Supplementary Materials Fig. microtome LEICA VT1000S and cultured in duplicate on a presoaked gelatin sponge (Johnson and Johnson, New Research, New Brunswick, NJ, USA) in six\well plates containing DMEM\F12 (Lonza; 10% FBS/1% PenStrep) (Centenera gene or scr control in T47D cells. Mammosphere formation assay was carried out on MCF7 receiving cells to verify the effect of CM from siESR1\knockdown cells. Results are expressed as relative mammosphere formation??SD, and statistical significance was tested using unpaired was used. When using CM from ER\knockdown cells, the mammosphere\forming capacity as well as holoclone formation in recipient cells was reversed, mimicking the ER\negative behaviour and suggesting a direct link between ER\ and HX\dependent secretion (Figs?1D and S1). In addition, overexpressing ER in the ER\negative cell line MDA\MB 231 results in a significant change on the effect of hypoxic secretion. However, ER overexpression did not fully revert the ER\negative behaviour to an ER\positive response (Fig.?1E). 3.2. Increased pluripotency signature of cells cultured in hypoxic conditioned media from ER\positive breast cancer cells To define and characterize subgroups of cells after treatment with CM from various cultures, we used single\cell gene expression profiling applying qPCR. In order to delineate subsets of cellular differentiation stages, we analysed genes involved in pluripotency (and and and and in cells treated with hypoxic CM compared to normoxic CM and a significant Chlorin E6 decrease in expression (Fig. S2A). These observations support the hypothesis of either an expansion of the or scr control followed by 48\h incubation in normoxic (NX) and hypoxic (HX) conditions. Progesterone expression levels were used as a functional control for the siESR1 knockdown. A holoclone assay was completed in MCF7 and MDA\MB 231 getting cells treated with CM from siESR1 knockdown MCF7 or T47D cells. Email address details are indicated as comparative holoclone development ?SD and statistical significance was tested using unpaired em t /em \check ( em n /em ?=?3). * em P /em ? ?0.05, ** em P? /em em ? /em 0.01 and *** em P? /em em ? /em 0.001 (c) Picture of MCF7 and MDA\MB 231 holoclone. Size pub represent 100?m. Just click here for more data document.(681K, pdf) Fig. S2. (a) Descriptive figures of MCF7 cells treated with normoxic (NX) and hypoxic (HX) CM from MCF7 cells for 48?h. Statistical significance was Chlorin E6 examined using unpaired em t /em \check between NX CM (shiny blue) treated MCF7 cells ( em n /em ?=?251) and HX CM (dark blue) treated MCF7 cells ( em n Chlorin E6 /em ?=?264) IL18R antibody and offered SEM. * em P /em ? ?0.05. (b) Relationship storyline for MCF7 cells treated with MDA\MB 231 CM NX (scarlet) and 231 CM HX (reddish colored) between differentiation genes and pluripotency genes. (c) An evaluation between NX CM and HX CM treated MCF7 cells shown as percentage positive cells in three different groups; Differentiation positive/pluripotency negative, double positive for differentiation and pluripotency and differentiation negative/pluripotency positive. Statistical significance was tested using Chi square test. ** em P? /em em ? /em 0.01. Click here for additional data file.(1.1M, pdf) Fig. S3. Biological processes involving the identified secreted proteins significantly changed between NX CM and HX CM from MDA\MB 468 (a) and T47D cells (b). Click here for additional data file.(5.0K, pdf) Table S1. Primer pairs. Click here for additional data file.(9.6K, xlsx) ? Chlorin E6 Click here for additional data file.(12K, docx) Acknowledgements We thank the patients from Sahlgrenska University Hospital who donated samples for this research and the Departments of Pathology and Surgery for patient consent and sample collection. This work was supported by grants from Knut and Alice Wallenberg Foundation; Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden; Swedish Cancer Society (2016\486 and 2016\438); Swedish Research Council (2012\05716, 2016\06074, 2016\01530, 015\03256 and 2017\01392); the Swedish state under the agreement between the Swedish government and the county councils; The ALF\agreement (721091 and 716321); VINNOVA; Assar Gabrielsson Research Foundation; BioCARE National Strategic Research Program at University of Gothenburg; Johan Jansson Foundation for Cancer Research; Wilhelm and Martina Lundgren Foundation for Scientific Research..