From a Road Less Travelled to a Worn Path: Three-Dimensional Tumour Models for Cancer Research and Therapeutics

Abstract: Cancer is the second leading cause of death among both men and women worldwide. During therecent years, three-dimensional (3D) tumour models have gained increasing interest as a pre-clinicalplatform for screening of compounds for potential use in cancer therapy. It is becoming recognizedthat two-dimensional (2D) cell culturing, in which cells are grown on physiologically irrelevant flatsurfaces, are not reliable tools for investigating chemo sensitivity of anti-cancer drug candidates.Therefore, the emerging miniaturized 3D tumour models in vitro are better representatives of thehuman tumour growing in the in vivo microenvironment. We have established a complex 3D humantumour outside the body using randomly oriented highly porous electrospun polycaprolactone (PCL)fibres, which mimic the collagen structure of the extracellular matrix (ECM). The data show that monoculturesof cancer cells grow as dense multicellular spheroids in the biocompatible 3D scaffolds,while normal cells show spread-out and elongated morphology. When co-cultured, JIMT-1 breastcancer cells and human dermal fibroblasts (HDFs) show a growth pattern similar to what is found ina tumour with the cancer cells growing in tight clusters surrounded by the fibroblasts. When grown inmono-culture or co-culture, the cells grow in the entire depth of the 3D PCL network. In addition, wecharacterized the proteins deposited by the cells in the 3D scaffolds incubated in the absence orpresence of transforming growth factor-β1 (TGF-β1), a tumour promoting cytokine. The data showthat the fibrous ECM proteins fibronectin, collagen I, and laminin are deposited throughout the depthof 3D structure. TGF-β1 treatment did not have a significant effect on protein deposition butsignificantly modulated the activity of matrix metalloproteinases and the level of interleukine-6cytokines in the medium of our 3D culture. In TGF-β1-treated co-cultures, the cancer cells changedthe growth pattern from tight clusters to be spread out along elongated HDFs. The 3D human tumourin vitro was utilized for evaluation of efficacy of two anti-cancer compounds; a well-known anti-cancerdrug paclitaxel and an experimental salinomycin analogue (SAEC). The experiments were performedin hypoxia and normoxia. Paclitaxel treatment was more toxic to the cancer cells while the SAEC wasmore toxic to the HDFs in normoxia and hypoxia. Furthermore, we fabricated and validated a 96-wellplates with 3D PCL fibre network as a high-throughput screening (HTS)-based assay. We comparedthe toxicity of paclitaxel and SAEC in 2D and 3D under normoxic and hypoxic condition. The datashow that the 96-well 3D system is a cost-efficient tool that can be used for assessing of new potentialchemotherapeutic drugs in an HTS manner. Thus, we have “proof of principle” that our tailor-designedhuman tumour outside the body has structure similar to a tumour in the body and that it can be usedfor investigation of chemical toxicity.