Skip to main content
Fig. 2 | Breast Cancer Research

Fig. 2

From: FGFR1 amplification or overexpression and hormonal resistance in luminal breast cancer: rationale for a triple blockade of ER, CDK4/6, and FGFR1

Fig. 2

Prolonged hormonal deprivation selects for cells with high FGFR1 mRNA levels which are resistant hormonal deprivation and fulvestrant. a Cell lines were exposed to estrogen deprivation by culturing and passaging them for > 24 months in regular culture media supplemented with 10% dextran charcoal-stripped (DCC) fetal bovine serum (FBS), what deprives FBS from estrogens. Cells undergo 4 progressive stages from the basal status (hormone-dependent) until the acquisition of resistance during a ~ 2-year process: quiescent long-term estrogen deprivation (LTED-Q; most cells have died and a number of cells remain but do not replicate), hypersensitive to estrogens (LTED-H; cells still do not replicate but if they are exposed to estrogens they replicate fast), estrogen independent (LTED-I; they are able to grow and replicate in absence of hormones but much slower than the parental), and hormonal-resistant (LTED-R; they behave in absence of hormones akin parental cells in presence of hormones). b Representative pictures of colony assays and quantification (relative plating efficiency—RPE) of MCF7, T-47D, and HCC1428 and their LTED-R variants in full or DCC media. For each cell line, RPE is depicted relatively to its own RPE in full media. c Same as in b, comparing vehicle versus fulvestrant at the depicted concentrations. d FGFR1 genetic (assessed by FISH—upper pictures), transcriptomic (assessed by RNAscope—lower pictures—and RTPCR—charts) and protein status (determined by western blot) of parental and LTED variants of MCF7 and T-47D. MCF7-FGFR1 is included in the western blot for control purposes. In FISH pictures: green dots: FGFR1 probe; red dots: chromosome 8 centromere probe. e FGFR1 copy number (FISH, above) and mRNA levels (RNAscope, below) in parental MCF7 and T-47D cell lines or mRNA levels (RNAscope) for engineered to overexpress FGFR1. The amplified HCC1428 cell lines are included for control purposes. f Colony assays and RPE of the same cell lines shown in e exposed to estrogen deprivation or fulvestrant (MCF7 cell lines, 0.5 nM; T47-D cell lines, 1.5 nM and HCC1428 cell line, 20 nM), indicating how FGFR1 mRNA high cell lines are resistant to fulvestrant regardless of amplification. Error bars: standard error. ***P < 0.001; **P < 0.01; *P < 0.05. Scale bars: FISH, 10 μm; RNAscope, 20 μm

Back to article page