, AZ, USA). Data were analyzed in
 PowerPoint. microCT images (Figs. 3, 5B, 5C):
 Micro-computed tomography was performed using Skyscan 1172
 (Bruker-MicroCT, Kontich, Belgium). Scans were performed at pixel size
 8.99 μm, and the images were reconstructed using a pair of software tools
 (nRecon v1.6.9.18, and CTan v1.13). Data were analyzed in
 Excel. Histology (Figure 3-figure supplement 1 and
 Figure 3-figure supplement 2): H&E staining was conducted on the
 sagittal sections, and images were taken with a microscope (U-TV0.63XG,
 OLYMPUS, Tokyo, Japan). The distribution of tumor cells in the tibial bone
 cavity was quantified by Image J software. Data were analyzed in
 Excel. Immunohistochemistry (Figure 3-figure
 supplement 3 and Figure 5-figure supplement 1): Immunohistochemistry
 staining was conducted on the sagittal sections, and images were taken
 with a microscope (U-TV0.63XG, OLYMPUS, Tokyo, Japan). The immunostained
 area was quantified in the tumor-invaded area by Image J software. Data
 were analyzed in Excel. <strong> Bioinformatics</strong> Survival rate (Figs. 5F, 6F, 6H): Patient survival analyses were obtained from a web-based tool, GEPIA (Gene Expression Profiling Interactive Analysis). Transcript levels (Figs. 6A, and Suppl. File 1): The TCGA (The Cancer Genome Atlas) database was used to predict tumor-suppressing protein candidates via GEPIA.  Protein interactions (Fig. 6G): The target protein-protein interaction network was shown by String (String Consortium; string-db.org/network/) via the Uniprot (Universal Protein Resource; uniport.org).
description: Data are provided as Excel, pdf, and ppt files.