Metastatic renal cell carcinoma cells growing in 3D on poly‑D‑lysine or laminin present a stem‑like phenotype and drug resistance

Abstract

3D spheroids are built by heterogeneous cell types in different proliferative and metabolic states and are enriched in cancer stem cells. The main aim of the study was to investigate the usefulness of a novel metastatic renal cell carcinoma (RCC) 3D spheroid culture for in vitro cancer stem cell physiology research and drug toxicity screening. RCC cell lines, Caki-1 (skin metastasis derived) and ACHN (pleural effusion derived), were efficiently cultured in growth-factor/serum deprived, defined, StemXvivo and Nutristem medium on laminin-coated or poly-D-lysine-coated plates. In optimal 3D culture conditions, ACHN cells (StemXVivo/poly-D-lysine) formed small spheroids with remaining adherent cells of an epithelial phenotype, while Caki-1 cells (StemXVivo/laminin) formed large dark spheroids with significantly reduced cell viability in the center. In the 3D structures, expression levels of genes encoding stem transcription factors (OCT4, SOX2, NES) and RCC stem cell markers (CD105, CD133) were deregulated in comparison to these expression levels in traditional 2D culture. Sunitinib, epirubicin and doxycycline were more toxic to cells cultured in monolayers than for cells in 3D spheroids. High numbers of cells arrested in the G0/G1 phase of the cell cycle were found in spheroids under sunitinib treatment. We showed that metastatic RCC 3D spheroids supported with ECM are a useful model to determine the cancer cell growth characteristics that are not found in adherent 2D cultures. Due to the more complex architecture, spheroids may mimic in vivo micrometastases and may be more appropriate to investigate novel drug candidate responses, including the direct effects of tyrosine kinase inhibitor activity against RCC cells.

The Role of Biomimetic Hypoxia on Cancer Cell Behaviour in 3D Models: A Systematic Review

“…EMT is a process identified as one of the key features in tumour invasion and metastasis. We identified a total of 19 publications [ 15 , 24 , 50 , 69 , 73 , 74 , 76 , 82 , 84 , 92 , 94 , 99 , 100 , 101 , 102 , 104 , 105 , 106 , 107 ] comparing markers of EMT in 2D vs. 3D tumour cultures, with 18 of those showing enhanced measurements of EMT in 3D compared to 2D cultures. In contrast with the generalised cohort of studies concerning biomimicry of in vitro cancer cultures, the studies specifically concerning EMT consisted of a majority of scaffold-based cultures at 67%, and 33% involving non-scaffold-based cultures.…”

“…We identified a total of 42 publications [ 16 , 19 , 22 , 23 , 30 , 41 , 42 , 45 , 49 , 52 , 56 , 59 , 61 , 64 , 68 , 72 , 74 , 80 , 85 , 89 , 100 , 101 , 102 , 103 , 105 , 106 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 ] comparing cell response to chemotherapy and other interventions between 2D and 3D in vitro tumour models, using a variety of anticancer agents and taking into account any publications which involved more than one type of anticancer agents, yielding in total 85 separate findings, including 79 outcomes concerning chemotherapy agents, three involving radiotherapy, and a further five exploring natural compound agents such as orange-peel flavones. The definitions for drug response varied throughout the studies in terms of markers used and duration over which drug responses were measured.…”

The anti-angiogenic tyrosine kinase inhibitor Pazopanib kills cancer cells and disrupts endothelial networks in biomimetic three-dimensional renal tumouroids

“…This reflects the biomimicry of drug penetration into a 3D mass rather than a cell sheet and has been described in other 3D models, for example, matrigel, 37 collagen hydrogels, fibronectin 38 and RCC spheres. 39 In tumouroids, drug penetration is even more challenging as the cancer mass is surrounded by a dense 10% collagen matrix. 24 Differential responses may also be linked to altered target receptor levels in 3D.…”

3D Cell Culture for the Study of Microenvironment-Mediated Mechanostimuli to the Cell Nucleus: An Important Step for Cancer Research

“…(C) . Cell culture-based examples of the impact of different types of matrices and forces that result in changes in gene transcription and angiogenesis as they relate to cancer aggressiveness ( 1 Novak et al, 2019 , 2 Bordeleau et al, 2017 , 3 Seidlits et al, 2011 , 4 Lin et al, 2015 , 5 Larson et al, 2014 , 6 Brodaczewska et al, 2019 , 7 Pankova et al, 2019 , 8 Jain et al, 2013 , 9 Sewell-Loftin et al, 2017 ).…”