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. Author manuscript; available in PMC: 2020 Jan 8.
Published in final edited form as: Nat Cell Biol. 2019 Jan 21;21(2):238–250. doi: 10.1038/s41556-018-0267-0

Figure 3: Perivascular niche-mediated chemoprotection does not depend on DTC cell cycle status.

Figure 3:

Analysis of single DTCs on stroma (MSC) or BM MVNs (MSC+EC) following treatment with a) 2500 nM doxorubicin or b) 120 nM paclitaxel. Data depict the percentage of TUNEL-positive single cells over n=3 independent experiments. In a) *** P=0.0004 and in b) P=0.0713 by unpaired, two-tailed t test. c) Schematic for experiment to determine how cell cycle status influences response to doxorubicin on BM stroma and BM MVNs. Here, cultures were seeded with T4–2 cells expressing tdTomato and a mVenus-p27K− reporter, treated with 2500nM doxorubicin and NucView405 to label apoptotic cells, and imaged for 72 hours. d) Stills from time-lapse videos on BM stroma (top) or BM MVNs (bottom). Q indicates a quiescent cell that subsequently undergoes apoptosis (white asterisk); P denotes a proliferative cell that subsequently undergoes apoptosis (white asterisk); both on stroma. Scale bar, 20 μm. e) Quantification of relative percentages of p27+ and p27− T4–2 cells that apoptose within 72h of doxorubicin treatment on BM stroma (MSC) and on BM MVNs (MSC+EC) from n=6 independent videos per condition. ** P=0.0027 comparing MSC to MSC+EC by Two-way ANOVA. f) Schematic for experiment to determine whether ‘priming’ MVNs with IGF-1 to trigger DTC proliferation sensitizes DTCs to doxorubicin. g) Representative images of quiescent, microvascular-associated DTCs at Day 10 (left), and after priming with vehicle or with 300 ng/ml IGF-1 at Day 17 (middle). Cultures were fixed and stained at this time point to analyze the proliferative status and viability of tumour cells (right). Scale bars, 50 μm. h) Percentage of TUNEL-positive DTCs per DTC cluster as a function of priming with vehicle or 300 ng/mL IGF-1, and treating with 2500 nM doxorubicin. n=30 and 32 cells from single cells or clusters of mostly 2–4 cells were analyzed for the vehicle- and IGF-1- primed conditions, respectively, over n =3 independent experiments. P=0.793 by unpaired, two-tailed t test. For a-b, e, h centre line represents the mean, and error bars the s.e.m. Statistical tests and P values are indicated. Source data are provided in Supplementary Table 1.