Figure 5.

Figure 5.

(A) Weekly time lapse photography of a representative MCR:SATB2 melanoma cell recipient. Black arrows indicate metastases, which can already be observed 1 week post-transplantation. Related to Figure 2E–I. (B) Subcutaneous primary melanoma limiting dilution transplantation model in transparent Casper zebrafish. 300,000, 3000, 300, or 30 primary pigmented primary melanoma cells were transplanted into a subcutaneous space in running along the dorsum of irradiated Casper recipients. Ten to 12 animals per condition were used, and a total of four MCR:EGFP and five MCR:SATB2 donors were transplanted over the course of two independent experiments. (C) Representative images of MCR:EGFP and MCR:SATB2 recipients transplanted with 30 cells. (D) Engraftment of surviving recipients at 3 weeks post-transplant was used to estimate the frequency of tumor propagating cell potential with Extreme Limiting Dilution Analysis (ELDA). Estimated frequency of tumor propagating cells between MCR:EGFP and MCR:SATB2 donors (Mann-Whitney, p<0.0159*). (D) In vivo drug treatment of allotransplanted primary MCR:EGFP or MCR:SATB2 tumors with FDA-approved BRAF inhibitor Vemurafenib shows confirms SATB2 as a resistance driver. Recipients casper fish were treated by daily oral gavage with 100 mg/kg of drug or vehicle control from day 10 post-transplant to day 24 post-transplant. Animals were imaged at day 10 and 24 and pigmented tumor area was estimated using digital calipers to assess treatment response. (E) Representative image of individual treated animals. (F) Mean normalized area % change in the treated cohorts: MCR:EGFP DMSO (n = 11) +12.45, SEM 2.55 vs. MCR:EGFP BRAFi (n = 12)–50.67, SEM 7.26 vs. MCR:SATB2 DMSO (n = 17) +11.06, SEM 1.84 vs. MCR:SATB2 BRAFi (n = 16) +22.06, SEM 2.81. Unpaired two-tailed t-test p values of pairwise comparisons are shown.