Osteosarcoma-enriched transcripts paradoxically generate osteosarcoma-suppressing extracellular proteins

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