Live imaging of TAK1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyI:C

Abstract

TGF-β activated kinase 1 (TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3LL cells implanted into the subcutaneous tissue of syngeneic C57BL/6 mice and treated with polyinosinic polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer (FRET)-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee-TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α (TNF-α), and interleukin1-β (IL-1β). The anisomycin-induced increase in FRET was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. TAK1 activity in 3LL cells was markedly increased by PolyI:C in the presence of macrophages. 3LL cells expressing Eevee-TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3LL cells at the periphery of the tumor exhibited higher TAK1 activity than the 3LL cells locating at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. PolyI:C injection, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within four days. These observations suggest that Eevee-TAK1 is a versatile tool to monitor cellular stress in cancer tissues.

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