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. 2023 Feb 15;13:2710. doi: 10.1038/s41598-023-29425-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Targeting PI3K/AKT/mTOR pathway in patient-derived cells. (a) Genomic alterations in PI3K/AKT/mTOR pathway, ER and cell cycle genes in PDXs. (b) Isolation of PDCs and ex vivo culture. (c) Sensitivity to tamoxifen and palbociclib. PDCs were treated with tamoxifen (0.1 μM), palbociclib (0.5 μM) or vehicle for 7 days. Waterfall plots represent the response of each PDC to treatment. The average area of treated spheres was relativized to the area of the control spheres (vehicle). PDCs were grouped as sensitive if significant differences in sphere area compared to their respective control were found; otherwise, they were classified as resistant. *p < 0.05, **p < 0.01, ****p < 0.0001. Data represent mean ± SD, one-way ANOVA followed by Tukey's test (independent replicates n = 2, with 2 experimental replicates in each group). (d) Activation of PI3K/AKT/mTOR pathway and expression of cell cycle proteins. Protein lysates from PDCs were analyzed by immunoblot with the indicated antibodies. PDCs harboring alterations in PIK3CA (red box), did not necessarily exhibit higher AKT or S6 phosphorylation levels. Tamoxifen-resistant PDCs showed an upward trend in cyclin E2 expression (blue box). Bands were quantified by densitometry and relativized to their loading control (bar graph). Data represent mean ± SD, two-sided Student’s t-test. (e) Sensitivity to alpelisib and everolimus. PDCs were treated with alpelisib (1 μM), everolimus (0.1 μM) or vehicle for 7 days. Waterfall plots represent the response of each PDC to treatment. The average area of treated spheres was relativized to the area of the control spheres (vehicle). PDCs were grouped as sensitive if significant differences in sphere area compared to their respective control were found; otherwise, they were classified as resistant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data represent mean ± SD, one-way ANOVA followed by Tukey's test (independent replicates n = 2, with 2 experimental replicates in each group). (f) Genomic alterations in PI3K/AKT/mTOR pathway. Alpelisib-resistant PDCs (474 and 313) presented PIK3CA-wt, while four out of seven alpelisib-sensitive PDCs harbored alterations in PIK3CA. All PDCs were sensitive to everolimus regardless of the presence of alterations in the PI3K/AKT/mTOR pathway. (g) Combined treatment with palbociclib, alpelisib/everolimus and fulvestrant. PDCs were treated with tamoxifen (0.1 µM), fulvestrant (0.1 µM), palbociclib (0.5 µM), alpelisib (1 µM), everolimus (0.1 µM) and their combinations for 7 days (sensitivity analysis, top panels) or 30 h (western blot, bottom panels). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data represent mean ± SD, one-way ANOVA followed by Tukey's test (independent replicates n = 2, with 2 experimental replicates in each group). Original blots/gels are presented in Supplementary Material, unprocessed western blots section.