Effects of SOX10 inhibition on melanoma cell invasion

found. Bcl-2 was found downregulated in SOX10-inhibited cells from all three cell lines (Figure 19 b). These data indicate that SOX10 inhibition can trigger intrinsic apoptosis.

To conduct this assay, melanoma cells were seeded in nutrition-free medium on top of a pored Matrigel-coated membrane. To stimulate melanoma cells to pass the Matrigel layer, the well below was filled with fibroblast-conditioned medium. This medium contains secreted factors that attract melanoma cells. Invaded melanoma cells at the bottom of the pored membrane can be stained and quantified. A schematic overview over this assay is presented in Figure 20.

Figure 20: Schematic overview of the Matrigel invasion assay.

To test invasion through an extracellular matrix- mimicking Matrigel layer, melanoma cells are seeded in nutrition-free medium on top of a Matrigel-coated transwell insert. The Matrigel layer is covering a pored membrane. Bellow the insert, the well contains fibroblast-conditioned medium, which attracts melanoma cells. Through migration and proteolysis, melanoma cells can reach the lower part of the insert. To quantify the invaded cells, the remaining cells and the Matrigel on the upper part of the insert are removed with cotton swabs and cells on the lower part are fixed and stained with a Diff-Quik staining set (Medion Diagnostics). Invaded cells can be counted by microscopic magnification.

As transfection of SOX10-targeting siRNAs reduced SOX10 expression already 24 hours after siRNA transfection (section 4.2, Figure 11), it was possible to determine the effect of SOX10 on invasion at a time point when cell death has not been induced yet.

Matrigel invasion assays with SOX10-inhibited and control WM278, WM1232, and 1205Lu cells revealed a significant reduction of melanoma cell invasion at an early time point after SOX10 inhibition (Figure 21 a-c). As expected due to their tumor’s origin, control cells of metastatic melanoma cell line 1205Lu showed the highest invasion capacity in this assay (Figure 21 c), while cells from the VGP cell line WM278 migrated slower, leading to a reduced cell yield under the membrane (Figure 21 a). Against expectations, control WM1232 invaded less than WM278 and 1205Lu (Figure 21 b).

Figure 21 d shows representative pictures of membranes with fixed and stained invaded melanoma cells.

Figure 21: Matrigel invasion assay with SOX10-inhibited melanoma cells.

Invasion through a Matrigel layer was assessed with cell lines WM278 (a), WM1232 (b), and 1205Lu (c) 24 hours after transfection of siSOX10a, siSOX10b, siControl, or without siRNA in three independent experiments. Compared to the control siRNA-transfected cells, SOX10 inhibition significantly reduced the number of invaded cells in three independent experiments. A significant difference compared to siControl with *P<0.05 is marked by an asterisk (one-way ANOVA). (d) Representative pictures of membranes after Matrigel invasion assay and cell staining with 1205Lu melanoma cells are shown.

Another in vitro model system for investigating melanoma cell invasion is the three-dimensional spheroid assay. In the first step, tumor cell aggregates are formed by cultivating melanoma cells on top of an agar-coated well. The agar prevents cell adhesion to the well’s bottom. These so-called spheroids are more representative for tumors in vivo in terms of morphology, cell-cell contacts, decreased proliferation rates, and a hypoxic core. It is a well accepted tumor model, which is broadly used for, e.g., screening of small molecule inhibitors [140].

As a second part of this assay, spheroids can be embedded in an ECM-mimicking collagen matrix that requires active migration and invasion for tumor cell spreading.

Nutrition for the cells is provided in the collagen matrix.

To perform this assay with SOX10-inhibited cells, siRNAs were transfected 24 hours before seeding the melanoma cells on top of the agar-coated wells. However, in contrast to control treatment, SOX10 inhibition impaired the formation of compact cell aggregates as tested in cell lines 1205Lu (Figure 22 a) and WM278 (Figure 22 b) at early time points (24 and 48 hours after seeding on the agar-coated wells).

Figure 22: Analysis of spheroid formation after SOX10 inhibition.

1205Lu (a) and WM278 (b) melanoma cells were subjected to spheroid formation by cultivation on agar-coated wells 24 hours post transfection with siSOX10a, siSOX10b, siControl, or no siRNA.

Representative pictures were taken 24 and 48 hours after seeding. Compared to the control cells, SOX10-inhibited cells did not form compact spheroids. Scale bar = 200 µm.

To analyze the molecular background of impaired spheroid formation, integrin expression was examined after SOX10 inhibition. Integrins mediate cell-matrix- as well

found in the cell line WM278 upon SOX10 inhibition while ITGA3 and ITGB1 were upregulated in all three analyzed cell lines (WM278, WM1232, 1205Lu) upon SOX10 inhibition (chapter 7, Figure S2). No tendency was found for ITGAV. Thus, impaired spheroid formation might be related to a change in cell adhesion proteins.

Furthermore, LIVE/DEAD staining of the forming spheroids demonstrated that no change in the amount of EthD-1-positive cells could be found in SOX10-inhibited compared to control cells (chapter 7, Figure S3 a). Even calcein-positive SOX10-inhibited cells did not form compact spheroids. Thus, impaired spheroid formation in SOX10-inhibited melanoma cells seems not to be related to increased cell death.

To further investigate the role of cell death in SOX10-mediated cell invasion cell death after SOX10-inhibition was blocked by treatment with Z-VAD and Nec-1 (chapter 7, Figure S3 b). Z-VAD is a caspase inhibitor and Nec-1 blocks necroptosis, which could be also activated by SOX10 inhibition due to an early increase of the AN- and PI-positive cell fraction (section 4.3.2, Figure 18 a). Due to this treatment, SOX10 inhibition did not induce cell death after 96 hours anymore but the invasion capacity of SOX10-inhibited melanoma cells remained significantly reduced (chapter 7, Figure S3 b). These data also indicate that reduced invasion after SOX10 inhibition is not only related to onset of cell death.

In the end, another assay was selected to further examine the effect of SOX10 on melanoma cell invasion. With the chick embryo invasion assay invasion capacity can be analyzed and quantified in vivo, located in an embryonic microenvironment [38]. This assay was performed in collaboration with Dr. Christian Busch at the Section Dermato-Oncology, University of Tübingen, Tübingen, Germany. In short, 1205Lu cells transfected with siSOX10a, siControl, or no siRNA were injected into brain vesicles at the hindbrain (rhombencephalon) of chick embryos at an early stage of their development (stages 12-13 according to Hamburger and Hamilton [94]). Melanoma cells form tumors in the dorsal neuroepithelium with single cells invading in the surrounding brain tissue. Histological analysis 96 hours after injection revealed the formation melanoma nodules in all embryos, also when SOX10-inhibited melanoma cells had been injected (Figure 23 a). Sections were stained for the proliferation marker MIB (Figure 23 b). More than 90% of tumor cells stained positive for MIB1, also tumor cells in nodules of SOX10-inhibited tumors, except for the central necrotic area.

Strikingly, the invasion of tumor cells in the surrounding host tissue was impaired upon SOX10 inhibition (Figure 23 a and b).

Figure 23: Chick embryo invasion assay with SOX10-inhibited and control 1205Lu cells.

1205Lu melanoma cells were injected into the rhombencephalic brain vesicle of chick embryos 24 hours after transfection with siSOX10a, siControl, or without siRNA. Tumor formation and invasion were analyzed 96 hours later. Representative chick embryo sections were stained with H&E (a), MIB1 (b), or anti-SOX10 antibodies (e), (f), (g). Scale bar sizes are depicted in the pictures. Arrows point to invading melanoma cells. (c) The maximum diameter of each tumor was measured microscopically in two directions. Tumor sizes did not change significantly when comparing siSOX10a- and siControl-tumors (P

= 0.1702 for diameter 1 and P = 0.5034 for diameter 2; t-test). (d) Quantification of invading cells was performed by counting three sections of the largest tumor diameter of each embryo (siSOX10a n = 6, siControl n = 4, no siRNA n = 7; one-way ANOVA versus siControl, *P < 0.001).

Regarding the control cells, invasion in single cells and clusters was evident (Figure 23 a and b, arrows). Nodules of the three experimental groups were measured in two directions and no significant difference was found indicating that the tumor formation and size was not affected by SOX10 inhibition (Figure 23 c). Quantification of invaded tumor cells in all embryos demonstrated a significant decrease of tumor cell invasion by SOX10 inhibition (Figure 23 d). SOX10 staining revealed strong nuclear signals in the tumor nodules and invading tumor cells in the control group (Figure 23 e and f) whereas SOX10 staining was reduced or absent in the tumors formed by SOX10-inhibited melanoma cells (Figure 23 g) demonstrating that the siRNA had still been preventing SOX10 expression. Thus, SOX10 inhibition seems to directly influence the invasion capacity of melanomas but not their proliferative potential at least in this model system.

Taken together, these data suggest that SOX10 has a critical influence on melanoma cell invasion independent of cell proliferation or survival.