Single cell sequencing of de-differentiated adipocytes induced by breast tumor

Purpose: The goals of this study are to determine adipocyte de-differentiation and the fate of these de-differentiated cells in breast tumor. Methods: Library prepared followed by 10X Genomics standard protocol. Transcriptome was generated by high throughput sequencing. Overall design: We previously described a doxycycline-inducible (Tet-On) 'pulse-labeling' adipocyte-tracer system, the 'inAd-Chaser-mTmG mice' (Adiponectin-rtTA; TRE-Cre; Rosa26RmT/mG). Prior to doxycycline treatment, all cells express membrane tdTomato from Rosa26 locus; whereas upon doxycycline treatment, Cre recombinase excises the loxp-flanked tdTomato cassette and it thus constitutively activates membrane EGFP expression specifically in adipocytes. Thus, it allows us not only to label adipocytes with EGFP, but also cells subsequently derived from these adipocytes. We hypothesized that adipocytes undergo de-differentiation during breast tumor progression. To this end, we induced breast tumor in the Ad-Chaser-mTmG mice, either by crossing to PyMT-mice or by implanting EO771 breast cancer cells into the mammary gland. After the 'pulse labeling', we isolated the tumor fibroblasts either from the PyMT-tumor or allograft-tumor and sorted the EGFP+/ tdTomato- cells by FACS, respectively. These EGFP+/ tdTomato- cells were then used for single-cell sequencing.