Effect of signaling pathway inhibition in cells expressing high levels of PD-L1.

<p><b>A</b>. Patient 1 pre cells were treated with DMSO (control) or BRAF inhibitors (dabrafenib 100nM/ml or vemurafenib 10μM/ml) or 10μM of MEK inhibitor UO126 or 40μM PI3K inhibitor LY29004 for 48 hours and the average PD-L1 expression determined by flow cytometry is represented as a fold difference of the mean fluorescence intensity compared to isotype levels. The plotted average mean fluorescence intensity and SEM are derived from three independent experiments. Western blots for indicated proteins after treatment for 24 hours with trametinib or cobemitinib, LY290024, BKM120 and BEZ235 are also shown. Lanes were from the same blot and a representative blot out of two independent experiments is shown. Patient 1 pre cells were further treated with 10μM I-BET151 (IBET) or 5μM BMS-345541 (BMS) for 48 hours and PD-L1 expression determined by western blotting. <b>B.</b> Patient 1 pre cells were transfected with siRNA against the p65 and p50 subunits of NF-κB or control silencer (-) for 72 hours. Cellular lysates were immunoblotted for the indicated proteins. One representative blot out of three independent experiments is indicated. <b>C</b>. Cells were transduced using lentiviral constructs of either control pSIH-copGFP or STAT3-copGFP for 72 hours. Immunoblot for the indicated proteins is shown. Cells were also independently transfected with the control or SMART Pool c-Jun silencer for 48 hours and the cellular lysates were immunoblotted for the indicated proteins. One representative blot out of two independent experiments is indicated.</p

Targeting NF-κB reduces PD-L1.

<p><b>A</b>. KMJR138 cells were treated with DMSO (control) or 100ng/ml IFN-γ (IFN) or indicated doses of BMS-345541 in the presence of IFN-γ for 48 hours before PD-L1 was analysed by flow cytometry. Mean Fluorescence Intensity (MFI) of PD-L1 expression is indicated; the average of two independent replicates is shown. <b>B</b>. Indicated cells were treated with either dimethyl sulfoxide (DMSO) (control) or 10μM of I-BET151 (IBET) or 5μM of BMS-345541 (BMS) for 48 hours in the absence or presence of 100ng/ml IFN-γ and PD-L1 cell surface expression was determined by flow cytometry. Mean fluorescence intensity (MFI) plotted is the average of three independent experiments. <b>C</b>. Sub-cellular fractionation of cellular lysates from a representative cell line (KMJR138) was probed for the indicated proteins. Cells were treated with DMSO (control), IFN-γ (IFN, +), I-BET151 (IBET), BMS-345541 (BMS) for 48 hours and separated into cytosolic (C) or nuclear (N) fractions. One representative blot out of two independent experiments is shown. <b>D</b>. KMJR138 cells treated with DMSO (control) or 100ng/ml IFN-γ, 5μM BMS-345541 or a combination of both for 48 hours in the absence or presence of 10μM pan-caspase inhibitor Q-VD-OPh were stained with Annexin V-APC, and PI to determine the number of apoptotic cells (left panel) and anti-PD-L1 antibody to determine the expression of PD-L1 (right panel) which is represented as the mean fluorescence intensity (MFI) over isotype controls. Average value from three independent experiments is indicated. <b>E.</b> Dual renilla-luciferase NF-κB reporter assay: KMJR138 cells were transfected with the control and NF-κB constructs for 24 hours and treated with the indicated inhibitors (10μM I-BET151 or 5μM BMS-345541) with or without 100ng/ml IFN-γ. Luciferase was measured and normalised to renilla values to determine promoter activity. Transfected cells were treated with the inhibitors for 24 hours and IFN-γ induction was carried out 1 hour prior to harvesting and recording luminescence. ***p-value = 0.0001; **p-value = 0.001; *p-value = 0.01. Average of two independent replicates experiments is indicated</p

STAT3 and c-Jun independent expression of inducible PD-L1.

<p><b>A</b>. KMJR138 cells were transduced using lentiviral constructs of either control pSIH-copGFP (-) or STAT3-copGFP (+) for 72 hours and treated with 5μM BMS-345541 (BMS) with or without 100ng/ml IFN-γ or DMSO (control) for an additional 48 hours. A representative immunoblot (out of three independent blots) for the indicated proteins is shown. <b>B</b>. KMJR138 cells were transfected with control siRNA (-) or with silencer (si) molecules against c-Jun for 48 hours and treated with DMSO (control) or IFN-γ (IFN) for an additional 48 hours and cellular lysates were immunoblotted for the indicated proteins. A representative blot out of two independent experiments is shown.</p

PD-L1 expression in melanoma cells.

<p><b>A</b>. KMJR138 cells were treated with indicated doses of IFN-γ for 48 hours and the cell surface expression of PD-L1 was determined by flow cytometry. The corresponding mean fluorescence intensity (MFI) with values is shown in the histogram and plotted in the graph below and corresponds to the relative fold increase over isotype levels. The time course graph represents the MFI at the indicated times upon treatment with 100ng/ml IFN-γ. All values are an average of two independent replicates. <b>B</b>. PD-L1 protein detection induced after IFN-γ treatment for 48 hours is indicated in the representative immunoblot. * corresponds to a non-specific band. <b>C.</b> A panel of melanoma cell lines were treated with 100ng/ml IFN-γ for 48 hours and cell surface expression of PD-L1 was determined by flow cytometry. MFI is represented in the graphs (average of three independent replicates) and MFI values are indicated along with the histograms. Control refers to dimethyl sulfoxide (DMSO) treatment. <b>D.</b> Quantitative real time PCR was performed to evaluate induction of PD-L1 transcripts on RNA isolated from cells treated with DMSO (control) or 100ng/ml IFN-γ for 24 hours. Levels were normalised to the 18sRNA transcript. Values from one of two independent replicates are shown. ***p-value = 0.0001; **p-value = 0.001; *p-value = 0.01 ns = non-significant.</p

MAPK and PI3K inhibition are not effective in abolishing PD-L1 expression.

<p><b>A</b>. Patient tumor derived cell lines KMJR138 or cell line from the Patient 3 post biopsy were treated with DMSO (control) or 10μM UO126 (MEKi) or 100nM dabrafenib (BRAFi) or 10μM vemurafenib (BRAFi) for 48 hours in the absence or presence of IFN-γ (+IFN) and PD-L1 expression was determined by flow cytometry. The mean fluorescence intensity (MFI) as a relative fold difference as compared to isotype controls is plotted. Average and SEM are derived from three independent experiments. KMJR138 cells were harvested 1 hour post treatment with the inhibitors and immunoblotted for the indicated proteins to detect decrease in p-ERK. <b>B</b>. Patient 3 post cells were treated with DMSO (control) or MEK inhibitors trametinib (100nM) and cobemitinib (1μM) and the immunoblot probed for PD-L1 and key proteins involved in the MAPK signalling pathway. Lanes have been depicted separately for clarity and are from the same representative blot. <b>C.</b> KMJR138 and patient 3 post cells were treated with 40μM PI3Ki LY294002 for 48 hours in the presence or absence of 100ng/ml IFN-γ (IFN) and PD-L1 expression was monitored using flow cytometry. An average of three independent experiments is indicated. The accompanying western blot indicates a decrease in p-AKT after treatment with LY294002. <b>D.</b> Patient 3 post cells were treated with DMSO (control), 100ng/ml IFN-γ (IFN) or 1μM BEZ235 or 1μM BKM120 for 24 hours and lysates were immunoblotted for the indicated proteins. *p-value = 0.01 ns = non-significant.</p

NF-κB is required for PD-L1 expression.

<p><b>A</b>. Indicated cells were transfected with control si (-) or siRNAs against p65 (+) for 48 hours and treated with 100ng/ml IFN-γ for an additional 48 hours before they were harvested for immunoblotting for the indicated proteins. V2 si RNA transfection was done independently with the accompanying control siRNA. One representative out of three independent blots is shown. <b>B</b>. <b>C</b>.KMJR138 cells were transfected with either control (-) or two independent siRNA targeting p105/p50 (+) for 48 hours and treated with 100ng/ml IFN-γ for an additional 48 hours before being harvested for immunoblotting for the indicated proteins. One representative out of three independent blots is shown. <b>C.</b> Cells were transfected with either IκBwt-GFP (control vector) or IκBmutant-GFP (super-repressor) using lipofectamine 2000 for 48 hours after which 100ng/ml IFN-γ was added. PD-L1 expression was detected by flow cytometry on GFP gated cells and is represented as MFI; the average of two independent experiments is indicated. *p-value = 0.01.</p

Illustration of the mean-shift model.

<div><p><i>A</i>. Initialization of the generic kernel based on the user-defined position (x’₀,y’₀).</p> <p>The kernel (here an octagon, ndir = 8) is divided into sectors (S1 to S8), each one containing two nested triangle-shaped regions (R_b and R_w), one sensitive to dark and the other to bright pixels (“b” means “black” and “w” means “white”). Here, R_b6 and R_w6 are shown, with a total of 16 regions (R_b1 to R_b8 and R_w1 to R_w8). Only the contours of sectors 2-5 are shown in order to lighten and better visualize the figure. The cell is not presented for clarity.</p> <p><i>B</i>. <i>Adjustment of the position of the center at t₀</i>. </p> <p>Sixteen mass centers (g_b1 to g_b8 and g_w1 to g_w8) are first calculated from the intensities of the pixels from each region, (g_b1 and g_w1 are shown). Sector mass centers (C) are calculated from g_wn and g_bn, (C₈ is shown as an example). The center (x₀,y₀) is defined as the centroid of the mass centers C₁ to C₈.</p> <p><i>C</i>. <i>Adaptation of the kernels to cell morphology at t₀</i>. </p> <p>The distances (d_i) between each mass center (g_wn) and kernel center (x₀,y₀) are calculated (d₈ is shown as an example). The new outer radii (r_wn) are calculated based on d_n, the average d_n distances, the expansion factor and the anisotropy factor. r_bn is assigned according to the ratio (r_b / r_w), which is initially defined by the user.</p> <p><i>D</i>. <i>Representation of the kernels at t₁</i>. </p> <p>Information is obtained applying the processes explained in B and C. The size of the sectors will increase or decrease (indicated by the arrows) as a function of cell shape modifications. </p></div

Time-dependent characteristics of cell trajectories extracted by manual and automatic tracking.

<div><p>Cells were imaged every four minutes for 12 hours and experiments were repeated three times. The same forty independent cells were tracked manually (M) and automatically (A). The following variables were extracted from the manually and automatically retrieved sets of coordinates:.</p> <p><i>A</i>. <i>Average migration speed of WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.0669 and p = 0.3266 for the average speed and standard deviation, respectively. </p> <p><i>B</i>. <i>Average acceleration of migration by WM852 human melanoma cells</i>. Manual and automatic methods were statistically significant (standard unpaired t-test comparing average acceleration, p = 10^-4). For the standard deviation of average acceleration, p = 0.2729. </p> <p><i>C</i>. <i>Percentage of pause by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.1783). </p> <p><i>D</i>. <i>Angles of displacement between two adjacent time frames (angle α) calculated for WM852 human melanoma cells</i>. Manual and automatic methods were statistically significant (standard unpaired t-test, p = 10^-4).</p> <p>E. Definition of variables .</p> <p>The polygon (gray hexagon) represents a cell migrating at three different time frames with three sets of coordinates. The angles α and β are defined relative to the horizontal line as a reference at two consecutive times. </p></div

Time benefit of automatic tracking <i>vs</i>. manual tracking.

<p>Cells were followed either manually (full circles) or using iTrack4U (white circles). It took about three minutes to track each single cell, corresponding to 181 frames, by manual tracking. Twenty cells can therefore be manually tracked in 1 hour and 200 cells during 10 hours of active work. Automatic tracking is performed in two major steps: (i) the establishment of the parameters for both pre-processing and tracking requires about two hours and (ii) the automatic tracking requires about four seconds to fully track a single cell. Using iTrack4U is beneficial for following over about 50 cells. </p

Geometric characteristics of cell trajectories associated with distances and extracted by manual and automatic tracking.

<div><p>Cells were imaged every four minutes for 12 hours and experiments were repeated three times. The same 40 independent cells were tracked manually (M) and automatically (A). The following variables were extracted from the manually and automatically retrieved sets of coordinates:.</p> <p><i>A</i>. <i>Total distance of migration by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.0774).</p> <p><i>B</i>. <i>Euclidian distance (start-end distance) of WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.9672).</p> <p><i>C</i>. <i>Persistence of migration by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.5012).</p> <p><i>D</i>. <i>Definition of migration variables used in this figure</i>. Total distance = d_ttl, Euclidian distance = d_S-E, persistence = d_ttl / d_S-E, minimum travelled distance = d_min, maximum travelled distance = d_max.</p> <p><i>E</i>. <i>Average distance of migration by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.0774 and p = 0.3913 for the average distance and standard deviation, respectively). </p> <p><i>F</i>. <i>Extreme values (minimum and maximum distances) of migration for WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant for the maximum distance (standard unpaired t-test, p = 0.2611). A significant difference for the minimum distance has no real meaning, as explained in the text (standard unpaired t-test, p = 0.001).</p></div