Investigation of Evolutionary Dynamics for Drug Resistance in 3d Spheroid Model System Using Cellular Barcoding Technology
Gizem Damla Yalcin, Kubra Celikbas Yilmaz, Tugce Dilber, Ahmet Acar
Abstract
Complex evolutionary dynamics governing the drug resistance is one of the major challenges in cancer treatment. Understanding these mechanisms requires a sequencing technology with higher resolution to delineate whether pre-existing or de novo drug mechanisms are behind the drug resistance. Combining this technology with clinically very relevant model system, namely 3D spheroids, better mimicking tumorigenesis and drug resistance have so far been lacking. Thus, we sought to establish dabrafenib and irinotecan resistant derivatives of barcoded 3D spheroids with the ultimate aim to quantify the selection-induced clonal dynamics and identify the genomic determinants in this model system. We found that dabrafenib and irinotecan induced drug resistance in 3D-HT-29 and 3D-HCT-116 spheroids are mediated by pre-existing and de novo resistant barcodes, indicating the presence of polyclonal drug resistance in this system. Moreover, whole-exome sequencing analysis found chromosomal gains and mutations associated with dabrafenib and irinotecan resistance in 3D-HT-29 and 3D-HCT-116 spheroids. Last, we show that dabrafenib and irinotecan resistance are also mediated by multiple drug resistance by detection of upregulation of the drug efflux pumps, ABCB1 and ABCG2, in our spheroid model system. Overall, we present the quantification of drug resistance and evolutionary dynamics in spheroids for the first time using cellular barcoding technology and the underlying genomic determinants of the drug resistance in our model system.
Introduction
Three-dimensional (3D) cell culture models, which provide a more physiologically relevant environment than typical two-dimensional (2D) cell culture, have emerged as significant tools in cancer research [1–3]. In recent years, 3D spheroids have received an increased interest among the many 3D culture systems [4, 5]. 3D spheroids are multicellular aggregates that more precisely mirror the structural and functional properties of tissues, providing useful models for investigating cancer progression and drug resistance [6]. Indeed, 3D cell culture models enable cells to maintain physiological characteristics such as cell polarization, differentiation, and cell-to-cell signalling, which are critical for understanding the processes in cancer progression and drug resistance [7, 8].
Materials and methods
Cell culture
HT-29 colorectal cancer cell line and its 3D spheroid derivative (3D-HT-29) were maintained in Dulbecco’s Modified Eagle Medium (Biological Industries, Israel) supplemented with 10% Fetal Bovine Serum (Gibco, USA), 1% (v/v) Penicilin-Streptomycin (Biological Industries, Israel) and 1% (v/v) L-Glutamine (Biological Industries). HCT-116 colorectal cell line and its 3D spheroid derivative (3D-HCT-116) were maintained in Roswell Park Memorial Institute‐1640 medium (Biological Industries, Israel) with 10% Fetal Bovine Serum, 1% (v/v) Penicilin-Streptomycin and 1% (v/v) L-Glutamine. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. The mycoplasma negativity in cell lines was routinely confirmed by a PCR-based method [40].
Results
Cellular barcoding and establishment of 3D spheroids of HT-29 and HCT-116 cell lines
First, utilizing the Cellecta lentiviral barcode library, cellular barcoding of the HT-29 and HCT-116 cell lines was achieved. In the initial HT-29 and HCT-116 cell populations, a multiplicity of infection (M.O.I) of 0.1 was employed to introduce a unique barcode per single cell. This was then followed by the establishment of 3D spheroids of HT-29 (3D-HT-29) and HCT-116 (3D-HCT-116) in a poly-HEMA coated flasks (Fig 1A). During in vitro culturing, 3D-HT-29 and 3D-HCT-116 spheroids exhibited full 3D forms and displayed spheroid characteristics (Fig 1B and 1C). Morphological characteristics examined under the light microscope showed compact and round morphology for both 3D-HT-29 and 3D-HCT-116 spheroids. Size of the spheroids formed by either cell type exhibited comparable results except only in a few areas where 3D-HCT-116 spheroids bigger in size were observed. Smooth surfaces observed in both spheroids limited to estimate total number of cells within the inner layers of these formations. Despite these observations which were made under the light microscope was informative, using scanning electron microscopy can provide more in-depth understanding to better characterize spheroids morphologically.
Discussion
In this study, we show quantitative measurements of clonal dynamics, via cellular barcoding technology, induced by dabrafenib- and irinotecan-resistance in 3D spheroids generated from HT-29 and HCT-116 cell lines. The cellular barcoding method used in the current 3D spheroid experimental system helped to identify the presence of both pre-existing and de novo barcodes under dabrafenib and irinotecan-mediated selection pressures, exhibiting polyclonal drug resistance. Furthermore, we discovered that dabrafenib and irinotecan-induced drug resistance were mediated by CNVs and SNVs observed in 3D-HT-29 and 3D-HCT-116 spheroids, which helped in understanding the underlying genomic alterations of drug resistance in our 3D-spheroidal experimental system. Overall, our study provides new insights into quantitative measurements and genomic determinants in the 3D-HT-29 and 3D-HCT-116 spheroid systems.
Acknowledgments
We thank The Scientific and Technological Research Council of Turkey (TUBITAK).
Citation: Yalcin GD, Yilmaz KC, Dilber T, Acar A (2023) Investigation of evolutionary dynamics for drug resistance in 3D spheroid model system using cellular barcoding technology. PLoS ONE 18(9): e0291942. https://doi.org/10.1371/journal.pone.0291942
Editor: Chien-Feng Li, National Institute of Cancer Research, TAIWAN
Received: July 18, 2023; Accepted: September 9, 2023; Published: September 26, 2023
Copyright: © 2023 Yalcin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are available at ArrayExpress, the accession number is E-MTAB-13216.
Funding: This study was funded by Türkiye Bilimsel ve Teknolojik Araştırma Kurumu, grant number: 118C197 to Ahmet Acar and Orta Doğu Teknik Üniversitesi, grant number: AGEP-108-2022-1094 to Ahmet Acar. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0291942#abstract0
