Cell Competition Drives the Formation of Metastatic Tumors in a Drosophila Model of Epithelial Tumor Formation  Teresa Eichenlaub, Stephen M. Cohen, Héctor Herranz  Current Biology  Volume 26, Issue 4, Pages 419-427 (February 2016) DOI: 10.1016/j.cub.2015.12.042 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 miR-8 Cooperates with EGFR to Induce Metastasis (A–D) Confocal micrographs of third-instar wing imaginal discs of the following genotypes: (A) apG4, UAS-GFP, (B) apG4, UAS-EGFR, UAS-GFP, (C) apG4, UAS-miR-8, UAS-GFP, and (D) apG4, UAS-EGFR, UAS-miR-8, UAS-GFP. Note that the optical section in (D) underestimates the volume of the tissue, in comparison to the comparatively flat imaginal discs in (A)–(C), which retained normal epithelial organization. DNA was labeled with DAPI (red). GFP is shown in green. Scale bars, 100 μm. Figure S5A controls for UAS-transgene number. (E and F) Low-magnification images showing larvae of the following genotypes: (E) apG4, UAS-EGFR, UAS-GFP and (F) apG4, UAS-EGFR, UAS-miR-8, UAS-GFP. Note the expansion of the GFP-expressing tissue in the anterior end of the larvae in (F), where the imaginal discs expressing apG4 are located, and the GFP-expressing metastases (arrows). Some of the GFP-expressing cells at the posterior ends of these animals reflect overgrowth of the apGal4 expression domain in the hindgut, which is visible as faint stripe of GFP expression toward the posterior side (right side of E). Current Biology 2016 26, 419-427DOI: (10.1016/j.cub.2015.12.042) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Giant Cells in EGFR+miR-8 Tumors (A) Time course of epithelial tumor formation in apG4, UAS-EGFR, UAS-ban, UAS-GFP imaginal discs (in days after transgene induction). Growth of the tumor was accompanied by loss of epithelial organization. DNA was labeled with DAPI (magenta). GFP is shown in green. Note that the images at successive days are not shown at the same magnification, so that the overgrowth of the tissue is underestimated, particularly at later stages when the disc has lost epithelial organization and has grown as a ball of cells. (B) Time course of tumor formation in apG4, UAS-EGFR, UAS-miR-8, UAS-GFP imaginal discs. DNA was labeled with DAPI (magenta). GFP is shown in green. Note that the area of the GFP-expressing tissue did not increase during the first few days. By 3 days, large cells with giant nuclei were visible and the number of normally sized GFP-expressing cells decreased. Few small GFP-expressing cells remained by day 4. The mass of GFP-expressing tissue increased thereafter by increasing number of giant cells and reappearance of small GFP-positive cells. Although the transgene were only expressed in dorsal compartment cells, tissue in the ventral compartment was gradually lost (Figure S1). Scale bars, 100 μm. See also Figure S1. Current Biology 2016 26, 419-427DOI: (10.1016/j.cub.2015.12.042) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Loss of Epithelial Polarity and Metastasis (A and B) Confocal micrographs showing giant cells in apG4, UAS-EGFR, UAS-miR-8, UAS-GFP imaginal discs. (A) The DNA resembles the banded pattern observed in polytene chromosomes. DNA is labeled with DAPI (magenta). GFP is shown in green. Figure S2 shows polytene chromosome squashes from giant cells. (B) Antibodies to DE-Cadherin (DE-cad, red) and Discs large (Dlg, white) were used to visualize apico-basal polarity. Polarized localization of DE-Cadherin and Dlg is seen in the normal epithelium (not expressing GFP). The giant cells show no indication of localized DE-Cadherin or Dlg expression. The right-hand panels show a z section of the same disc. (C–E) Metastatic tumors in ap-Gal4, UAS-EGFR, UAS-miR-8, UAS-GFP larvae. GFP is shown in green. Actin is shown in red. DNA is shown in blue. (C) Invasion of a group of GFP-expressing cells through the wall of the gut. (D) Invasion of GFP expressing cells into the muscles of the proventriculus. (E) A single giant GFP-expressing cell partially wrapped around a tracheal airway. Many examples of this type were observed, raising the possibility that the giant cells might move along the trachea. (F) MARCM clones expressing UAS-EGFR, UAS-miR-8, UAS-GFP were produced in the eye-antenna discs using the eyeless-FLP system. DNA is labeled with DAPI (magenta and blue). GFP is shown in green. EGFR is shown in red. Expression of EGFR+miR-8 caused overgrowth of the eye disc. GFP- and EGFR-expressing cells were observed invading the ventral nerve cord (the boxed area is shown at higher magnification below). Scale bars, 100 μm. See also Figure S2. Current Biology 2016 26, 419-427DOI: (10.1016/j.cub.2015.12.042) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Apoptosis and Cell Engulfment apGal4, UAS-EGFR, UAS-miR-8, UAS-GFP tumorous imaginal discs. (A–C) Discs were labeled with antibody to the activated form of Caspase 3 (red) to visualize apoptotic cells. EGFR+miR-8 expression is shown by co-expression of GFP in green. Nuclei were labeled by DAPI (blue). (A) Caspase-positive cells were numerous and often located close to the GFP-expressing giant cells (e.g., arrow). (B) Time course showing progressive loss of small cells. Many small GFP-expressing cells showed activated Caspase. Caspase activation was not observed in the giant cells. Activated Caspase was also observed in wild-type cells outside the transgene expression domain (not GFP positive). (C) Higher-magnification views of giant cells to illustrate the presence of many small Caspase-positive cells adjacent to the giant cells. In some cases, Caspase-positive cells appear to have been internalized (arrows). Note also what appear to be other small GFP-expressing cells inside the giant cells. We interpret these as engulfed cells that were not Caspase positive at the time of fixation. DNA can still be detected in some of the small GFP cell corpses. (D) The disc was labeled with antibody to dMyc (red). Myc levels can be compared in the single channel image at right. Note the difference between the level of Myc in the giant cells compared to the normal tissue. Arrows highlight nascent giant cells with intermediate Myc levels. Additional examples are presented in Figure S3. (E) The disc was labeled with antibody to pMAD to visualize Dpp signaling activity (red). Note the higher level of pMAD in the nuclei of the giant cells. Additional examples are presented in Figure S3. Scale bars, 100 μm. See also Figure S3. Current Biology 2016 26, 419-427DOI: (10.1016/j.cub.2015.12.042) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Clones Expressing EGFR+miR-8 (A) MARCM clones expressing GFP (WT), EGFR, miR-8, or both EGFR and miR-8, as indicated. (B) EGFR+miR-8 clones labeled with antibody to activated Caspase 3 (red). White arrowheads (top) and red arrows (bottom) indicate nearby wild-type GFP-negative cells undergoing apoptosis. (C and D) Time-course comparing growth or EGFR+miR-8 clones after 1, 2 and 3 days in a wild-type background versus a background in which the surrounding cells expressed a tubulin>dmyc transgene. A time course of giant cell formation in EGFR+miR-8 clones shown in Figure S4. Clones were marked by coexpression of GFP. DNA was labeled with DAPI. Scale bars, 100 μm (A, C, and D) and 20 μm (B). See also Figure S4. Current Biology 2016 26, 419-427DOI: (10.1016/j.cub.2015.12.042) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Role of Engulfment and Apoptosis in the Formation of Giant Tumor Cells apGal4, UAS-EGFR, UAS-miR-8, UAS-GFP tumorous discs co-expressing the indicated UAS transgenes. (A) Control for the appearance of giant cells in EGFR+miR-8 discs. (B–D) Note the absence of giant cells in the samples expressing ELMO RNAi, Mbc RNAi, and Draper RNAi transgenes. These discs were restored to a normally polarized epithelial morphology. Metastases were not found in animals of these genotypes. Figure S6 shows controls for RNAi efficacy and controls for the effects of increased UAS copy number. This did not suppress the tumor phenotype. Figure S5 shows controls and depletion of Draper in MARCM RNAi clones. (E) Control for the appearance of giant cells in EGFR+miR-8 discs. (F and G) Giant cells did not form in the samples expressing the apoptosis inhibitors p35 or DIAP1. These discs were restored to a normally polarized epithelial morphology. Metastases were not found in animals of these genotypes. Figure S5D shows a control for Caspase activation in discs expressing UAS-p35. Scale bars, 100 μm. See also Figure S5. Current Biology 2016 26, 419-427DOI: (10.1016/j.cub.2015.12.042) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 miR-8 Acts via Peanut to Induce Cytokinesis Failure (A) Predicted target site for miR-8 in the peanut transcript (http://www.targetscan.org). Pairing to the seed region of the miRNA is indicated. (B–D) Peanut protein expression in wing discs expressing the indicated transgenes (B) under apGal4 control. Expression of miR-8 (C) and the Peanut RNAi transgene (D) reduced Peanut protein levels. (E and F) High-magnification views of wing discs showing dividing cells with centrosomes labeled with antibody to CNN protein. Note the presence of cells with three or four centrosomes in the disc expressing the Peanut RNAi transgene. (G and H) apGal4, UAS-EGFR, UAS-miR-8, UAS-GFP tumorous discs (G), with co-expression of a UAS-Peanut transgene (H) to restore Peanut expression in the UAS-EGFR, UAS-miR-8 cells. The right-hand panels in (H) show an optical cross-section the same disc. DE-Cadherin (red) and nuclei (DNA, blue) are shown to illustrate epithelial polarity. Note the absence of giant cells and the restoration of normal apico-basal polarity in these discs. Current Biology 2016 26, 419-427DOI: (10.1016/j.cub.2015.12.042) Copyright © 2016 Elsevier Ltd Terms and Conditions