Fig 2: Secretome analysis in sera and MAC cultureMass spectroscopy analysis exclusively detected APN A. or AZGP1 B. expression only in the breast cancer patients with nontuberculous Mycobacterium (BCa-NTMnb) infection. The spectra (panel 2) and 2 unique peptide sequences of APN (a, shaded yellow, panel 3) and AZGP1 (b, yellow, panel 3) was detected in the exosomes. C. ECM1 expression was not detectable in the MAC culture derived exosomes of NTMnb cases (N=7) but detectable in all BCa-NTMnb subjects (N=10). Human breast cancer cell lines MDA-MB-231, SUM-149 and one BCa-NTMnb subject were used as positive control for ECM1 detection. One healthy and one NTMnb subject were used as negative controls. Syntenin was used as an exosome marker and loading control. D. Model suggesting the possible role of ECM1, APOC4, APN and AZGP1. Exosome derived ECM1 in concert with other secretory and lypolysis associated proteins APOC4, APN and AZGP1 might have influenced BCa development and immune suppression leading to increased NTM susceptibility.

Fig 3: ECM1 detection and validationA. Mass spectroscopy analysis detected ECM1 expression only in the breast cancer patients with Bronchiectasis non tuberculous Mycobacterium (BCa-NTMnb) infection, but not in the healthy, normal (N1) or individuals with Bronchiectasis non tuberculous Mycobacterium (NTMnb) infection. The spectra (panel 2) and 4 unique peptides (shaded yellow, panel 3) of ECM1 detected in the circulating exosomes. B. Western blotting analysis in the circulating exosomes of the breast cancer patients with Bronchiectasis non tuberculous Mycobacterium (BCa-NTMnb) infection, healthy, normal (N) or individuals with Bronchiectasis non tuberculous Mycobacterium (NTMnb) infection. Syntenin was used as an exosome marker and loading control. C. Western blot analysis of ECM1 in additional 40 BCa subjects.

Fig 4: Role of ECM1+ exosomes on cellular transformationA. Delivery of the exosome cargo to normal human mammary epithelial cell line HMLE (arrows). Exosomes were purified from sera, labelled with exo-glow green and co-cultured with the HMLE cells for 24 hours followed by imaging under a fluorescent microscope. Magnification X 200. B. Marked epithelial to mesenchymal transition (arrows) in the HMLE cells following co-culture with the ECM1+ exosomes compared to the ECM1- exosomes. Magnification X 200. C. Increased Vimentin/CDH1 ratio (red/green signal intensity) treated cells with disrupted cell-cell contact (yellow arrowheads) and mesenchymal differentiation (white arrowheads) in the cells treated with ECM1+ exosome. Magnification X 200. D. Western blot analysis confirming decreased expression of CDH1 and augmented expression of Vimentin in HMLE cells treated with ECM1+ exosomes. E. Higher ECM1 expression in HMLE cells treated with ECM1+ exosomes compared to the control. Actin was used as loading control (D-E). F. Glutamate production was higher (p=0.001) in the HMLE cells treated with ECM1+ exosomes compared to the ECM1- exosomes.