Fig. 5.

Development of an estrogen-independent HCI-013 PDX line (HCI-013EI). a HCI-013 PDX tumors were grown in mice with intact ovaries and exogenous E2 supplementation (0.4 mg E2 pellet, and E2 water starting 4 weeks post tumor implant). At end point, tumors were harvested and re-implanted into OVX mice with cleared fat pads, without E2 supplementation. Once fully grown, tumors were harvested, digested, and tumor cells were expanded in tissue culture for 2 weeks in estrogen deprived conditions (phenol red-free medium, with charcoal stripped serum and without estrogen supplementation). Tumor cells were then re-implanted into OVX mice with cleared fat pads, without E2 supplementation. The estrogen-independent tumors that grew out under these conditions were expanded, validated and named HCI-013EI. b Histological comparison of ER, PR, CAM5.2, hCD45, and mouse (ms) CD31 stains in HCI-013 and HCI-013EI PDX tumors. c Detection of Y537S homozygous ESR1 mutation in HCI-013 and HCI-013EI PDX tumors using droplet digital PCR. Graph shows 2D scatter plots with fluorescent detection of individual droplets with either gDNA or cDNA. Dots represent droplets with WT (green), or mutant ESR1 (blue) genotypes, or droplets containing both WT and mutant ESR1 (orange). Droplets without DNA content are indicated in black. d Tumor growth curves of HCI-013 and HCI-013EI under fulvestrant treatment (200 mg/kg). All data shown as mean ± SEM. e Histological comparison of ER and CAM5.2 IHCs in HCI-013EI PDX tumors treated with long-term fulvestrant or vehicle