Abstract B48: Modeling of EMT process in 3D cultures and proteomic analysis of differential protein expression in EMT related drug resistance in colorectal cancer cells
Authors: Yue Xiaoshan , Hummon Amanda 
Published: May 2015

Abstract
The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Resistance exists against almost every effective anticancer drug and can develop by numerous mechanisms including decreased drug uptake, increased drug efflux, activation of detoxifying systems, activation of DNA repair mechanisms, and evasion of drug-induced apoptosis. Previous reports showed that Epithelial-Mesenchymal Transition (EMT) and loss of CDH1 are closely related to MDR. However, it is still unclear about the mechanisms and molecular events accompanied with EMT-related drug resistance. 2D- cell cultures are commonly used model systems for carrying out in vitro assays. However, recent studies have demonstrated that cells maintained in 2D-cultures display phenotypic differences from those grown in 3D-culturing systems, and the latter has more similarities to primary tumor tissues and would be a better system for in vitro studies. However, traditional EMT inducing methods, such as treating with TGF-beta, does not work well in 3D cultures. In this research, we established 3D-culture models for studying EMT process and EMT-related MDR. We first established an inducible CDH1 knock-down colon adenocarcinoma cell line (HT29) by transfecting a vector with inducible expression of shRNA against CDH1. Three shRNA sequences targeting different sites of CDH1 gene were evaluated. The vector containing a non-specific targeting shRNA sequence was used as negative control. Stable transfected cell lines were established by treating transfected cells with puromycin for at least 14 days to eliminate non-transfected cells from the culture. shRNA expression was initiated by treatment with doxycyclin and CDH1 knockdown was confirmed by RT-PCR at mRNA level and western blot at protein level. The inducible CDH1 knockdown cell lines were cultured to form 3D spheroids and CDH1 knockdown was initiated at day4. Boyden chamber assays were utilized to evaluate changes in migratory capabilities of cells with reduction of CDH1. Cell viability after drug treatment was used for evaluating drug resistance. For the proteomic study, at the optimized time point and drug concentration, SILAC 3D-cultured EMT and no-EMT cells are treated with drugs and collected and mixed with 1:1 ratio and digested with trypsin. High-pH-Low-pH-RP two-dimensional fractionation is used to reduce sample complexity. Peptides are detected through Q-Exactive mass spectrometer. Our data shows that the optimized seeding density for starting 3D cultures is 7000-9000 cells/well for HT29 colorectal cancer cells, and the culturing time should be within 14 days in order to maintain good quality 3D spheroids. By treatment with doxycyclin, the CDH1 level decreased by 50% in CDH1 knockdown HT29 cells. The cells were able to maintain in spheroid structures even with down-regulated CDH1 levels. However, the CDH1-knockdown cells clearly show higher migration ability and morphological changes on the surface of 3D spheroids. Cell viabilities after treatment with three different drugs (irinotecan, 5-fluorouracil, and oxaliplatin) were tested in inducible CDH1-knockdown 3D models with or without initiating CDH1 down-regulation. In CDH1-knockdown 3D models, the cells developed very weak drug resistance. We hypothesis that the MDR accompanied with EMT might not only result from CDH1 loss but be a synergistic effect from activation of TGF-beta pathways as well. We are currently collecting data after adding TGT-beta to our CDH1-knockdown 3D models. In conclusion, the inducible CDH1-knockdown 3D-cultures are considered to be a good model for studying EMT process and is promising to reveal the mechanisms for EMT-related MDR in combination with TGF-beta treatment.
Citation Format: Xiaoshan Yue, Amanda Hummon. Modeling of EMT process in 3D cultures and proteomic analysis of differential protein expression in EMT related drug resistance in colorectal cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B48.