MGL recognises CD45RA on human Tregs and modulates their suppressive capacity.

<p>(A) Flow cytometry analysis of purified Treg stained with anti- CD4, CD25, FOXP3 and CD45RA antibodies. (B) Competition studies of rhMGL-Fc binding to CD45RA expressed by Tregs using GalNAc polymer. Filled histograms represent the untreated Tregs, while the open histograms show Tregs treated with rhMGL-Fc or rhMGL-Fc+GalNAc. (C) Western blot analysis of Treg lysate immunoprecipitated with rhMGL-Fc. Samples were run in 4–12% SDS-PAGE gel and were analysed with anti-CD45RA and rhMGL-Fc. These results are representative of one donor out of three. (D) Immunosuppression capacity of Tregs alone and Tregs treated with rhMGL-Fc after four days of co-culture with CD4⁺ CD25^- T cells (ratio 1:5, Treg:CD4⁺CD25^- T cells), in presence of anti-CD3 and anti-CD28. (E) IFNγ spots produced by CD8+ T cells (5×10⁴/well) stimulated with mDCs in presence or not of Tregs ± rhMGL. The results correspond with the mean of three independent experiments ± standard deviation (SD). * corresponds to <i>p</i><0.05 and ** to p<0.01.</p

Immunofluorescence for vimentin in cell cultures treated with the TGF-ΒR1 inhibitor LY2157299.

<p>Expression of vimentin was unchanged by LY2157299. However, IF for vimentin (red) highlighted evident morphological changes in mucin- and mixed-IHCCA cell cultures treated for 72 hrs with the TGF-ΒR1 inhibitor LY2157299 (50 μM). LY2157299 induced the loss of typical mesenchymal features, instead favoring the epithelial phenotype with formation of cell clusters (white arrows). No morphologic change was induced by LY2157299 in CK19-mucin cell cultures. Cell clusters with undefined edges, a typical feature of the epithelial phenotype, were observed in mucin- and mixed- IHCCA cells treated with LY2157299 (white arrows) and in CK19-mucin (yellow arrows, similar in treated and untreated cells). Mean +/- SD of N = 3–5 independent experiments.</p

Apoptosis (Annexin V-FITC/PI by FACS) of primary cell cultures exposed to TGF-ΒR1 inhibitor (LY2157299) and CK2 inhibitor (CX4945), and RT-PCR analysis of snail-1 and vimentin in primary cell cultures exposed to CX4945.

<p><b>A:</b>After 72 hrs of treatment, CX4945 10 μM induced apoptosis in both mucin- and mixed-IHCCA primary cell cultures ** = p<0.01 vs control. <b>B:</b>When CX4945 was combined with MK2206, an enhanced number of apoptotic cells was seen with respect to CX4945 or MK2206 alone. * = p<0.05 vs control and MK+CX; & = p<0.05 vs MK and CX alone. <b>C:</b> CX4945 30 μM did not induce apoptosis in CK19 mucin primary cells. <b>D:</b> CX4945 (10μM) treatment for 72h induced vimentin expression in all three primary cell cultures, whereas snail-1 expression was induced only in CK19-mucin, being down-regulated in mixed-IHCCA and unaffected in mucin-IHCCA. mRNA expression was normalized to control cells exposed to CX4945 carrier and considered equal to 1. * = p<0.05 vs control. Mean +/- SD of N = 3–5 independent experiments.</p

RTK human phosphoarrays in mucin-IHCCA cells resistant to CX4945 treatment.

<p>After 72h of CX4945 (10 μM) treatment, surviving cells showed contemporary deactivation of both EGFR and HGFR signalling. The black lines indicate the spots positive for EGFR and HGFR activation.</p

Clonogenic assays.

<p>The number of colonies (A and B) and the absorbance of the extracted dye (crystal violet) measured at 595 nm (C) indicate the clonogenic potential of CCA cell cultures. We tested the clonogenic capacity of mucin-IHCCA cells after sequential treatment with CX4945 (72 hrs) and MK2206 (48 hrs) versus their combination. The sequential treatment of CX4945 followed by MK2206 showed the highest inhibitory effect on mucin-IHCCA cell growth. We calculated the numbers of colonies and the absorbance of the extracted dye at 595 nm. & = p<0.05 vs non pre-incubated conditions. $ = p< 0.05 vs others columns. Mean +/- SD of N = 3–5 independent experiments.</p

Immunofluorescence for γH2ax and MTS assays.

<p><b>A-C:</b> mucin-IHCCA primary cell cultures were investigated by IF for γ-H2ax nuclear foci, a biomarker of DNA double-strand breaks. After 72hrs of treatment, CX4945 (10μM) increased the number of γ-H2ax positive (red) nuclei vs control. A potent DNA-PK inhibitor (NU7026, 10 μM) reduced γ-H2ax positive nuclei as indicated by fluorescent intensity vs controls and blocked the effects of CX4945 on γ-H2ax ** = p<0.01 vs other columns;*** = p<0.001 vs other columns; & = p<0.01 vs CX4945 and NU7026 alone. <b>D</b>: NU7026 alone or in combination with CX4945 failed to influence cell viability in mucin-IHCCA cultures vs control and CX4945 alone.** = p<0.01 vs control and NU7026 alone. Mean +/-SD of N = 3–5 independent experiments.</p

Sequences of primer pairs used for amplifying the genes of interest (GOI) and the internal reference gene (GAPDH).

<p>Primers were designed by the PROBEFINDER software (<a href="https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp" target="_blank">https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp</a>).</p

Wounding-healing assays.

<p>Mucin-IHCCA, mixed-IHCCA and CK19-mucin cell cultures were treated with LY2157299 (50 μM), CX4945 (10μM) or LY2157299 (50 μM) + CX4945 (10μM). <b>A:</b> pharmacological inhibition of TGF-ΒR1 by LY2157299 produced inhibitory effects on cell migration. In mucin-IHCCA the effects of LY2157299 were evident at 120 hrs, while in mixed-IHCCA and CK19-mucin effects were already visible at 48hrs. & = p<0.05 vs CTLR;* = p<0.05 vs CTLR. <b>B:</b> CX4945 exerted more evident inhibitory effects on cell migration in mucin-IHCCA and the effects were higher with respect to LY2157299. & = p<0.05 vs CTLR and LY2157299 alone; * = p<0.05 vs CTLR <b>and</b> LY2157299 alone; = p< 0.05 vs mixed- and CK19-mucin. C: the combination of CX4945 andLY2157299 exerted relatively more evident additive effects on cell migration in mixed-IHCCA than in CK19-mucin, but no additive effect was seen in mucin-IHCCA. & = p<0.05 vs CTLR;* = p<0.05 vs CTLR; = p< 0.05 vs CX4945 alone. <b>D</b>: representative images of mixed-IHCCA cell cultures where the synergistic effect of CX4945+LY2157299 was more evident with respect to the other cell lines. The wound area was calculated with respect to control at T0, considered as 1. Mean +/- SD of N = 3–5 independent experiments.</p

Immunofluorescence (IF) and RT-PCR analyses of primary cell cultures.

<p><b>A</b>: By IF, vimentin was expressed in mucin- (a, red), mixed-IHCCA (e, red) and CK19-mucin (i, red) primary cell cultures. CK19 was only expressed in CK19-mucin primary culture (l, red). N-cadherin was expressed in all three primary cultures (c,g and m, red). β-catenin has nuclear and cytoplasmic localization in primary cultures (d,h and n, red). <b>B:</b> By RT-PCR, CK2 was more expressed in mucin- and mixed-IHCCA than in CK19-mucin cell cultures or hBTSCs while TGF-ΒR1 and E-cadherin genes were more expressed in CK19-mucin. The E-cadherin gene was virtually unexpressed in mucin- and mixed-IHCCA cell cultures. mRNA expression levels were normalized with respect to hBTSCs, considered equal to 1. * = p<0.05 vs other cell cultures; & = p<0.05 vs mixed-IHCCA. Mean +/- SD of N = 3–5 independent experiments.</p

Effect of TGF-ΒR1 inhibitor (LY2157299) and CK2 inhibitor (CX4945) on cell viability (MTS assay).

<p>Primary cell cultures were treated with CX4945, LY2157299/5 or MK2206 and MTS was performed at 72 hrs. <b>A:</b> TGF-ΒR1 inhibition by LY2157299 did not influence cell viability in any type of primary cell culture. <b>B:</b> CK2 inhibition by CX4945 significantly inhibited cell viability in both mucin- and mixed-IHCCA cell cultures starting at a 10 μM concentration, with a more pronounced effect in mucin-IHCCA cells. In CK19-mucin cell cultures expressing high levels of epithelial markers (CK19-mucin and E-cadherin mRNA), the anti-survival effect of CX4945 was observed starting at 30 μM. * = p<0.05 vs other cell cultures;** = p< 0.01 vs 10μM; & = p<0.001 vs 10μM in mixed-IHCCA. <b>C:</b> The combination of CX4945 + LY2157299 failed to increase the inhibitory effect of CX4945 on viability of mucin- and mixed- IHCCA cell culture. * = p<0.05 vs control and LY2157299. <b>D:</b> LY2157299 and CX4945 treatment, either alone or in combination, did not affect hBTSCs survival. <b>E:</b> The combination of CX4945+MK2206 (AKT inhibitor) showed additive inhibitory effects on viability of mucin- and mixed-IHCCA with respect to a single treatment; this was not observed in CK19-mucin. * = p< 0.05 vs controls; & = p<0.05 vs control and MK; $ = p<0.05 vs MK and CX alone. Mean +/- SD of N = 3–5 independent experiments.</p