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.

Head and Neck Cancer Stem Cell-Enriched Spheroid Model for Anticancer Compound Screening

“…The MCTS model, on the other hand, is designed in microtiter formats, has shorter cultivation times, is compatible with automated and multimodal detection systems, and is thus often employed in the search for anticancer compounds using HTS [ 18 , 50 ]. Many anticancer compounds have been analyzed using MCTS, including a dual phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) inhibitor dactolisib, a tyrosine kinase inhibitor sunitinib, and others [ 15 , 17 ]. However, as MCTS employs FBS-supplemented medium with a known differentiation effect, only partial enrichment of CSC is possible.…”

“…Among the most commonly used spheroid forming techniques in HNSCC are cultures of free-floating spheres and MCTS [12][13][14][15][16]. Cultures of free-floating spheres are technically quite challenging as they demand long-term cultivation (2-3 weeks), multi-step cell culture manipulations, and growing cells on flat low-adherent surfaces, causing the generation of multiple non-uniform spheroids with no spatial separation, which limits the use of this model for compound screenings [17].…”

In vitro models as tools for screening treatment options of head and neck cancer

“…The success rate of MCTS ranges between 50 and 100% of HNSCC originated at different locations with a range of culture of time between 4 and 21 days ( 60 ). Compared to 2D cultures, MCTS better recapitulate the characteristics of in vivo HNSCC by phenotyping the original tumor regarding cell heterogeneity and genetic variability ( 61 , 62 ), so that in vitro drug responses of the model can be traced back to genetic alterations of the primary tumors ( 63 ). Furthermore, MCTS also allowed an enrichment of cancer stem cells as shown for FaDu cells as a model system, thereby offering a suitable tool for active screening for drugs targeting cancer stem cells ( 64 ).…”

Preclinical models in Head and Neck Squamous Cell Carcinoma

“…To assess the performance advantage of MCTS versus 2D cultures, they tested the set of 19 drugs with established 2D monolayer GI assays using the same HNSCC cell lines. MCTS drug responses could be stratified into high-, intermediate-, and low-impact tiers using a cumulative multiparameter drug impact score, maximizing the usefulness of these more physiologically relevant tumour cultures to establish models of resistance [15].…”