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Figure 2 (A) Phalloidin staining for F-Actin in primary zebrafish melanoma cell culture, reveals the presence of F-actin-positive foci in MCR:SATB2 (45–3) cells, which are not present in MCR:EGFP (zmel1) cells. Scale bar is 20 μm. Primary tumor immunohistochemistry shows MCR:SATB2 tumors abundantly express Cortactin. Scale bar 50 μm. (B) MCR:SATB2 (63–4) cells show increased Oregon green 488-conjungated gelatin degradation, compared to MCR (CK5) cells 24–25 hr post-seeding. Scale bar is 50 μm. (C) Percentage of cells with degraded gelatin after SATB2 induction in iSATB2 human melanoma cell lines A375, SKMEL2, and SKMEL28 transduced with pInducer20-SATB2. Cells were seeded on gelatin in media +/- doxycycline after 48 hr +/- doxycycline induction. (D) Upon SATB2 induction in human melanoma cell line SKMEL2, cells form invadopodia and show increased matrix degradation Scale bar is 20 μm. (E) Orthotropic allograft migration assay in transparent Casper zebrafish. A total of 300,000 primary pigmented primary melanoma cells were transplanted into the dorsum of irradiated Casper recipients, which are monitored for the formation of pigmented distant metastasis that have spread past the anatomical midline. Metastases are represented as black circles. (F) Pooled recipient data at the experimental end point at 3.5 weeks post-transplantation, 59.4 ± 2.3% (SEM; n = 76 total recipients, grafted from seven individual donor tumors) of MCR:SATB2 transplants formed distant metastasis, compared to 21.8±4.5% (SEM; n = 37 total recipients grafted from five individual donor tumors) of EGFP-control transplants (p<0.0001). MCR:EGFP transplants developed an average 1.1 ± 0.4 (SD) distant metastasis per fish, versus 4 ± 4.2 (SD) in MCR:SATB2, where a maximum of 20 metastases per fish was observed. (G) MCR:SATB2 recipients more frequently developed bilateral (28.9%), and internal metastases (22.4%) compared to MCR:EGFP donors (5.4%), showing spreading along the neural tube (14.5% versus 2.7%). (H) At 3.5 weeks-post transplantation, compared to MCR:EGFP donor transplants, MCR:SATB2 transplants develop distant and internal metastases. Histopathology of MCR:SATB2 Casper recipients showing a (I–I) hypodermal metastasis, and (I–II) internal metastasis with spreading along neural tube.