Fig 2: Immunohistochemical analysis of pluripotency, differentiation, and epithelial-to-mesenchymal transition markers in H9 cells cultured on Matrigel. (a) Immunohistochemistry with no permeabilisation using antibodies for vimentin (VIM), SSEA-4, TRA-1-60, R-10G, E-cadherin (E-cad), and SSEA-1. (b) Immunohistochemistry with permeabilisation using antibodies for VIM, OCT3/4, NANOG, ZEB1, SNAI1/2, and TWIST. Nuclei were counterstained with DAPI. The inset shows a 3 × enlargement of the figure. The scale bar represents 200 µm.

Fig 3: 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.

Fig 4: mRNA and miRNA expression analysis for cells in substate 1 and substate 2. (a) mRNA microarray analysis. Analysis of significance was performed using Z-score of substate 2 versus substate 1 (see Supplementary Fig. S1). The symbols of the obtained mRNAs were converted to NCBI Entrez Gene IDs by BioMart based on GRCh38.p12. GO enrichment analysis was performed using the IDs (false-discovery rate [FDR] = 0.05, fold enrichment = 1.5; see Supplementary Tables S2–S3). (b) Heat map showing pluripotency, epithelial-to-mesenchymal transition, and trilineage markers. The map was constructed using the mRNA microarray data of pluripotency markers (CDH1 [E-cadherin], CDH2 [N-cadherin], LIN28A, LIN28B, NANOG, POU5F1 [OCT3/4], and SOX2), epithelial-to-mesenchymal transition markers (SNAI1, SNAI2, TWIST1, TWIST2, ZEB1, ZEB2, and VIM), and trilineage markers (ACTA2 [aSMA], AFP, OTX2, SOX17, T [brachyury], and TUBB3 [TUJ1]) (see Supplementary Table S4). The red and blue coloured fold change (FC) numbers indicate significantly higher and lower expression in substate 2 than in substate 1, respectively. (c and d) miRNA microarray analysis of the cells (c) and exosomes (d). The miRNAs that showed a 2-fold difference in expression between substate 1 and substate 2 cells were subjected to analysis of significance using paired t-test with multiple testing correction (q < 0.05; Supplementary Tables S5–8). The miRNA target prediction was performed by Targetscan 7.2. The predicted up- and downregulated mRNAs were derived from the down- and upregulated miRNAs, respectively (see Supplementary Tables S9–S12). The symbols were converted to NCBI Entrez Gene IDs using BioMart based on GRCh38.p12 (see Supplementary Tables S9–S12). GO enrichment analysis was performed using the IDs of genes targeted by more than 50% miRNAs (false-discovery rate [FDR] = 0.05, fold enrichment =1.5; see Supplementary Tables S13–S16).