Abstract
One of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for successful treatment. One of the ways cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3-dimensional (3D) multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistance (MDR) and non-MDR cells does not yet exist. In the present report we describe MCSs obtained from post-diagnosed, pre-treated patient-derived (PTPD) cell lines from head and neck squamous cancer cells (HNSCC) that often develop resistance to therapy. We employed an integrated approach combining response to clinical drugs and screening cytotoxicity, monitoring real-time drug uptake, and assessing transporter activity using flow cytometry in the presence and absence of their respective specific inhibitors. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and two-dimensional (2D) monolayer cultures of the same set of cell lines. We show that MCSs provide a robust and reliable in vitro model to evaluate clinical relevance. Our proposed strategy can also be clinically applicable for profiling drug resistance in cancers with unknown resistance profiles, which consequently can indicate benefit from downstream therapy.
Highlights
One of the hallmarks of cancers is their ability to develop resistance against therapeutic agents
We further studied the reactive oxygen species (ROS) generation in both in vitro models using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay, in order to have a better understanding of the multicellular tumor spheroids (MCSs) microenvironment of the pre-treated patient-derived (PTPD) head and neck squamous cancer cells (HNSCC) cell lines, which we used for further assessment of multi drug resistance (MDR) status
Large differences could not be observed between the drug responses of 2D cell cultures and MCSs obtained from LK0902 and LK0917 cell lines to cisplatin and methotrexate, significant differences were observed between the drug responses of LK1108 2D cell cultures and MCSs to doxorubicin
Summary
One of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and twodimensional (2D) monolayer cultures of the same set of cell lines. The second strategy uses a genotype-based assay to focus on the identification of genetic anomalies arising in the treatment-resistant cell lines[19] These two tactics have been exploited to integrate numerous MDR pump inhibitors into cancer treatment modalities; the outcomes were not sufficiently effective for clinical translation[20]. We describe a fast and robust in vitro model and assay system for the profiling of drug resistance status in cancer cells using MCSs obtained from PTPD HNSCC. We validated our findings with a flow cytometry-based (FACs) assay for functional detection and profiling of MDR phenotypes in 2D cell cultures and MCSs by assessing calcein-AM uptake in the presence of specific efflux pump inhibitors
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