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.

Here are the procedures for collecting and analyzing in vitro and in vivo data and conducting bioinformatics analysis.

 In vitro assays

• 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.

In vivo assays (mouse model)

• 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.

 Bioinformatics

• 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).

Data are provided as Excel, pdf, and ppt files.