Page 63 - Haematologica Vol. 109 - July 2024

ARTICLE - Leukemia-induced interferon signature in stroma M.W.E. Smeets et al.
ture (false discovery rate [FDR]=1.08x10-16, FDR=2.23x10-15, FDR=3.81x10-11, and FDR=1.44x10-15, respectively.) (Figure 1B and Online Supplementary Figure S7). Other IFN-re- lated genes such as IFITM1, IFI44L, IFIT1 and ISG15 were 3.1- to 4.3-fold upregulated (FDR=1.08x10-6, FDR=5.21x10-11, FDR=2.99x10-12, and FDR=4.73x10-12, respectively (Online Supplementary Figure S7). STAT1, IRF7, and IRF9 were also upregulated in MSC (FDR=9.61x10-06, FDR=1.47x10-10, and FDR=0.00137, respectively), but to a lesser extent (1.5- to 1.9-fold) compared to the previously mentioned genes (On- line Supplementary Figure S7). Vice versa, no MSC-induced change in expression of IFN-signature genes in BCP-ALL cells was found (Online Supplementary Figures S8 and S9). We observed, however, that the intrinsic IFN-related gene profile was slightly higher in mono-cultured ETV6-RUNX1 ALL cells compared to B-other or high hyperdiploid ALL samples (Online Supplementary Figures S8 and S9). Expression of IFN-related genes was remarkably higher in MSC after co-culture with ETV6-RUNX1-positive BCP-ALL cells (n=8) compared to their expression in MSC sorted after co-culture with B-other BCP-ALL cells (n=6) as ex- emplified for IFI6 (3-fold increase; P=0.031), MX1 (2.6-fold increase; P=0.048) and OAS3 (2.1-fold increase; P=0.04) in Figure 2A and for a set of 20 IFN signature genes in Figure 2B. Of particular interest is case ALL#1, which induced a similar IFN-related gene signature in MSC as ETV6-RUNX1 cases. ALL#1 turned out to be an ETV6-RUNX1-like case as defined by gene expression.20 In correspondence with
the observation for patients’ ALL cells (Figure 2A, B), we noticed that MSC exposed to the ETV6-RUNX1-positive cell line REH consistently upregulated IFNα/b genes where- as this was more variable for the non-ETV6-RUNX1 cell lines (Online Supplementary Figure S10). Exposure of MSC to healthy immune cells did not induce the typical IFN signature (n=5) (Figure 3A-C). However, we did observe induction of CXCL10 expression (P=0.018), which was most likely due to an interaction with monocytes since this in- duction was mainly observed in samples containing larger numbers of monocytes. No other IFN signature genes were induced. Thus, we found that the IFN signature was selectively induced by BCP-ALL cells, most profoundly by ETV6-RUNX1-positive ALL cells, as co-culture of MSC with healthy immune cells did not provoke a similar IFN signature.
In previous studies we showed that the viability of leuke- mic cells depends on a close and direct contact with MSC mediated via tunneling nanotubes,10 as can be visualized by reduced DiI dye transfer from pre-labeled ALL cells in the upper compartment to MSC in the bottom compartment of a transwell setting (87.6% decrease, P<0.0001) (Figure 4A, B). Although preventing the formation of tunneling nanotubes significantly reduced the expression levels of CXCL10 (3.3-fold, P=0.016), IFI44L (1.57-fold, P=0.009) and IFITM1 (1.5-fold, P=0.005), not all tested genes were affected (i.e., IFI6, IFITM1, MX1, IFI27) (Figure 4C). This suggests that tunneling nanotube-dependent signaling
AB
C
Figure 3. Normal cord blood, with the exception of mono- cyte-containing preparations, has a limited effect on expression levels of interferon-related genes in mesenchymal stromal cells. IFI6, ISG15, IFIT1, MX1, CXCL10, IFI44L, IFI27, IFITM1, and OAS3 gene expression levels measured by reverse transcriptase quantitative polymerase chain reaction in mesenchymal stromal cells (MSC) (MSC#2) sorted after co-culture with mononuclear cells from five different cord blood samples: cord blood with relatively (A) low (N=2) or (B) high (N=2) percentages of monocytes (>20%), or (C) an unknown cellular composition (N=1). Bars are means of triplicate measurements ± standard error of mean for two independent co-culture experiments. The dashed line (---) in- dicates mRNA expression levels of MSC after mono-culture, set at 100%. P<0.05 MSC: mesenchymal stromal cells.
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