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C. Martín-Cortázar et al.
FBS contains numerous potentially chemoattractant stimuli for lymphoma cells whose receptors could be downregulated upon CDCA7 silencing, thereby account- ing for the inhibition of cell migration towards FBS. To challenge this hypothesis, we used the stomal cell-derived factor 1 (SDF1) chemokine as chemoattractant for BL-2 and Toledo cell instead of FBS. SDF1 activated BL-2 and Toledo cells migration in fibronectin-coated transwell plates (Online Supplementary Figure S4A) and this migration was markedly inhibited by CDCA7 silencing (Online Supplementary Figure S4B). However, the expression of CXCR4, the SDF1 receptor, was not modified in these cells upon CDCA7 silencing, as determined by flow cytometry analysis (Online Supplementary Figure S5). CDCA7 knockdown could potentially inhibit migration towards SDF1 by modulating the binding of lymphoma cells to fibronectin. However, SDF1 did not regulate this interaction and CDCA7 silencing also failed to modulate the binding of SDF1-treated lymphoma cells to fibronectin (Online Supplementary Figure S6).
Disruption of the tubulin and actomyosin cytoskeletons by CDCA7 silencing
Since cytoskeleton reorganization is critical for cell migration, we next investigated the role of CDCA7 in the
AC
reorganization of the microtubule and actomyosin cytoskeletons in lymphoma cells. Confocal microscopic imaging of lymphoma cells stained with fluorescently- labeled phalloidin showed a polarized distribution of fila- mentous actin (F-actin) in >40% control-transduced Toledo and BL-2 lymphoma cells (Figure 5A-D). CDCA7 silencing reordered F-actin around the cells, decreasing the percentage of cells with polarized distribution of F-actin (Figure 5A-D). In addition, CDCA7 knockdown markedly increased F-actin levels (Figure 5A, B, E). Staining of the microtubule cytoskeleton revealed its marked polarization in control-transduced lymphoma cells and that CDCA7 knockdown elicited its redistribution around the cells (Figure 5A-D). Moreover, while actin and microtubule cytoskeletons were located in opposite ends of most con- trol-transduced cells, their distribution overlapped in CDCA7-silenced cells (Figure 5A, B, F). Of note, we could not assess the polarization of tubulin and actin cytoskele- tons in DG-75 cells because these cells did not attach to the fibronectin-coated coverglasses used for these studies.
The actin-binding protein α-actinin is an important organizer of the actomyosin cytoskeleton.32 Four α-actinin isoforms have been identified (ACTN1-ACTN4), but non- muscle cells express only ACTN1 and ACTN4.32 Staining of lymphoma cells with a monoclonal antibody specific
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Figure 3. CDCA7 silencing impairs lymphoma inva- sion in vivo. (A,B) DG-75 and (C,D) Toledo cells lentivirally transduced with short hairpin (sh) con- trol (Ctl) or the CDCA7-spe- cific shRNA, sh25 and sh83, were stained and injected into the yolk sac of zebrafish embryos. Images of representative zebrafish embryos in which sh-Ctl- or sh-25- transduced (A) DG-75 or (C) Toledo cells have invaded (sh-Ctl) or not (sh- 25) their caudal region. Long scale bars, 500 mm; short scale bars, 100 mm. Percentage of embryos with >5 (B) DG-75 or (D) Toledo cells in the caudal region shown as the mean + standard error of mean of four independent exper- iments. ***P<0.001 and ****P<0.0001 (χ2 and two-tailed Fisher exact test). More than 100 embryos were examined for each condition, in four independent experiments.
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haematologica | 2020; 105(3)