Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function

Abstract

Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.

Photothermal Therapy of Glioblastoma Multiforme Using Multiwalled Carbon Nanotubes Optimized for Diffusion in Extracellular Space

“…Little is known about the role of the heat shock response with regards to the efficacy of thermal therapy for GBM, and no studies have examined the use of CNMTT or other nanoparticle-based thermal therapies in the context of induced expression of HSPs. Furthermore, few studies have used three dimensional cell cultures such as multicellular tumor spheroids, which may more closely mimic tumors in vivo 34 , to evaluate nanoparticle-based thermal therapy. Therefore, the purpose of this study was: (i) to evaluate the feasibility of using slow infusion techniques similar to CED to deliver CNTs into brain extracellular matrix (ECM)-mimicking phantoms; (ii) to determine the efficacy of CNMTT in comparison to conventional heat delivery in GBM cell lines and multicellular tumor spheroids; and (iii) to determine if CNMTT could overcome the protective effects of induction of the heat shock response prior to initiation of CNMTT.…”

Isolation of spheroid-forming single cells from gastric cancer cell lines: enrichment of cancer stem-like cells

“…Moreover, the microwell mimics the in vivo environment and contains CSC-secreted factors [32]. One recent study reported that round spheroids showed a signature of high mRNA expression for secreted molecules, which activate cell cycle-related signaling pathways and their downstream transcriptional regulators [33]. This finding supports the idea that the microwell is more effective at mimicking the in vivo environment by sharing the medium.…”

Prediction of tumor response of rectal cancer cells via 3D cell culture and in�vitro cytotoxicity assay before initiating preoperative chemoradiotherapy

“…The resulting spheroids were classified into four distinct groups based on morphology as 'round', 'mass', 'aggregate' and 'none' (Fig. 1) (19). For further analysis, the 'round', 'mass', and 'aggregate' types were grouped into the category 'mass-forming group' for efficient statistical analysis.…”

“…With regards to environmental comparison, typical cells in 3D have ~100% of their surface area exposed to other cells or matrix, whereas cells in 2D have ~50% of their surface area exposed to fluid, ~50% exposed to the flat culture Prediction of tumor response of rectal cancer cells via 3D cell culture and in vitro cytotoxicity assay before initiating preoperative chemoradiotherapy surface or intermediate, and very little surface area exposed to other cells. A previous data analysis of spheroid-based drug screening systems revealed that tumor spheroid size and morphology in a 3D culture system was associated with in vivo drug sensitivity (19). Previous studies demonstrated significantly increased resistance to radiation in tumor cells in 3D cell culture, in comparison with that of 2D culture (20,21).…”