Abstract 3165: 3D culture may better represent trastuzumab resistance associated with PIK3CA mutation than 2D culture
Authors: Tatara Takashi , Mukohara Toru , Tanaka Rina , Shimono Yohei , Toyoda Masanori , Kiyota Naomi , Hirai Midori , Kakeji Yoshihiro , Minami Hironobu 
Published: July 2017

Background: It is becoming clear that presence of PIK3CA mutations is associated with lower pathological complete response rate in patients with HER2-overexpressing breast cancer when treated with trastuzumab-based chemotherapy in neo-adjuvant settings. On the other hand, in in vitro studies using traditional 2-dimentional (2D) cell culture, differential cellular or biochemical response to trastuzumab between PIK3CA-mutant (mt) and -wild-type (wt) cells has not been clearly demonstrated. Further, while tumor shrinkage is occasionally observed in breast cancer patients who are treated with trastuzumab as a single agent, cyto-toxic effect of trastuzumab is not simulated in 2D culture models. Recently, many studies reported 3-dimensional (3D) cell culture mimics in vivo environment better than 2D culture. Therefore, we hypothesized that 3D culture better represents clinically-observed trastuzumab resistance associated with PIK3CA mutation than 2D culture, and decided to comparatively investigate cellular and biochemical response to trastuzumab in HER2-amlified PIK3CA-mt and -wt cell lines cultured in 2D and 3D environments.

Method: HER2-amplified breast cancer cell lines, BT474 (PIK3CA-wt), and UACC893 and MDA-MB361 (PIK3CA-mt) were seeded (day 0) and allowed to grow in 2D and 3D (NanoCluture Plate®, ORGANOGENIX, Kanagawa, Japan) cell culture plates. On day 3, trastuzamab (10 µg/ml) and/or BKM120 (1 and 5 µM), a PI3K inhibitor, were added. The effect of the drugs on cell growth was evaluated with WST-8 assay on days 3 through 7. Apoptosis and cell signaling were evaluated using Western blot on day 6 and days 3 through 5, respectively.

Result: In PIK3CA-wt BT474, treatment with trastuzumab led to decrease in cell number, indicating cyto-toxic effect, only in 3D culture but not in 2D culture. In PIK3CA-mt UACC893 and MDA-MB-361 cell lines, treatment with trastuzumab resulted in no cellular reduction either in 2D or 3D cultures. Consistently, increase in cleaved PARP, indicative for apoptosis, was observed only in 3D-cultured BT474 but not in 2D-cultured BT474 or two PIK3CA-mt cell lines. Furthermore, in BT474, greater decrease in phosphorylation of AKT (p-AKT) was observed in 3D culture than in 2D culture. In PIK3CA-mutant cell lines, trastuzumab did not change level of p-AKT regardless of cell culture conditions. In PIK3CA-mutant UACC893, combined treatment with trastuzumab and BKM120 resulted in greater increase in expression of cleaved PARP than either drug alone.

Conclusion: Trastuzumab-induced inhibition of PI3K/AKT pathway and resultant apoptosis in HER2-overxpressing PIK3CA-wt cells may be observed in 3D culture, which may be simulating cyto-toxic effect of trastuzumab by itself observed in clinic. Furthermore, 3D cell culture represents the resistance to trastuzumab associated with PIK3CA mutation better than 2D cell culture.

Citation Format: Takashi Tatara, Toru Mukohara, Rina Tanaka, Yohei Shimono, Masanori Toyoda, Naomi Kiyota, Midori Hirai, Yoshihiro Kakeji, Hironobu Minami. 3D culture may better represent trastuzumab resistance associated with PIK3CA mutation than 2D culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3165. doi:10.1158/1538-7445.AM2017-3165