Mechanical Confinement Promotes Heat Resistance of Hepatocellular Carcinoma via SP1/IL4I1/AHR Axis

Mechanical stress can modulate the fate of cells in both physiological and extreme conditions. Recurrence of tumors after thermal ablation, a radical therapy for many cancers, indicates that some tumor cells can endure temperatures far beyond physiological ones. This unusual heat resistance with unknown mechanisms remains a key obstacle to fully realizing the clinical potential of thermal ablation. By developing a 3D bioprinting-based thermal ablation system, we demonstrate that hepatocellular carcinoma (HCC) cells in this 3D model exhibit enhanced heat resistance as compared with cells on plates. Mechanistically, the activation of transcription factor SP1 under mechanical confinement enhances the transcription of Interleukin-4-Induced-1, which catalyzes tryptophan metabolites to activate the aryl hydrocarbon receptor (AHR), leading to heat resistance. Encouragingly, the AHR inhibitor prevents HCC recurrence after thermal ablation. These findings reveal a previously unknown role of mechanical confinement in heat resistance and provide a rationale for AHR inhibitors as neoadjuvant therapy. To investigate the specific factors endowed by a bona fide 3D environment and reveal the mechanism of heat resistance in the 3D model, we performed RNA sequencing to delineate the transcriptional states of HCC cells in the 2D, 3D, and animal models. To interrogate the key factor among 11 metabolic genes that altered tumor metabolism to induce heat resistance, we performed an additional RNA-sequencing to explore the transcriptional state of HCC cells in the 3D model after heat stress compared with untreated control.