Figure 7

LSD1 correlates with MBC CTC mesenchymal- and CSC-like characteristics. (a) CTCs were isolated from MBC patient liquid biopsies through double expression of cytokeratin (CK) and vimentin (VIM) and absence of CD45 expression on the DEPArray. Representative image from DEPArray and workflow shown. (b) CD45⁻/VIM⁺/CK⁺ CTC cell counts were determined in two samples collected six weeks apart (sample 1 and sample 2, respectively) (n = 10). (c) Immunofluorescence microscopy was performed on all fixed CTCs collected from 10 MBC patients at each time point and compared to normal donors. Representative images showing DAPI staining in CTCs is shown (n = 5 healthy donors and 10 MBC patients). (d) CTCs were separated based on LSD1-s111p TNFI relative to negative controls, primary and secondary antibody controls as well as healthy donor expression into phenotypes 1, 2 and 3 (high, moderate and low expression, respectively) and Snail TNFI and CSV TCFI were determined (n = 10). Representative images are shown for each dataset. (e) Average percentage of phenotype 1, 2 and 3 cells (n = 10). (f) CTCs were separated based on LSD1 TNFI relative to negative controls, primary and secondary antibody controls as well as healthy donor expression into phenotypes 1, 2 and 3 (high, moderate and low expression, respectively) and ALDH1A TNFI and ABCB5 TNFI were determined (n = 10). Representative image is shown for each dataset. (g) Average percentage of phenotype 1, 2 and 3 cells (n = 10). (h) Graph depicts the PCC for LSD1 and ALDH1A (n = 10). −1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. (i) Graph depicts LSD1-s111p TNFI, CSV TCFI and CD45⁻/VIM⁺/CK⁺ CTC cell count in five samples collected at indicated time points. Representative images are shown for each time point (n = 1 patient). Isolated CTCs were treated with either vehicle alone, pargyline or phenelzine and then immunofluorescence microscopy or qPCR was performed. Total florescence intensity of CSV and LSD1-s111p and Snail after treatment with (j) pargyline; or (k) phenelzine (n > 75 cells from 10 patients). (l) ALDH1 TNFI after treatment with pargyline or phenelzine (n = 20 cells from 10 patients). (m) VIM and CD44 mRNA transcript levels after treatment with pargyline or phenelzine (n = 10 pooled). Scale bars = 10 μM. ns = p > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Mann-Whitney test.