Abstract 2024: Resistance of colorectal cancer cells to 5-FU treatment in three dimensional cell culture models under perfusion involves BCL-2
Authors: Hirt Christian , Papadimitropoulos Adam , Panopoulos Evangelos , Mele Valentina , Muraro Manuele , Cremonesi Eleonora , Droeser Raoul , Mengus Chantal , Heberer Michael , Oertli Daniel , Iezzi Giandomenica , Zajac Paul , Eppenberger-Castori Serenella , Tornillo Luigi , Terracciano Luigi , Martin Ivan , Spagnoli Giulio 
Published: May 2015

Abstract
Assessment of the effectiveness of chemotherapeutic compounds in bidimensional cell cultures poorly mimics resistance development and the tumor-microenvironment complexity in vivo. In this study we addressed the suitability of a perfused bioreactor to sustain colorectal cancer cell growth and to test established treatment regimens in an in-vitro setting as compared to normal cell culture or xenografts of the same cell line.
By culturing the HT29 colorectal cancer cell line in 3D on collagen scaffolds tissue-like structures characterized by a heterogeneous pattern of proliferating and apoptotic cells closely resembling xenografts from the same cell line as generated in immunodeficient mice could be successfully established. CDX2, a colorectal cancer tumor-marker , was expressed in 3D static and perfused cultures, but not in 2D cultures. Upon perfusion, homogeneous seeding on scaffolds could be obtained and significantly higher numbers of tumor cells were recovered, as compared to static cultures (13.7 fold increase, p&lt;0.0001).
After 48 hours treatment with clinically relevant concentrations of 5-FU no effect on numbers of cells cultured in 3D under perfusionand as xenografts was observed, as compared to a 55.8% inhibition of 2D cultures. The fraction of Ki67 negative cells (G0 cell phase), was increased after a 48 hours treatment in the 3D perfused and xenografts, but it reached again levels similar to untreated cells after 96 hours. In contrast,cells surviving n 2D cell cultures remained largely positive for Ki67. Importantly, in perfused cultures we could only observe a marginal effect on the expression of BCL-2, TRAF1, FLIP apoptosis resistance genes, as compared to a significant down regulation of their expression in 2D cell cultures and 3D static conditions. In the same line, a combination of ABT-199, a new clinically approved BCL-2 inhibitor, and 5-FU induced additional cytostatic and cytotoxic effects in 3D perfused but not in 2D cell cultures (83.3% inhibition vs. 39.8% p=0.003).
Notably, we found that BCL-2 expression in tissues from neoadjuvant treated patients with resistance to treatment was higher than in stage and grade matched colorectal cancers from untreated patients or patients responding to treatment.
Cultures in perfused bioreactors offer the possibility of generating tumour tissue-like structures over a short time period. These structures are characterized by sensitivity to chemotherapeutic treatments similar to that of xenografted tumors and significantly different from standard 2D cellular assays.In particular, our data suggest that inhibition of BCL-2 in combination with 5-FU treatment in colorectal cancer could be endowed with therapeutic potential.
Citation Format: Christian Hirt, Adam Papadimitropoulos, Evangelos Panopoulos, Valentina Mele, Manuele Muraro, Eleonora Cremonesi, Raoul Droeser, Chantal Mengus, Michael Heberer, Daniel Oertli, Giandomenica Iezzi, Paul Zajac, Serenella Eppenberger-Castori, Luigi Tornillo, Luigi Terracciano, Ivan Martin, Giulio Spagnoli. Resistance of colorectal cancer cells to 5-FU treatment in three dimensional cell culture models under perfusion involves BCL-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2014-2024