Supplementary Table 1 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Supplementary Table 1 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumor

Supplementary Table 2 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Supplementary Table 2 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumor

Additional file 1 of Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

Supplementary material, approximately 2.13 MB

Data_Sheet_1_Association Between Response to Nivolumab Treatment and Peripheral Blood Lymphocyte Subsets in Patients With Non-small Cell Lung Cancer.docx

Immune checkpoint blockade represents a major breakthrough in advanced non-small cell lung cancer (NSCLC) therapy. However, success is limited to a subset of patients and there is a critical need to identify robust biomarkers associated with clinical response. In this study, we assessed whether pre-existing immunological characteristics, as well as immune parameters measured during treatment, might provide such clinical guidance. We studied blood samples collected at baseline and during treatment in a cohort of advanced NSCLC patients (n = 74) treated with nivolumab. Several lymphocyte subsets and biomarkers were then correlated with overall survival (OS) as well as clinical response, assessed using RECIST criteria. We found that patients characterized by longer OS had higher levels of CD3+, CD4+, and CD8+ T cells but lower levels of NK cells at baseline. Moreover, that they displayed a statistically significant lower expression of PD-1 on both CD3+ and CD8+ T cells (p = 0.013 and p = 0.033, respectively). The pre-treatment level of exhausted T cells (CD8+PD1+Eomes+) was significantly lower in patients with controlled disease (CD), defined as partial response (PR), and stable disease (SD), compared to those with progressive disease (PD) (p = 0.046). In CD patients, the frequency of exhausted CD8+ T cells further decreased during treatment cycles (p = <0.0001, p = 0.0032, and p = 0.0239, respectively). In conclusion, our results suggest that the distribution of lymphocyte subsets and expression of PD-1 on T cells before treatment may help predict the outcome of anti-PD-1 treatment in NSCLC patients. In addition, assessing the initial levels of exhausted T cells as well as their decrease upon treatment may also predict response and clinical outcome.</p

Silencing of <i>SDCBP</i> by siRNA inhibits uveal melanoma cell migration.

<p><b>A:</b> Western blot analysis of MEL 270 and 92.1 cell lines upon 72 hrs treatment with scrambled siRNA (C), and <i>SDCBP</i> targeting siRNA (Synt). <b>B:</b> wound-healing assay on MEL 270 and 92.1 cell lines treated with scrambled siRNA (C) or with <i>SDCBP</i> targeting siRNA (Synt). Mean of migration distance of MEL 270 cells (<b>C</b>) and 92.1 (<b>D</b>) treated with scrambled siRNA (black bars) or with <i>SDCBP</i> targeting siRNA (grey bars), P values are indicated.</p

Analysis of Mda-9/syntenin protein expression in uveal melanoma cell lines.

<p><b>A:</b> Immunostaining of fixed and permeabilized cell lines (left) and primary cultures (right). Original magnification 400×. The insets show the negative control performed by the use of non-immune rabbit Ig.</p

<i>SDCBP</i> mRNA is expressed in uveal melanoma cells.

<p><b>A:</b> Conventional RT-PCR analysis of <i>SDCBP</i> gene expression in cell lines derived from primary tumors (MEL 270 and 92.1), cell lines derived from metastatic lesions (OMM1 and OMM2.5) and from four primary cultures derived from patients' ocular tumors (1,2,3,4). The lane identified by “C-” indicates negative control in the absence of cDNA. <b>B:</b> qPCR analysis of <i>SDCBP</i> mRNA expression in uveal melanoma cell lines and primary cultures. Expression values are normalized on the mean of <i>GAPDH</i> gene expression.</p

Gene expression profile of primary uveal melanomas reveals high but heterogeneous expression of <i>SDCBP</i>.

<p><b>A:</b> Bars indicate intensitiy of <i>SDCBP</i> expression in 29 primary uveal melanoma analyzed by gene expression profiling in the present study. <b>B:</b> Heat map showing the expression levels of syndecan (<i>SDC</i>)-1, -2, 3 and -4 genes, <i>SDCBP</i> and syntenin-2 (<i>SDCBP2</i>). <b>C:</b> Comparrison of <i>SDCBP</i> expression in metastatic and non-metastatic patients (n = 29) in our cohort showed a trend to higher <i>SDCBP</i> expression in metastatic patients (p = 0.07). The same type of comparison performed on gene expression profile data from Onken et al. (D), between class1 (low-risk) and class 2 (high risk) patients (n = 27) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029989#pone.0029989-Onken1" target="_blank">[13]</a> and on gene expression profile data from Laurent et al. (E) between metastatic and non-metastatic patients (n = 63) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029989#pone.0029989-Laurent1" target="_blank">[35]</a> showed significantly elevated levels of <i>SDCBP</i> in high risk and metastic patients, respectively.</p

Immunohistochemical analysis of mda-9/syntenin in tissue sections of primary uveal melanomas shows correlation with metastatic progression.

<p><b>A:</b> mda-9/syntenin expression in a choroidal metastasis of colon adenocarcinoma <b>A′:</b> primary uveal melanoma stained by secondary antibody in the absence of anti-mda-9/syntenin antibody (negative control). <b>B, C, D:</b> representative primary uveal melanomas displaying low, medium or high levels of mda-9/syntenin expression, respectively (original magnification 400×). <b>E:</b> liver metastasis of uveal melanoma (original magnification 100×). <b>F:</b> the same section at higher magnification (200×). Arrows indicate single cells of UM positive for mda-9/syntenin, which infiltrate the mda-9/syntenin-negative liver parenchima. <b>G:</b> liver metastasis of uveal melanoma from a different patient. <b>H:</b> the same at higher magnification. <b>I:</b> Kaplan-Meier analysis of Mda-9/syntenin protein expression and disease-free survival in patients with primary tumors. Patients with low mda-9/syntenin expression (dark line) showed longer survival than patients with high expression (gray line) (P<0.014). Patients were stratified according to a combination of qualitative/semi-quantitative grading. Censored patients are indicated in each curve.</p

Silencing of mda-9/syntenin in 92.1 uveal melanoma cells inhibits in vitro invasion and HGF mediated signaling.

<p>A: Expression of c-MET in 92.1 cells detected by indirect immunofluorescence and flow-cytometry; c: negative control. B: Invasion of matrigel membranes by 92.1 cells towards different stimuli: medium with 10% serum (C), 50% conditioned medium from MG63 cell line (CM), 100 ng/ml recombinant HGF in 0.1% serum. C: Silencing of <i>SDCBP</i> (Synt-siRNA) in 92.1 cells inhibits their ability to invade matrigel membranes in response to conditioned medium from MG63 cell line (CM) or recombinant HGF (100 ng/ml). Data are presented as percentage of invading 92.1 cells treated with scrambled siRNA (C-siRNA). * p<0.04. D: Western blot showing inhibition of FAK, AKT and Src phosphorylation in <i>SDCBP</i>-silenced 92.1 cells compared to cells treated with scrambled siRNA. The same membrane was also stained for unphosphorylated FAK, AKT and Src , mda-9/syntenin and β-actin as protein loading control. E: Silencing of mda-9/syntenin in 92.1 uveal melanoma cells does not effect c-MET expression and p-MET phosphorylation. Western blot analysis of c-MET, p-MET, mda-9/syntenin and and β-actin as protein loading control in in 92.1 <i>SDCBP</i> silenced cells and control siRNA.</p