Fig 2: Characterization of human epidural adipose tissue-derived mesenchymal stem cells (hEpi AD–MSCs): (a) representative image of passage 5 of human epidural AD–MSCs; (b) expression of positive markers (CD105, CD90, and CD73) of human AD–MSCs analyzed by flow cytometry (upper). Expression of negative markers (CD45, CD34, and CD14) of human AD–MSCs analyzed by flow cytometry (below). The horizontal axis represents the fluorescence intensity (FL1 = FITC, FL2 = PE), and the vertical axis indicates the cell count.

Fig 3: Induction of SNAI1 in TNBC MDA-MB-468-iSNAI1 cells upregulates NT5E/CD73 mRNA and protein expression. (A) mRNA expression of SNAI1, ZEB1, VIM, CDH1 NT5E and NT5E in EPI, EMT, and MET conditions. The expression of each gene in EMT and MET conditions was calculated relative to the EPI condition. Bars represent means from four independent experiments ± SEM (*P < 0.05 calculated by Mann Whitney). (B) Representative Western-blot showing the protein expression of SNAI1, Vimentin, and E-cadherin in MDA-MB-468 cells cultured under EPI, EMT, and MET conditions. Actin was used as a loading control. (C, D) Immunofluorescence images of SNAI1 (C), Actin, E-cadherin, and Vimentin (D) staining (in green) in MDA-MB-468 cells cultured under EPI, EMT, or MET conditions. Nuclei are stained with DAPI (blue). The images shown are representative of two independent experiments. Scale bar: 50 µm. (E) Flow cytometry analysis of the percentage (%) of MDA-MB-468 cells cultured under EPI, EMT, and MET conditions that are positive for both CD73/SNAI1 (CD73+ SNAI1+) and either CD73 (CD73+) or SNAI1 (SNAI1+). CD73 MFI is reported. Bars represent means from three independent experiments ± SEM (*P < 0.05, **P <0.01, ***P < 0.001 by unpaired t-test). (F) CD73 surface expression in MDA-MB-468 cultured under EPI, EMT, and MET conditions by flow imaging cytometry. Left panels: representative images of 104 acquisitions for each condition on brightfield (BF), CD73 and live/dead (L/D) channels. The scale bar and event number are shown. Right panel: Violin plot quantification of CD73 mean fluorescence intensity (MFI) in MDA-MB-468. Results are the average of 104 events for each condition. (***P < 0.001 calculated by unpaired t-test).

Fig 4: SNAI1 directly regulates CD73 expression in human TNBC MDA-MB-468-iSNAI1 cells. (A) Schematic representation of the different E-boxes identified in silico in the human CD73 promoter (CD73 mRNA, NCBI Reference Sequence: NM_002526). The transcription Start Site (TSS) and the ATG start codon are reported at positions +1 and +58, respectively. The control motif (+800 to +995) corresponds to a region containing no E-box and is used as a negative control. (B) ChIP was performed on MDA-MB-468-iSNAI1 cells cultured under EPI or EMT conditions using anti-SNAI1 antibodies followed by five pairs of primers flanking the identified E-boxes (E-box-1-5) or primers flanking control region. E-cadherin (E-CAD) primers were used as a positive control. For each gene, the RT-qPCR signals were normalized to control IgG. SNAI1 ChIP signal was reported as fold enrichment over IgG control. Two individual experiments (done in triplicate) were performed (*P < 0.05, **P <0.01, ***P < 0.001 calculated by unpaired t-test). (C) Correlation between NT5E/CD73 and SNAI1 in tumor mRNA from 12 TNBC patients. Pearson correlation coefficient (r) and P-value are shown. (D) Analysis process of METABRIC dataset. (E) Correlation between NT5E, SNAI1, VIM, and CDH1 gene expression in TNBC patients from METABRIC dataset (n = 258). For each association: Upper panel: correlation between NT5E/CD73 and the indicated gene. Pearson coefficients and P values are shown. Lower panel: TNBC samples were separated according to NT5E/CD73 expression level to form high (Z score = +0.5) and low (Z score =-0.5) NT5E/CD73 groups. In each group. SNAI1, VIM, and CDH1 gene expression were evaluated. Dots represent mRNA level ± SEM (**P <0.01, ****P < 0.0001 calculated by unpaired t-test). ns, not significant.

Fig 5: Expression of NT5E/CD73 mRNA and protein in mesenchymal-like MDA-MB-231 and epithelial like MDA-MB-468 TNBC cells. (A) RT-qPCR measurement of NT5E mRNA in mesenchymal-like MDA-MB-231 and epithelial-like MDA-MB-468 TNBC cell lines. NT5E expression was calculated relative to MDA-MB-468 cells. Bars represent means from four independent experiments ± SEM; *P < 0.05 calculated by Mann Whitney. (B) Flow cytometry analysis of cell surface CD73 in TNBC cells. Left panel: percentage of CD73-positive (CD73+) cells; Middle panel: representative FACS histograms of indicated cells stained with control isotype or anti-CD73 antibody. Right panel: mean fluorescence intensity (MFI) of cell surface CD73 in MDA-MB-468 and MDA-MB-231 cells. Bars represent means from three independent experiments ± SEM, **P <0.01, ***P < 0.001 calculated by unpaired t-test. (C) CD73 cell surface expression in MDA-MB-468 and MDA-MB-231 cells acquired by imaging cytometry. Left panels: representative images for each cell line acquired on brightfield (BF), CD73-PE, and live/dead (L/D) channels. The scale bar and event number are shown. Right panel: Violin plot quantification of CD73 mean fluorescence intensity (MFI) acquired by imaging cytometry in MDA-MB-468 and MDA-MB-231 cells. Results are the average of 104 acquisitions, ***P < 0.001 by unpaired t-test. (D) Left panels: Morphology of untreated (UNT)- and EGF (EGF)-treated MDA-MB-468 cells. Bar: 300µm. Middle panels: mRNA expression of CD73 and EMT markers (SNAI1, ZEB1, VIM and CDH1) in MDA-MB-468 cells treated with rhEGF (50ng/ml for 6 days). The expression level of each gene in treated (EGF) cells was calculated relative to untreated (UNT) cells. Bars represent means from four independent experiments ± SEM; *P < 0.05 calculated by Mann Whitney. Right panel: Representative Western-blot showing the protein expression of E-cadherin, SNAI1, Vimentin in (UNT)- and EGF (EGF)-treated MDA-MB-468 cells. Actin was used as a loading control. (E). Flow cytometry quantification of surface CD73. Left panel: Percentage of CD73 positive MDA-MB-468 cells treated as described in (D). Middle panel: representative FACS histograms of untreated or rhEGF-treated MDA-MB-468 cells stained with isotype or anti –CD73 antibody. Right panel: Mean fluorescence intensity (MFI) of cell surface CD73 in MDA-MB-468 treated as described in (D). Bars represent means from four independent experiments ± SEM; *P < 0.05, and ***P < 0.001 are calculated by unpaired t-test.