Figure 1.

Secreted protein screening reveals modulators of response to anti-estrogens, PI3Ki, and mTORC1i in ER+ breast cancer. (A) Flowchart of screening approach. (B) In the discovery screen, MCF-7/GFP and T47D/GFP cells were treated with or without 1 µM fulv and each of 297 recombinant secreted proteins (at the documented ED₅₀) for 7 d. Medium, protein, and drug were refreshed on day 4. GFP fluorescence was measured on days 1 and 7. Background- and baseline-subtracted fluorescence values (day 7 fluorescence − day 1 fluorescence) were used to calculate a rescue z-score for each protein: (sample well fluorescence − mean fluorescence across plate)/SD. Red, pink, light blue, and dark blue points represent proteins that rescued from fulv (z ≥ 1), partially rescued from fulv (0.3 ≤z <1), partially sensitized to fulv (−0.3 ≥ z>-1), or sensitized to fulv (z ≤ 1), respectively. (C) In the validation screen, MCF-7 and T47D cells were treated with or without 1 µM fulv and dose ranges of 24 cytokines for 5 d. Relative viable cell numbers were measured using an SRB assay. Each square represents mean of duplicates. Rescue (%) is calculated as (cytokine-treated sample/no-cytokine sample) − 1. Cytokine names are colored by ability to rescue in B. (D) In the expansion screen, MCF-7, T47D, ZR75-1, and HCC-1500 cells were treated with or without 1 µM 4-hydroxytamoxifen (T), 1 µM fulv (F), 1 µM pictilisib (P), 20 nM everolimus (E), and dose ranges of 13 validated cytokines (from C) for 5 d. Cytokine names are colored by ability to rescue in B. Data were analyzed as in C. (E) Nine rescue hits from the discovery screen with complete mRNA and protein expression data were used to derive a TME Expression Score, which is the mean of z-scores calculated based on relative mRNA and protein (IHC) expression levels in ER+ breast TME tissues and cell types.