Abstract 29: Activity of trastuzumab emtansine (T-DM1) in 3D cell culture
Authors: Boyer Jean Z. , Phillips Gail Lewis , Nitta Hiro , Garsha Karl , May Eric , Admire Brittany , Kraft Robert , Peccarelli Megan , Zamorano Andre , Gill Scott , Dennis Eslie , Vela Liz , Towne Penny 
Published: August 2019

Abstract
Introduction: Cell spheroids/aggregates generated from three dimensional (3D) cell culture methods are similar to real tumors in terms of tissue morphology, biology, and gene expression. We performed a unique 3D cell culture drug efficacy study with Trastuzumab emtansine (T-DM1) across a number of breast cancer cell lines that were previously investigated in 2D cell culture (Lewis Phillips, et al, 2008). We obtained significantly different results for some cell lines grown as 3D spheroids/aggregates when compared to those grown as 2D cultures.
Methodology: We performed 3D cell culture and produced FFPE blocks (using novel methods) from 3D spheroids/aggregates to determine HER2 (IHC) protein expression levels for five cell lines: SK-BR-3; BT-474; MDA-MB-361; MDA-MB-175 and MCF-7. We also performed HER2 gene-protein assay (HER2 GPA) to determine HER2 gene amplification along with IHC protein expression status in four cell lines: SK-BR-3; BT-474; MDA-MB-361 and MDA-MB-175. Drug (T-DM1) activity testing using CellTiterTM-Glo 3D cell viability assay was performed on 3D cell spheroids/aggregates for comparison with 2D cells. Images were obtained of T-DM1 internalization in BT-474 cells and spheroids using pHrodo™ iFL Human IgG Labeling Reagent.
Results: In 3D spheroids/aggregates, HER2 IHC staining and GPA assay showed for SK-BR-3 and BT-474 HER2 3+ expression and HER2/CEP17 of ≥ 2; MDA-MB-361 cells with HER2 2+ expression and HER2/CEP17 of ≥ 2.0; MDA-MB-175 cells with HER2 1+ expression and HER2/CEP17 &lt; 2.0 and MCF-7 cells with HER2 0+(IHC staining only without HER2 GPA data). Some of the 3D spheroids/aggregates in MDA-MB-361 cells showed heterogeneous expression of HER2 protein. The IC50 values of 3D spheroids/aggregates for some cell lines were significantly higher than were demonstrated for cell lines grown in 2D cell cultures. The fold changes between 3D spheroids and 2D cells (72h T-DM1 treatment time) are: 4.2 for SK-BR-3; ≳ 10 for BT-474 and 22 for MDA-MB-361. Additionally, the fluorescent images showed that a longer incubation time was required for the T-DM1 drug (3 µg/ml) to be internalized effectively into BT-474 3D spheroids; for example, about 120h for 3D spheroids in comparison to about 36h in 2D cells. Interestingly, the 3D spheroids incubated for 120h with T-DM1 (470 µm) are smaller in size than 3D spheroids in the control group (600 µm) incubated for 120h without T-DM1 treatment.
Conclusions: Drug efficacy studies performed on 3D cultured spheroids/aggregates are expected to be very important and biologically relevant for determining drug activity in tumor tissue. Our drug efficacy study using 3D cell culture demonstrated greater concentrations of T-DM1 and longer incubation times were required than for cells grown as 2D in some cell lines, likely due to less efficient internalization.
Citation Format: Jean Z. Boyer, Gail Lewis Phillips, Hiro Nitta, Karl Garsha, Eric May, Brittany Admire, Robert Kraft, Megan Peccarelli, Andre Zamorano, Scott Gill, Eslie Dennis, Liz Vela, Penny Towne. Activity of trastuzumab emtansine (T-DM1) in 3D cell culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 29.