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Aberrant expression of SCF in CLL
patients with low SCF levels (SCFlow) (Figure 1D and E, Online Supplementary Table S4).
Microenvironmental triggering through immune receptors induces stem cell factor expression in chronic lymphocytic leukemia
We investigated the impact of BcR activation on SCF expression after stimulating negatively selected CLL cells from U-CLL clones with anti-IgM or anti-IgG (depending on the heavy chain isotype expressed by the malignant cells) for 24 h. Western blotting and flow cytometry analy- sis revealed that BcR crosslinking significantly induced SCF expression in U-CLL (n=9, FD:1.6; P=0.0039 and n=6, FD:1.5; P=0.03 respectively), while, as expected, no signif- icant change was detected in M-CLL (n=4), likely due to their overall weak responsiveness to BcR stimulation (Figure 2A, Online Supplementary Figure S3A-C).29
Subsequently, we studied the effect of TLR9 signaling on SCF expression by stimulating negatively selected malig- nant cells from U-CLL clones (n=7) and M-CLL clones (n=5) with CpG for 24 h. Flow cytometry analysis unveiled that, compared to untreated controls, TLR9 activation con-
ferred a significant increase in SCF+ cells in U-CLL (FD: 1.7; P=0.0156) as opposed to a (non-significant) decrease in M- CLL (FD: 1.65) (Figure 2B, Online Supplementary Figure S3D). Furthermore, enzyme-linked immunosorbent stud- ies revealed that TLR9 activation by CpG also triggered higher secretion of soluble SCF into the supernatants of U- CLL cell cultures compared to the amount secreted by untreated controls (FD: 9.3; P=0.0156) (Figure 2C).
We next investigated whether SCF expression is related to CLL proliferation. To this purpose, negatively selected malignant cells from eight CLL cases were stimulated with CpG/CD40L for 72 h and the percentages of SCF+ and pro- liferating (Ki-67+) CLL cells were determined. The antici- pated increase in proliferating cells (FD: 7.1; P=0.0078) (Figure 2D) was accompanied by an increase in SCF+ cells (FD: 3.7; P=0.0078) (Figure 2E). Most Ki-67+ CLL cells were also SCF+ (85% Ki-67+/SCF+ CLL cells, 15% Ki-67+/SCF- CLL cells; P=0.0078) (Figure 2F, Online Supplementary Figure S3E).
Prompted by this observed correlation between SCF and proliferation in CLL, we suppressed SCF expression in the proliferating MEC1 CLL cell line by short interfering
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Figure 2. Microenvironmental triggering induces stem cell factor in chronic lymphocytic leukemia cells. (A) Comparison of stem cell factor (SCF)+ cells (flow cytom- etry for viable/SCF+ chronic lymphocytic leukemia [CLL] cells) from IG-unmutated CLL (U-CLL) and IG-mutated (M-CLL) cases, untreated (Control) cases and cases treated with IgM/IgG. (B) Comparison of SCF+ cells (flow cytometry for viable/SCF+ CLL cells) from U-CLL and M-CLL cases, untreated (Control) cases and cases treat- ed with CpG. (C) Comparison of secreted SCF (pg/mL; determined by enzyme-linked immunosorbent assay in cellular supernatants) from U-CLL cells, untreated (Control) and CpG-treated cells. (D, E) Comparison of proliferating cells (Ki-67+) (D) and SCF+ cells (%) (E) in untreated (Control) and CpG/CD40L-treated CLL cases, as determined by flow cytometry. (F) The majority of proliferating (Ki-67+) CLL cells were also SCF+ as determined by flow cytometry. (G) Lymph node (left) and the corresponding bone marrow biopsy (right) from the same CLL patient showing CLL invading cells with aberrant SCF expression. (H) Reactive, non-CLL lymph node (left) exhibiting SCF positivity mostly in mantle zones but not in germinal centers and reactive, non-CLL bone marrow (right) exhibiting scant SCF positivity in scarce immune and endothelial cells. Interconnected dots represent one case in two different conditions while bars represent median values. The Wilcoxon P test was applied to evaluate statistical significance; *P<0.05, **P<0.01, ***P<0.001. FD: fold difference.
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