Supplementary Figure 5: NKILA is an effective target for adoptive T-cell therapy (ACT) for breast cancer patient-derived xenografts (PDX).
From: NKILA lncRNA promotes tumor immune evasion by sensitizing T cells to activation-induced cell death
(a) Representative ER−PR−HER2−/ER−PR−HER2+ PDXs are shown in comparison with the original tumor samples. Tumor identification numbers and molecular subtyping are shown on the left. Tissue sections of the primary breast tumors from the patients and of the corresponding PDXs from the same individuals were stained in H&E and immunostained for ER, PR, HER2, Ki67 and FasL (n = 7 cases of PDX). Scale bars, 100 μm. (b) The transduction efficacy of CD8+ T cells was determined by flow cytometric staining of firefly luciferase. The numbers indicate the percentages of luciferase+ T cells (mean ± SEM, for n = 3, P<0.0001). (c, d) NKILA expression in the T cells before injection into PDX-bearing mice (c) and those isolated from the harvested PDX 4 weeks after injection (d) was evaluated by qRT-PCR (mean ± SEM, n = 4 cases of PDX per group; ***P<0.001). (e) PDX growth rates determined by tumor volume with indicated treatment are shown (mean ± SEM, n = 3 mice per group per PDX case; **P < 0.01, ***P < 0.001 compared to the PDX-bearing mice without ACT treatment). (f) The expression of BCL2, BCL2L1, IER3 and GADD45B in the CD8+ T cells isolated from the indicated PDXs. Bars correspond to mean ± SEM (n = 7 cases of PDX per group). **, P<0.01, ***, P<0.001. BCL2, P<0.0001; BCL2L1, P<0.0001; IER3, P=0.0065; GADD45B, P=0.0003. (g) Immunohistochemistry staining for MUC1 in the PDX and the primary tumor of the case SYMH005. Scale bars, 100 μm. (h) PDX-infiltrating CTLs (SYMH005) were incubated with deep red cell tracker-labeled T2, T2/MUC1 or T2/HER2 cells for 12 h and T2 cell death was assessed by PI uptake using flow cytometry. The numbers shown indicate the percentages of PI+ T2 cells (mean ± SEM, n = 3 mice; ***, P<0.001 compared with the percentages of unloaded T2 cell death). (i) Intracellular perforin levels and surface-marker CD107a expression in the isolated PDX-infiltrating CTLs (SYMH005) challenged by the T2, T2/MUC1 or T2/HER2 were determined by flow cytometry. Numbers indicate the percentages of gated cells stained for perforin or CD107a (mean ± SEM, n = 3 mice; **, P<0.01 compared with the untreated PDX-infiltrating CTLs (-). Perforin: P=0.0036; CD107a: P=0.0086). P-values, unpaired two-tailed Student’s t-test (b-f, h, i).
