A liquid culture cancer spheroid model reveals low PI3K/Akt pathway activity and low adhesiveness to the extracellular matrix

Three‐dimensional (3D) cultures of cancer cells in liquid without extracellular matrix (ECM) offer in vitro 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 in vitro 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. Cancer spheroids present their apical plasma membrane outside when grown in liquid culture without anchorage. By directly comparing them with gel matrix‐grown spheroids, we demonstrate the origin of the apical‐out polarity at the first cleavage, low endogenous Akt activity and low attachment capacity to ECM. Thus, agar‐based culture supplement LA717 is a useful tool for anchorage‐independent cultures while allowing clonal growth of spheroids.