Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity (2020)
Figure 7

Figure 7. Estrogen and Palbociclib sensitivity of CTC-ITB-01:

A Equal numbers of CTC-ITB-01 cells were seeded on 6-well culture plates and grown in the presence or absence of different 17-β-estradiol concentrations as indicated. After 10 days cells were fixed and stained with Coomassie blue staining (right panel). Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations (right panel). Scale bar 200 µm.

B Stable knockdown of ER-alpha within CTC-ITB-01 cells was performed by using lentiviral transfer of non-targeted (scramble) or two different targeted shRNAs against ER-alpha. CTC-ITB-01 cells were seeded on 6-well plates. After 10 days cells were fixed and stained (left upper panel). Macroscopic photos are shown of the fixed cells (left lower panel), scale bar 200 µm. Western blots of lysates from knockdown of ER-alpha expression in CTC-ITB-01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies. The extracts were obtained 72h after lentiviral transduction (right panel). Where indicated, western blots were visualized at long exposure (l.e.) and/or short exposure (s.e.).

C CTC-ITB-01 and MCF-7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated. 24h after E2 deprivation or E2-treatment cells were harvested. Western blots of the protein lysates were probed with antibodies targeting ER-alpha, FOXM1, Bcl-2, PR-A, PR-B and ID1, actin was used as loading control, visualized at long exposure (l.e.) and/or short exposure (s.e.).

(D,E) (D) Growth curves of CTC-ITB-01 and (E) MCF-7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.

(F,G) (F) Influence of Palbociclib on CTC-ITB-01 growth after 300 hours and (G) MCF-7 after 80 hours. Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.

(H,I) (H) Effect of Palbociclib on the growth of CTC-ITB-01 cells and (I) MCF-7 cells. Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.

Table 1: Functionally relevant mutations in CTC-ITB-01, vaginal metastasis and primary tumors identified by whole-exome sequencing

TP53

All four samples were analyzed for rare (MAF, minor allele frequency, <1%), functionally relevant variants with an allele frequency of 10% of reads and higher in at least one of the samples. 219 cancer-associated genes curated from the COSMIC, HGMD and OMIM databases were analyzed and 9 pathogenic mutations in 6 genes were identified. Gene symbol were used as approved by the HGNC, and location of variants on cDNA and protein (one letter code) level, and allele frequency is shown. del, deletion; ins, insertion; *, stop codon; fs, frameshift.

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