This study included 181 randomly selected patients with primary OS who underwent radical resection at the First Affiliated Hospital of Shantou University Medical College, First Affiliated Hospital of Zhejiang University Medical College, First Affiliated Hospital of Sun Yat-sen University Medical College and Sun Yat-sen University Cancer Center from 2010 to 2019. None of the patients received preoperative radiotherapy or chemotherapy (Table S1). OS samples (n = 181), non-tumor tissues (n = 44) and lung metastasis tissues (n = 9) were used to analyze COL6A1 protein expression by immunohistochemistry. 181 OS cases included tissues with metastatic (n = 29) and without metastatic (n = 152). 98/181 (54%) were male and 83/181 (46%) were female. The median age was 24.5 years (range 8-79 years). 41 pair of case-matched OS and non-tumor tissues were obtained to evaluate COL6A1 expression by western blot and 22 fresh OS tissues and 7 non-tumor tissues were used to analyze COL6A1 expression at mRNA level. Human serum specimens were collected from healthy donors and OS patients before resection in the Sun Yat-sen University Cancer Center. The serum samples included 10 healthy controls, 25 OS patients without lung metastasis, and 5 OS patients suffering lung metastasis. This study was approved by the ethical review committees of the Sun Yat-sen University Cancer Center. All participants involved in our study provided written informed consents.

Three human OS cell lines MG63, U2OS and Saos-2 were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1× antibiotic mixture (Invitrogen, Carlsbad, CA, USA). Osteoblast cell line hFOB1.19 was purchased from American Type Culture Collection (Manassas, VA, USA). MRC5 was purchased from Sciencell Company (USA) and cultured in Eagle's minimum essential medium (Gibco) supplemented with 10% FBS (Gibco). Cell lines were authenticated by short tandem repeats (STR) profiling and confirmed to be mycoplasma negative.

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