Cell-in-cell structures are observed in doxorubicin-treated mouse mammary tumors and breast cancer cell lines. (A) Cells from an MMTV-Wnt1 mammary tumor were plated and infected ex vivo in separate dishes with lentiviruses expressing either H2B-GFP or farnesylated-mCherry. Cells were orthotopically transplanted into the #4 mammary fat pad of C57BL/6j mice, and after tumors formed, cells were mixed and transplanted again in more mice. After these mosaic tumors formed with cells expressing two different markers, mice were treated with doxorubicin. Tumors were harvested during senescent response, sectioned, DAPI stained, imaged on a confocal microscope in three channels, and merged. Z-stack projections display 14 images taken 2 µm apart with a total z range of 24.12 µm. (B) Cells from a second MMTV-Wnt1 tumor were separately infected with lentiviruses expressing GFP and H2B-mCherry as in A, and mosaic transplants composed of these cells were treated, harvested, and imaged. Z-stack projections display 21 images taken 2 µm apart with a total z range of 38.82 µm. Scale bars in A and B represent 50 and 25 µm for wide-field and zoomed images, respectively. Representative images of cell-in-cell structures are depicted. (C and D) Populations of breast cancer cell lines expressing GFP or mCherry were mixed and treated with doxorubicin (DOXO) for 24 h as stated in Materials and methods. 8 d after DOXO addition, 4226 (C) and MCF-7 (D) cells were fixed and DAPI stained, imaged on a fluorescent microscope in three channels, and merged. White arrowheads denote mCherry vesicles present within GFP-positive, senescent cells. Scale bar represents 50 µm.