Additional file 3 of Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma

Additional file 3

Additional file 2 of Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma

Additional file 2

Additional file 1 of Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma

Additional file 1

Overexpression or silencing of B7-H3 expression regulates the expression of MMP-2 in osteosarcoma cells with western blot analysis.

<p>GAPDH was used as an internal control. Histogram represents densitometric analysis of the ratio of B7-H3, MMP-2 and GAPDH bands. Experiments were repeated at least 3 times and the mean value was calculated. *<i>p</i><0.05, compared with control cells. <i>p</i> values were determined by Paired Student’s t test.</p

Overexpression or silencing of B7-H3 expression enhances or suppresses invasive ability of osteosarcoma cells in vitro.

<p>*<i>p</i><0.05, compared with control cells. <i>p</i> values were determined by Paired Student’s t test.</p

Representative immunostaining for B7-H3 expression in osteosarcoma, osteochondroma and bone fibrous dysplasia tissue.

<p>B7-H3 immunostaining in osteosarcoma tissues (A) strong positive, (B) moderate positive, (C) weak positive. B7-H3 immunostaining in osteosarcoma adjacent tissues (D), osteochondroma tissues (E) and fibrous dysplasia tissues (F). ×200 magnification.</p

Correlation between infiltrating T lymphocytes and B7-H3 expression in osteosarcoma tissues.

<p>Values in bold signify <i>p</i><0.05.</p

Constitutive gene expression of B7-H3 in three osteosarcoma cell lines.

<p>(A) validation of B7-H3 mRNA level in osteosarcoma cells with RT-PCR analysis. GAPDH was used as an internal control. (B) validation of B7-H3 expression in osteosarcoma cells with western blot analysis. GAPDH was used as an internal control. Histogram represents densitometric analysis of the ratio of B7-H3 and GAPDH bands. Experiments were repeated at least 3 times and the mean value was calculated. *<i>p</i><0.05, compared with the other two kinds of cells. <i>p</i> values were determined by one-way ANOVA.</p

Overall survival of 61 osteosarcoma patients in relation with B7-H3 protein expression.

<p>Cumulative survival time was calculated using the Kaplan-Meier method and analyzed using the log-rank test. Patients with high tumor B7-H3 protein expression had a significantly poorer prognosis than patients without or with low tumor B7-H3 protein expression (<i>p</i> = 0.005). The median survival of patients with high tumor B7-H3 protein expression was 47.1 months in contrast to 58.3 months in patients with low tumor B7-H3 protein expression.</p

B7-H3 is Overexpressed in Patients Suffering Osteosarcoma and Associated with Tumor Aggressiveness and Metastasis

<div><p>B7-H3 is a member of the B7-family of co-stimulatory molecules, which has been shown to be broadly expressed in various tumor tissues, and which plays an important role in adaptive immune responses. The role of B7-H3 in osteosarcoma, however, remains unknown. In this study we used immunohistochemistry to analyze B7-H3 expression in 61 primary osteosarcoma tissues with case-matched adjacent normal tissues, and 37 osteochondroma and 20 bone fibrous dysplasia tissues. B7-H3 expression was expressed in 91.8% (56/61) of the osteosarcoma lesions, and the intensity of B7-H3 expression in osteosarcoma was significantly increased compared with adjacent normal tissues, osteochondroma and bone fibrous dysplasia tissues (<i>p</i><0.001). Patients with high tumor B7-H3 levels had a significantly shorter survival time and recurrence time than patients with low tumor B7-H3 levels (<i>p</i><0.001). Moreover, tumor B7-H3 expression inversely correlated with the number of tumor-infiltrating CD8⁺ T cells (<i>p</i><0.05). In vitro, increasing expression of B7-H3 promotes osteosarcoma cell invasion, at least in part by upregulating matrix metalloproteinase-2 (MMP-2). In conclusion, our study provides the first evidence of B7-H3 expression in osteosarcoma cells as a potential mechanism controlling tumor immunity and invasive malignancy, and which is correlated with patients’ survival and metastasis.</p></div