Fig. 2. Exosomes from T2D patient-derived adipocytes induce greater EMT- and CSC-associated gene expression in breast cancer cell models than exosomes from non-diabetic donor-derived adipocytes.

(A) Human primary subcutaneous pre-adipocytes from T2D and non-diabetic (ND) patients were differentiated and exosomes were isolated from conditioned media. MCF7 cells were treated with exosomes for 5 days and IPA was performed on EMT gene expression array data (fig. S2A, data file S3). Data are IPA-generated Z scores from N=3 independent cultures (fig. S2A and data file S3). Results are compared to vehicle control. (B) Immunofluorescence staining for vimentin and E-cadherin in MCF7 cells cultured with exosomes from ND or T2D adipocyte conditioned media, compared to control (vehicle-treated). DAPI, nuclear counterstain. Scale bar, 10 μm. For each of N=3 independent experiments, 25 images were examined by immunofluorescence. One representative image is shown, out of 25 images collected for each of the three experimental conditions with three replicates. (C) Pathways associated with cancer stem-like cell (CSCs) [N=3, as in (A)] were tested for induction by exosomes purified from adipocyte conditioned media of ND or T2D patients, and compared to vehicle control. Corresponding data in fig. S2B and data file S4. (D and E) Individual EMT genes from (A) and CSC genes from (C), respectively, were validated with PCR TaqMan probes. Data are means ± SEM from 3 independent experiments. (F) Quantitation of immunofluorescence signals in (B), as means ± SEM. Expression in each exosome experimental (exo) purified from either ND or T2D adipocyte-conditioned media was compared to vehicle control (con). * P <0.05, ** P <0.01, *** P <0.005, and ns not significant by unpaired, two-tailed t-test.