Page 64 - Haematologica Vol. 109 - July 2024

ARTICLE - Leukemia-induced interferon signature in stroma M.W.E. Smeets et al.
only partially contributes to the BCP-ALL-induced gene expression changes in MSC.
To examine whether the upregulation of IFN-related genes in MSC had a causal effect on the viability of primary BCP-ALL cells, IFI6, MX1, IFI27, IFI44L and ISG15 were len- tivirally silenced resulting in an efficient knockdown of 67-93% for individual genes (Online Supplementary Figure
S11A-E). The viability of MSC upon knockdown of several IFN-related genes was largely unaffected compared to controls, except for shIFI27-treated MSC (P<0.0001) (On- line Supplementary Figure S11F). Efficient knockdown of CXCL10 in MSC could not be achieved by four different short hairpin constructs tested (data not shown). Silencing of the strong IFN gene signature observed in MSC upon
AC
B
Figure 4. Direct cell-cell contact contributes to induction of Interferon-related gene expression in mesenchymal stromal cells.
(A) Schematic overview of the experimental setup of direct co-culture and transwell co-culture of REH cells with mesenchymal stromal cells (MSC) (MSC#2) using 0.4 μm pore-size transwells. (B) Percentage of DiI-positive MSC after direct (circles, white bars) and transwell (squares, gray bars) co-culture with REH leukemic cells for 40 hours. Dots represent averaged duplicate measure- ments; bars represent means ± standard error of mean (SEM) for three independent experiments. (C) Interferon-related gene expression levels, measured by reverse transcriptase quantitative polymerase chain reaction, in MSC sorted after direct (circles, white bars) or transwell (squares, gray bars) co-culture with REH cells for 40 hours. Dots represent averaged triplicate measure- ments; bars represent means ± SEM for four independent experiments. The dashed line (---) indicates mRNA expression levels of MSC after mono-culture, set at 100%. *P<0.05, **P<0.01, ****P<0.0001. MSC: mesenchymal stromal cells.
AB
Figure 5. Silencing of interferon-related genes in mesenchymal stromal cells does not affect the viability of primary B-cell pre- cursor acute lymphoblastic leukemia cells. Fold-change in percentage of viable leukemic cells after 72 hours of co-culture with transduced mesenchymal stromal cells (MSC) (MSC#2). A non-silencing control - short-hairpin luciferase (NSC-shLuc) is used as reference. (A) ETV6-RUNX1 (N=3), and (B) B-other (N=2) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Five shR- NA per gene were tested for knockdown efficiency; only shRNA resulting in >60% knockdown (shIFI6 #4, shISG15 #4, shMX1 #3/5, shIFI44L #1/3, and shIFI27 #2, see Online Supplementary Methods) were used. Bars represent means ± standard error of mean of duplicate measurements for three independent experiments. A dashed line (---) indicates that viability of ALL cells is equal to the viability of ALL cells co-cultured with NSC-shLuc-MSC (fold-change = 1; gray bars). ALL: acute lymphoblastic leukemia; NIC: non-infected control; NSC-shLuc: non-silencing control - short-hairpin-luciferase; sh: short hairpin.
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