Abstract
Three-dimensional (3D) cultures of cancer cells in liquid without extracellular matrix (ECM) offer invitro models for metastasising conditions such as those in vessels and effusion. However, liquid culturing is often hindered by cell adhesiveness, which causes large cell clumps. We previously described a liquid culture material, LA717, which prevents nonclonal cell adhesion and subsequent clumping, thus allowing clonal growth of spheroids in an anchorage-independent manner. Here, we examined such liquid culture cancer spheroids for the acquisition of apical-basal polarity, sensitivity to an Akt inhibitor (anticancer drug MK-2206) and interaction with ECM. The spheroids present apical plasma membrane on the surface, which originated from the failure of polarisation at the single-cell stage and subsequent defects in phosphorylated ezrin accumulation at the cell boundary during the first cleavage, failing internal lumen formation. At the multicellular stage, liquid culture spheroids presented bleb-like protrusion on the surface, which was enhanced by the activation of the PI3K/Akt pathway and reduced by PI3K/Akt inhibitors. Liquid culture spheroids exhibited slow proliferation speed and low endogenous pAkt levels compared with gel-cultured spheroids and 2D-cultured cells, explaining the susceptibility to the Akt-inhibiting anticancer drug. Subcutaneous xenografting and invitro analysis demonstrated low viability and adhesive property of liquid culture spheroids to ECM, while migratory and invasive capacities were comparable with gel-cultured spheroids. These features agree with the low efficacy of circulating tumour spheroids in the settling step of metastasis. This study demonstrates the feature of anchorage-independent spheroids and validates liquid cultures as a useful method in cancer spheroid research.
Highlights
Adherent cells, both cancerous and noncancerous, grow in the form of clusters when cultured in threedimensional (3D) condition in vitro [1]
The apical plasma membrane was detected by antibodies against phosphorylated ezrin/ radixin/moesin, which is highly enriched on the apical side of polarised epithelial cells [12], whereas the basal plasma membrane was detected by antibodies against b1-integrin, which interacts with extracellular matrix (ECM) [13]
One of the major applications has been to MDCK cells using Matrigel, which has served as the benchmark model system of epithelial cells, whereas, in cancer cell studies, both gel-based and liquid-based cultures may be required to reflect the metastatic property, which potentially locate cells in both fluidal and matrix environment in vivo
Summary
Both cancerous and noncancerous, grow in the form of clusters when cultured in threedimensional (3D) condition in vitro [1]. If sufficient extracellular matrix (ECM) is provided and cells maintain a strong apical–basal polarity, the clusters present a cyst-like structure, known as acini or spheres, comprising a closed cell monolayer surrounding a lumen [2]. Abbreviations 2D, two-dimensional; 3D, three-dimensional; CK, cytokeratin; ECM, extracellular matrix; pERM, phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558); qPCR, quantitative polymerase chain reaction. The FEBS Journal published by John Wiley & Sons Ltd on behalf of.
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