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Maximizing the Value of Cancer Drug Screening in Multicellular Tumor Spheroid Cultures: A Case Study in Five Head and Neck Squamous Cell Carcinoma Cell Lines.

ABSTRACT: With approval rates <5% and the probability of success in oncology clinical trials of 3.4%, more physiologically relevant in vitro three-dimensional models are being deployed during lead generation to select better drug candidates for solid tumors. Multicellular tumor spheroids (MCTSs) resemble avascular tumor nodules, micrometastases, or the intervascular regions of large solid tumors with respect to morphology, cell-cell and cell-extracellular matrix contacts, and volume growth kinetics. MCTSs develop gradients of nutrient and oxygen concentration resulting in diverse microenvironments with differential proliferation and drug distribution zones. We produced head and neck squamous cell carcinoma (HNSCC) MCTSs in 384-well U-bottom ultra-low-attachment microtiter plates and used metabolic viability and imaging methods to measure morphologies, growth phenotypes and the effects of 19 anticancer drugs. We showed that cell viability measurements underestimated the impact of drug exposure in HNSCC MCTS cultures, but that incorporating morphology and dead-cell staining analyses increased the number of drugs judged to have substantially impacted MCTS cultures. A cumulative multiparameter drug impact score enabled us to stratify MCTS drug responses into high-, intermediate-, and low-impact tiers, and maximized the value of these more physiologically relevant tumor cultures. It is conceivable that the viable cells present in MCTS cultures after drug exposure arise from drug-resistant populations that could represent a source of drug failure and recurrence. Long-term monitoring of treated MCTS cultures could provide a strategy to determine whether these drug-resistant populations represent circumstances where tumor growth is delayed and may ultimately give rise to regrowth.

SUBMITTER: Kochanek SJ

PROVIDER: S-EPMC7343231 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture (3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers, served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67, cleaved caspase-3, pan-cytokeratin, p16INK4a, CD45, ∝smooth muscle actin and vimentin over time, immunohistochemistry and immunofluorescence staining were performed Patient checkup data were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and 3D-OTCs (day 14) of HPV-driven tumors. However, p16INK4a expression levels were varying. Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth and might, in the future, serve as a platform to perform sensitivity testing upon treatment to predict therapy response.

Project description:Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer worldwide. Radiotherapy, with or without surgery, represents the major approach to curative treatment. However, not all tumors are equally sensitive to irradiation. It is therefore of interest to apply newer system biology approaches (e.g., metabolic profiling) in squamous cancer cells with different radiosensitivities in order to provide new insights on the mechanisms of radiation response. In this study, two cultured HNSCC cell lines from the same donor, UM-SCC-74A and UM-SCC-74B, were first genotyped using Short Tandem Repeat (STR), and assessed for radiation response by the means of clonogenic survival and growth inhibition assays. Thereafter, cells were cultured, irradiated and collected for subsequent metabolic profiling analyses using liquid chromatography-mass spectrometry (LC-MS). STR verified the similarity of UM-SCC-74A and UM-SCC-74B cells, and three independent assays proved UM-SCC-74B to be clearly more radioresistant than UM-SCC-74A. The LC-MS metabolic profiling demonstrated significant differences in the intracellular metabolome of the two cell lines before irradiation, as well as significant alterations after irradiation. The most important differences between the two cell lines before irradiation were connected to nicotinic acid and nicotinamide metabolism and purine metabolism. In the more radiosensitive UM-SCC-74A cells, the most significant alterations after irradiation were linked to tryptophan metabolism. In the more radioresistant UM-SCC-74B cells, the major alterations after irradiation were connected to nicotinic acid and nicotinamide metabolism, purine metabolism, the methionine cycle as well as the serine, and glycine metabolism. The data suggest that the more radioresistant cell line UM-SCC-74B altered the metabolism to control redox-status, manage DNA-repair, and change DNA methylation after irradiation. This provides new insights on the mechanisms of radiation response, which may aid future identification of biomarkers associated with radioresistance of cancer cells.

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Maximizing the Value of Cancer Drug Screening in Multicellular Tumor Spheroid Cultures: A Case Study in Five Head and Neck Squamous Cell Carcinoma Cell Lines.

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