Quantitative Size-Based Analysis of Tumor Spheroids and Responses to Therapeutics

Abstract

Drug resistance remains a major clinical problem despite advances in targeted therapies. In recent years, methods to culture cancer cells in three-dimensional (3D) environments to better mimic native tumors have gained increasing popularity. Nevertheless, unlike traditional two-dimensional (2D) cell cultures, analysis of 3D cultures is not straightforward. Most biochemical assays developed for 2D cultures have to be optimized for use with 3D cultures. We addressed this important problem by presenting a simple method of quantitative size-based analysis of growth and drug responses of 3D cultures of cancer cells as tumor spheroids. We used an aqueous two-phase system to form consistently sized tumor spheroids of colorectal cancer cells. Using spheroid images, we computed the size of spheroids over time and demonstrated that growth of spheroids from this analysis strongly correlates with that using a PrestoBlue biochemical assay optimized for 3D cultures. Next, we cyclically treated the tumor spheroids with a MEK inhibitor, trametinib, for 6-day periods with a recovery phase in between. This inhibitor was selected because of mutation of colon cancer cells in the MEK/ERK pathway. We used size measurements to evaluate the efficacy of trametinib and predict development of resistance of colon cancer cells during the cyclical treatment and recovery regimen. This size-based analysis closely matched the biochemical analysis of drug responses of spheroids. We performed molecular analysis and showed that resistance to trametinib emerged due to feedback activation of the PI3K/AKT signaling pathway. Therefore, we combined trametinib with a PI3K/AKT inhibitor, dactolisib, and demonstrated that size-based analysis of spheroids reliably allowed quantifying the effect of the combination treatment to prevent drug resistance. This study established that size measurements of spheroids can be used as a straightforward method for quantitative studies of drug responses of tumor spheroids and identifying drug combinations that block resistance.

RETRACTED ARTICLE: A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function

“…Other classes of compounds that killed tumor spheroids were those that interfered with MAPK signaling. HCT116 cells are mutant KRAS-driven colon cancer, so compounds that antagonize RAF, MEK, or ERK signaling would be expected to act selectively against this tumor type 21 . Intriguingly, many compounds whose targets and mechanisms act through ECM remodeling could be distinguished by their unique phenotypes.…”

Challenges of applying multicellular tumor spheroids in preclinical phase

“…Second, it provides a means to quantify treatment plans and estimate the impact of treatment uncertainty on the results. In most circumstances, compact spheroids are more resistant to the drug than aggregated cells, and smaller spheroids are more sensitive to both chemotherapy and radiotherapy [ 11 , 75 – 80 ]. This is because the degree of drug penetration is poor where there are tight cell-to-cell adhesions, and the presence of hypoxic cells in larger MCTs may increase resistance to the therapy.…”

“…In addition, various methods have been suggested to get uniform-sized MCTs. The microprinting of MCTs using the aqueous two-phase system (ATPS) generates size-controlled HT-29 spheroids with an average diameter of 440 ± 24 µm [ 11 ]. Herein, ATPS comprises aqueous solutions of polyethylene glycol (PEG) and dextran (DEX).…”

“…MCTs closely mimic in vivo solid tumors' main features, such as structural organization and the gradients of oxygen, pH, and nutrients [ 11 , 12 ]. Beyond a critical size of about 500 µm, MCTs show characteristic features of avascular tumors with an external proliferating zone, an internal quiescent zone, and a necrotic core due to gradients of nutrient and oxygen concentration [ 13 – 18 ].…”

Rapid Evaluation of Novel Therapeutic Strategies Using a 3D Collagen-Based Tissue-Like Model

“…This parameter was directly used to characterize the reproducibility of the sample preparation and compared with the values of the literature. A quantitative comparison of CV obtained with 3D-CCMs and the ones reported in different studies is illustrated in Figure 1B ( Gong et al, 2015 ; Kang et al, 2015 ; Chen et al, 2016 ; Zanoni et al, 2016 ; Kwak et al, 2018 ; Sarkar et al, 2018 ; Shi et al, 2018 ; Thakuri et al, 2019 ).…”