Fig 2: Upregulation of B7-H3 in OSCC specimens. A B7-H3 protein expression in human OSCC and adjacent healthy tissues was assessed by IHC (weakly expressed in normal tissues, moderately expressed in OSCC1, intensely expressed in OSCC2). B B7-H3 mRNA expression in human OSCC and adjacent healthy tissues was assessed by qRT-PCR. B7-H3 mRNA expression was upregulated in human OSCC than adjacent healthy tissue. C and D The association between B7-H3 mRNA expression and the clinicopathological characteristics in human OSCC was analyzed. High expression of B7-H3 mRNA in human OSCC was associated with advanced T stage and lymph node metastasis. E B7-H3 mRNA expression in OSCC cell lines was upregulated than normal epithelial cell lines. F B7-H3 protein expression in OSCC cell lines was upregulated than normal epithelial cell lines.

Fig 3: B7-H3 regulates glycolysis through HIF-1a and its downstream targets. QRT-PCR and western blot were used to determine the mRNA and protein expression of HIF-1a and key factors regulated by B7-H3 silencing and overexpression. A Silencing B7-H3 downregulated the mRNA level of PFKFB3 and Glut1 without affecting HIF-1a mRNA level. B Overexpression of B7-H3 upregulated the mRNA of PFKFB3 and Glut1 without affecting HIF-1a mRNA level. C Silencing B7-H3 downregulated the protein level of HIF-1a and its downstream targets PFKFB3 and Glut1. D Overexpression of B7-H3 upregulated the protein level of HIF-1a and its downstream targets PFKFB3 and Glut1. E Western blot analysis of HIF-1a, PFKFB3 and Glut1 expression in B7-H3-overexpressing cells when HIF-1a was silenced by siRNA. Silencing HIF-1a downregulated the expression of PFKFB3 and Glut1 in B7-H3-overexpressing cells. F, G Glucose uptake and intracellular lactate production were detected in B7-H3-overexpressing cells when HIF-1a was silenced. Silencing HIF-1a decreased glucose uptake and lactate production in B7-H3-overexpressing cells.

Fig 4: B7-H3 regulates aerobic glycolysis of OSCC through PI3K/Akt/mTOR signaling. A The protein expression of PI3K, Akt, and mTOR 72 h after siRNA treatment using siB7-H3 1 or siNC was determined via western blot. Silencing B7-H3 downregulated the expression of p-PI3K, p-Akt and p-mTOR. B The protein expression levels of PI3K, Akt, and mTOR in B7-H3-overexpressing and normal control cells were determined via western blot. Overexpression of B7-H3 upregulated the expression levels of p-PI3K, p-Akt and p-mTOR. C Protein expression of Akt, mTOR, HIF-1a and downstream glycolysis key factors in B7-H3-overexpressing cells was determined via western blot 3 h after blockade of the PI3K/Akt signaling pathway using LY294002, API-2 and rapamycin.

Fig 5: B7-H3 promotes glucose uptake and tumor growth in OSCC tumor xenografts. Cal27 B7-H3-overexpressing OSCC cells were subcutaneously implanted in athymic nude mice (n=4/group). A Images of the tumor specimens. B Tumor volume was monitored by caliper measurements for 6 weeks after injection. C Average tumor weight measured at 6 weeks after injection. Mice in the B7-H3-overexpressing group had a larger tumor weight. D Glucose uptake was measured in vivo at 6 weeks after tumor cell injection using the fluorescent probe 2-DG-750, as described in Materials and Methods (n=3/group; two of the mice died before fluorescent imaging). IVIS images of 2-DG-750 uptake in each individual mouse. Average of total flux (photons/second) indicates the intensity of 2-DG-750 uptake at the primary site of cell injection. Higher 2-DG-750 uptake in the B7-H3-overexpressing group was detected. E The expression levels of B7-H3, HIF-1a and PFKFB3 in implanted tumors were detected by IHC. Intense B7-H3 staining in the B7-H3-overexpressing group and weak B7-H3 staining in the control group were observed at the end point. Similarly, the expression levels of HIF-1a and PFKFB3 in the B7-H3-overexpressing group were higher than those in the control group.