Fig 1: Tumor-siRNA-CD155 transfected cells modulate DNAM-1 expression. (a) Frequency of DNAM-1+ NK cells and (b) DNAM-1 expression (MFI) in PBMCs from 5 HDs exposed to siRNA-CD155- or siRNA-control-transfected HepG2 cells. (c) Frequency of DNAM-1 positive NK cells and (d) MFI were evaluated in NK cells of PBMCs from five HDs after exposure to primary HCC cells transfected with siRNA-CD155 or siRNA-control. Matched data were analyzed by a parametric ANOVA test.
Fig 2: Soluble CD155 correlates with advanced disease in HCC patients. (a) Soluble CD155 (sCD155) was tested in sera of 30 HD, 88 HCC and 22 no HCC subjects. HCC patients with an advanced disease are indicated as red dots. (b) DNAM-1 expression (mean fluorescence intensity, MFI) in NK cells of 16 HDs, 30 HCC subjects, and 16 no HCC subjects. (c–e) Correlation between sCD155 concentration in serum form HCC patients and FIB-4, APRI and number of platelets (PLT). (f,g) sCD155 in HCC stratified according to Child–Pugh (A, n = 75; B n = 11) and MELD (6–8, n = 46; 9–17, n = 34) disease indicators. (h) sCD155 in HCC stratified according to the grade of tumor differentiation: G1, G2, and G3 (well, moderately and poorly differentiated, respectively); G1: n = 21; G2, n = 43; G3, n = 22. Middle bars represent median values, box plots are 25% and 75% percentiles, whiskers are minimum and maximum values. The non-parametric Kruskal–Wallis or Mann–Whitney U tests were used to compare data in (a,b) and (f–h) panels. The non-parametric Spearman’s test was used to correlate sCD155 and clinical parameters ((c–e) panels).
Fig 3: CD155 expression on tumor tissues. (A) Distribution of CD155pos and CD155neg HCC specimens and representative images of immunohistochemical analysis (n = 51). Representative images of CD155 positive (upper row) and negative (lower row) expressions in HCC tumor tissues at 10X magnification, bars: 100 μm. (B) Distribution of the same CD155pos and CD155neg HCC patients stratified according to tumor grading (moderate and poorly differentiated, G2 and G3 respectively). (C) Expression of CD155 in HCC primary cells from G2 (n = 7) and G3 tumors (n = 10) evaluated by flow cytometry (MFI, calculated as CD155 MFI-isotype control MFI). Representative histograms of CD155 expression in HCC primary cells derived from a G2 and a G3 tissue. (D) Overall survival of HCC patients according to CD155 expression.
Fig 4: DNAM-1 is downregulated upon exposure to tumor cells. (a) Proportion of DNAM-1+ NK cells in LIL and matched TIL and DNAM-1 expression (as MFI) measured ex vivo by flow cytometry. The paired t-test was used to compare data. (b) The frequency of DNAM-1+ NK cells and DNAM-1 MFI was analyzed in NK cells from seven HDs in the presence or absence of Huh 7.5 cells or cell-free supernatant (SN) from Huh 7.5 cell cultures. (c) The frequency of DNAM-1+ NK cells and DNAM-1 MFI were determined in PBMCs, after exposure to Huh 7.5 cells or in the Transwell® system to prevent contact with Huh 7.5 cells (HD, n = 6). (d) Downregulation was assessed after co-culture of PBMCs from 13 HDs with 5 primary HCC cells or with HCC cell culture SN. (e) DNAM-1 downmodulation in NK cells from four HDs co-cultured with primary HCC cells expressing CD155. (f) The experiments were repeated using primary HCC cells expressing low levels of CD155. The non-parametric Friedman test for matched data was used for comparisons.
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