Abstract 3165: 3D culture may better represent trastuzumab resistance associated with PIK3CA mutation than 2D culture

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