Figure 6

LSD1 inhibition suppresses chemotherapy-induced EMT, CAFs and promotes a M1 macrophage infiltration. Balb/c nude mice were treated with either vector only, nab-paclitaxel 30 mg/kg, phenelzine 40 mg/kg, or nab-paclitaxel 30 mg/kg + phenelzine 40 mg/kg. Tumours were excised, cut in half, digested with collagenase IV into single cell suspensions and immunofluorescence microscopy was performed on pooled samples and TNFI or TCFI was determined after probing with: (a) anti-LSD1, anti-LSD1-s111p; or (b) anti-Snail, anti-EGFR, anti-CSV, anti-ABCB5, or anti-ALDH1A1 (n ≥ 15 individual cells from 5 pooled). (c) PCC was determined for FAP and LSD1 (n = 20 individual cells from 5 pooled). −1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. (d) Graphs indicate FAP TNFI and CCL2 TCFI (n = 20 individual cells from 5 pooled). Representative image is shown for each data set. Scale bars = 10 µM. (e) Graph represents the total number of F4/80⁺ cells in individual tumours for each group as determined by flow cytometry (n ≥ 4). (f) TCFI of F4/80 (n = 60 individual cells from 5 pooled). Representative image is shown for each data set. Scale bars = 20 µM. (g) All cells were probed with anti-F4/80 and either anti-CCR7, anti-CD38, anti- CD206 or anti-EGR2. Cells that were determined to be F4/80-positive were then analyzed for their expression of M1- or M2-activated macrophage markers. TCFI was then determined for (h) CCR7, CD38; (i) CD206 and EGR2 (n ≥ 40 individual cells from 5 pooled). Black = control; red = nab-paclitaxel; blue = phenelzine; green = nab-paclitaxel + phenelzine. All data represents the mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Mann-Whitney test.