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. 2020 Jun 23;10:1012. doi: 10.3389/fonc.2020.01012

Figure 5.

Figure 5

(A) Quantification of Ki67-positive nuclei stained on LAPC9 ex vivo tissue slices treated with the indicated compounds; a minimum of 5 fields per condition were analyzed. Data are reported as mean ± SD (B). Representative images of Ki67-stained uncultured and untreated tissue, as well as of tissue treated with Rapalink-1 (RLK), rapamycin (RPM), and doxorubicin (DOX) at the reported concentrations are included, with full section enclosed in caption. Scale bar, 50 μm. (C) Schematic of the in vivo mouse experiment. R and V indicate administration of Rapalink-1 or vehicle, respectively, at the indicated day. (D) Picture of LAPC9 tumors explanted from Rapalink-1-treated (bottom) and vehicle-treated groups (top) at the end of experiment. Mice weight curves (E) and tumor size measurement (F) of bilateral LAPC9 PDX tumors in mice treated with vehicle (blue) or Rapalink-1 (red). For each group, n = 3 mice; Data are reported as mean ± SEM. (G) Representative HE stainings of LAPC9 tumors from vehicle-treated (top) and Rapalink-treated (bottom) mouse groups. Scale bar, 100 μm. (H) Quantification of Ki67-positive nuclei stained on FFPE sections of LAPC9 tissues from mice treated with Rapalink-1 or vehicle; a minimum of 9 fields per sample were analyzed. (I) Quantification of western blots of LAPC9 lysates from mice treated with Rapalink-1 or vehicle. Signal from assessed targets was normalized to loading control (beta actin) for each lane (J). Summary table of viability values and of CD44+ and ALDHHi cells in LAPC9 tumors from mice treated with vehicle (purple circles) or Rapalink-1 (orange circles), analyzed by flow cytometry. Data are reported as mean ± SEM, N = 2–4. Representative flow cytometry plots of viability and CD44 expression are reported in (K). (L) Representative ALDEFluor staining dot plots for ALDHHi determination reported in (J); open histogram, unstained; orange, Rapalink-1; purple, vehicle. LAPC9 cells from tumors of mice treated with Rapalink-1 showed a non-significant increase of ALDHHi cells. A population bearing DEAB-resistant ALDH isoforms is detected in LAPC9 tumors of mice treated with vehicle (top, left panel) that is not evident in the tumors of mice treated with Rapalink-1 (top, right panel). *p < 0.05; **p < 0.01; ***p < 0.001.