Supplementary Data from Bevacizumab plus Fotemustine as First-line Treatment in Metastatic Melanoma Patients: Clinical Activity and Modulation of Angiogenesis and Lymphangiogenesis Factors

Supplementary Figures S1-S3 and Supplementary Table S1.</p

CD107a degranulating assay on distinct NK cell subsets expressing single inhibitory receptors.

<p>CD107a degranulating ability of activated NK cell subsets from each patient was analyzed in the presence of autologous melanoma cells, either adding or not an anti-pan HLA class I blocking mAb. E/T ratio = 1.5∶1. A representative analysis of patient CA is shown in panel <b>A</b>. In order to assess the link between HLA class I expression on melanoma cells and their susceptibility to autologous NK cell-mediated lysis, the degranulation of NK cell subsets, expressing a single inhibitory receptor for distinct HLA class I molecules, was analyzed. Staining NK cells with mAbs against a specific KIR or NKG2A and with a mixture of mAbs against the other inhibitory receptors allowed to gate on the specific subset and evaluate its CD107a expression after co-culture with autologous melanoma cells. <b>B</b>: Melanoma specific deletion of a HLA class I allele in the same patient CA. HLA-Bw4 was here detected by flow cytometry on the surface of lymphocytes but not of the autologous melanoma. As a comparison, another conserved HLA-A allele is shown. In accordance with Bw4 deletion, KIR3DL1+ NK cell subset exerts a strong cytolytic activity against autologous melanoma cells, which is not significantly increased following anti-HLA class I masking.</p

Melanoma surface molecules and NK cell activating receptors involved in the lysis of melanoma cells by autologous NK cells.

<p><b>A:</b> Melanoma cell expression of molecules able to trigger NK cell activity. Representative data are shown for the six melanoma cell lines whose autologous lymphocytes were also available for performing the functional experiments summarized in panel B. Melanoma cells were stained with mAbs against ligands of NKG2D and DNAM-1 and with mAbs against adhesion molecules LFA-3 and ICAM-I. <b>B:</b> Contribution to the lysis by different activating NK cell receptors. Cytotoxic activity of IL-2 activated NK cells from each patient against the autologous melanoma cells was assessed in the presence of anti-DNAM-1, -NKp30, -NKp44, -NKp46 and -NKG2D (black columns) and isotypic control (grey column) mAbs. *: p≤0.05; **: p≤0.01; n.s.: not significant. E/T ratio = 20∶1. Data shown represent mean values of the results obtained from three to six independent experiments.</p

Comparative HLA class I typing in normal lymphocytes and melanoma cells.

<p>Comprehensive HLA class I typing for each patient are shown. HLA-A,-B and -C alleles relevant for the NK cell inhibition are in bold. (X) indicates that the gene coding for the antigen was present but the HLA protein was not expressed on the cell surface (detected by serologic typing – Terasaki method and, when possible according to antibody availability, also by flow cytometry), whereas (XX) indicates that the gene coding for the antigen was deleted.</p

Cytolytic activity against autologous melanoma of NK cell subsets expressing no inhibitory receptors for self-HLA class I.

<p>The cytolytic activity of NK cell subsets expressing no inhibitory receptors for self-HLA class I (“HLA non-inhibited”) was compared with the counterpart expressing at least one receptor for self-HLA. <b>A:</b> Cytolytic activity was assessed by CD107a degranulation assay for total “HLA-inhibited” and total “HLA non-inhibited” NK cell subsets. A representative experiment is shown. Briefly, triple fluorescence staining with a mixture of PE-conjugated anti-KIR2DL1 mAb and a mixture of other anti-KIR (KIR2DL2/3 and KIR3DL1) and anti-NKG2A mAbs allowed to gate on the KIR⁻/NKG2A⁻ subset plus NK cell subset expressing receptors for HLA class I alleles not expressed by the related patient. On the other hand, the staining permitted also to gate on all “HLA-inhibited” subsets. Thus, as shown in panel A, CD107a expression of the whole “HLA non-inhibited” subset was analyzed and compared with that of whole “HLA-inhibited” subset, in the presence of absence of anti-HLA class I blocking mAb. In agreement with the absence of inhibitory receptors for self HLA, “HLA non-inhibited” NK cell subset did not increase their cytotyitc activity in the presence of anti-HLA class I masking mAb. A summary of all experiments performed in each patients is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008132#pone-0008132-g003" target="_blank">figure 3B(ii,iii,iv)</a> . <b>B:</b> The effectiveness of “HLA non-inhibited” NK cell subsets in killing autologous melanoma was also assessed by a standard ⁵¹chromium release assay. The whole “HLA non-inhibited” NK cell populations derived by three representative patients were sorted out by flow cytometry as double negative cells and compared with the unstained total bulk NK populations. The comparison was performed with the unstained total NK cells, rather than with HLA-inhibited NK cell subets, to avoid possible interference caused by antibody binding to functional receptors. Results shown are from three patients and data correspond to mean values±SD of experiments performed in triplicate.</p

Role of HLA class I alteration in melanoma susceptibility to lysis by autologous NK cells.

<p>Summary of CD107a degranulating assays (performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008132#pone-0008132-g002" target="_blank">figure 2A</a>) analyzed on NK cell subsets in the presence of autologous melanoma cells from six patients. Degranulation was analyzed on (i) NK cell subsets expressing the single inhibitory receptors, (ii) NK cell subsets expressing one or more inhibitory receptors (“Total HLA-inhibited”), (iii) NK cell subsets expressing no inhibitory receptors for self HLA (“Total HLA non-inhibited”) and (iv) the whole NK cell population. Data represent percentages of CD107a expression in the presence of autologous target (grey columns) or in the presence of autologous target plus anti-HLA class I mAb (black columns). Values were calculated by subtracting the spontaneous CD107a expression in the absence of the target. NK cell subsets expressing no receptors for self-HLA are depicted on a grey background. n.d. indicates that the specific NK cell subset was not detectable in a given patient. For each patient, on the right side of the figure, patient HLA class I antigens that are relevant for inhibiting NK cell activity are shown and antigens that have undergone deletions on autologous melanoma cells are marked by a slash. For HLA class I A,B,C, both molecular and serological typing were performed (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008132#pone-0008132-t001" target="_blank">Table 1</a>), while HLA-E was detected only at mRNA level. For each HLA class I antigen, matched inhibitory receptor is also specified. Results are expressed as means of 3 to 6 independent experiments. E/T ratio was 1.5∶1.</p

Cytolitic capabilities of human NK cells against autologous melanoma.

<p><b>A:</b> Susceptibility of melanoma cells to autologous NK cell-mediated lysis. Freshly isolated or IL-2 activated NK cells were used as effector cells in a specific ⁵¹chromium release assay. The effector/target (E/T) ratio used was 20∶1. Four representative patients are shown and results are expressed as means±SD derived from experiments in triplicate. <b>B:</b> Flow cytometry evaluation of total HLA class I expression (W6/32 mAb) on melanoma cells and, as a comparison, on autologous lymphocytes. <b>C:</b> Susceptibility to NK-mediated lysis of melanoma cells compared with susceptibility of HLA class I-deficient FO-1 cell line and autologous lymphocytes stimulated with PHA (PHA lymphocytes). Cytolitic activity was assessed by ⁵¹chromium release tests. In order to evaluate the inhibitory effect caused by melanoma HLA class I expression on NK cell activity, lysis of FO-1, autologous melanoma cells and PHA lymphocytes was also analysed in the presence of the anti-pan HLA class I blocking mAb (A6-136, IgM) at the indicated E/T ratios. At least three experiments were performed for each patients. Representative experiments of three patients out of six are shown. Data represent mean value±SD of experiments performed in triplicate.</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