DLK1, delta-like 1 homolog (Drosophila), regulates tumor cell differentiation in vivo

Abstract

The stem cell-like characteristics of tumor cells are not only essential for tumor development and malignant progression, but also significantly contribute to therapy resistance. However, it remains poorly understood how cancer cell differentiation or stemness is regulated in vivo. We investigated the role of the stem cell gene DLK1, or delta-like 1 homolog (Drosophila), in the regulation of cancer cell differentiation in vivo using neuroblastoma (NB) xenografts as a model. We found that loss-of-function mutants of DLK1 significantly enhanced NB cell differentiation in vivo likely by increasing the basal phosphorylation of MEK and ERK kinases, a mechanism that has been shown to facilitate neuronal differentiation. We also found that DLK1(+) cells are preferentially located in hypoxic regions. These results clearly demonstrate that DLK1 plays an important role in the maintenance of undifferentiated, stem cell-like phenotypes of NB cells in vivo.

Figure 1. DLK1 loss-of-function in NB cells increases the potential for neuronal differentiation in vivo

Parafin-embedded sections of (a) BE(2)C xenografts (Ctrl, DLK1-DM or DLK1-ΔCyto) or (b) ER xenografts (Ctrl or DLK-FL) were incubated with an anti-neurofilament (NF) antibody as described in Materials and Methods. Bar = 100 µm. NF⁺ cells were counted from three random fields (mean ± sem, **p<0.001; ***p<0.0001 versus control). (c) Co-immunofluorescence of DLK1 (red) and neurofilament (green) was performed in frozen tumor sections as described in Materials and Methods (bar = 100 µm). DLK1⁺ cells (white triangles) do not express NF whereas NF⁺ cells (yellow arrowheads) do not express DLK1.

Figure 2. DLK1 loss-of-function in NB cells enhances the potential for glial differentiation in vivo

Parafin-embedded sections of (a) BE(2)C xenografts (Ctrl, DLK1-DM or DLK1-ΔCyto) or (b) ER xenografts (Ctrl or DLK-FL) were incubated with an anti-glial fibrilar acidic protein (GFAP) antibody as described in Materials and Methods. Bar = 100 µm. GFAP⁺ cells were counted from three random fields (mean ± sem, *p<0.01; ***p<0.0001 versus control).

Figure 3. DLK1 promotes NB cell proliferation in vivo

Parafin-embedded sections of (a) BE(2)C xenografts (Ctrl, DLK1-DM or DLK1-ΔCyto) or (b) ER xenografts (Ctrl or DLK-FL) were incubated with an anti-Ki67 antibody as described in Materials and Methods. Bar = 100 µm. Ki67⁺ cells were counted from three random fields (mean ± sem, *p<0.01; **p<0.001 versus control).

Figure 4. DLK1 enhances angiogenesis in NB xenografts

Parafin-embedded sections of (a) BE(2)C xenografts (Ctrl, DLK1-DM or DLK1-ΔCyto) or (b) ER xenografts (Ctrl or DLK-FL) were incubated with an anti-von Willebrand Factor (vWF) antibody as described in Materials and Methods. Bar = 100 µm. vWF⁺ cells were counted from three random fields (mean ± sem, **p<0.001 versus control).

Figure 5. DLK1⁺ NB cells are preferentially localized in hypoxic regions

Serial frozen sections of a BE(2)C xenograft tumor were subjected to double immunofluorescence staining of (a, i, ii) Hypoxyprobe (green) and CD31 (red) or b, I’, ii’) DLK1 (green) and CD31 (red) as described in Materials and Methods. Bars: 200 µm (a, b); 100 µm (i, ii, i’, ii’).

Figure 6. DLK1 loss-of-function increases ERK phosphorylation

(a) Cell lysates were prepared from BE(2)C-Ctrl, DLK1-DM and DLK1-ΔCyto cells either under normal growth conditions (control) or after 16-hour serum deprivation. Phosphorylated and total proteins were examined using Western blots as described in Materials and Methods. One of three independent experiments is shown. (b) BE(2)C cells with stable expression of DLK1-DM, DLK1-ΔCyto or vector control were seeded at a density of 1 × 10⁴ cells per well in 96-well plates in the presence or absence of 1 nM retinoic acid (RA). Cell growth was analyzed by the MTS assay (Promega). Data shown are mean ± sem from three independent experiments. *p<0.02; **p<0.007 versus control at each time point. (c) BE(2)C cells with stable expression of DLK1-DM, DLK1-ΔCyto or vector control were incubated with 1nM, 100nM, 1µM RA for 48 hours. Phosphorylated and total ERK proteins were examined using Western blots. (d) Ratios of phophorylated to total p42 ERK protein were calculated using density measurements by NIH Image J. One of three experiments was shown.