Researchers have discovered that tumor cells undergoing mesenchymal-amoeboid transition (MAT) activate an RIPK3-independent, MLKL-mediated programmed necroptosis pathway under persistent physical confinement.

This pathway not only induces cell death but also suppresses the amoeboid migration of tumor cells, acting as an endogenous tumor-suppressive checkpoint. Under the selective pressure of tumor evolution, escape clones with low MLKL expression are selected, which drive tumor metastasis with enhanced migratory and invasive capacities.

The study was published in Developmental Cell by a research team led by SUN Liming from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, in collaboration with LIU Yanjun from the Institutes of Biomedical Sciences, Fudan University.

As tumors grow, mechanical compressive stress builds up within the tumor mass, driving tumor cells to undergo MAT. These cells can migrate rapidly through confined spaces, including gaps in the extracellular matrix and blood vessel walls, facilitating distant metastasis. Yet how these cells respond to prolonged, intense confinement was previously unclear.

Traditionally, MLKL, the executioner protein of necroptosis, has been thought to require phosphorylation by the upstream kinase RIPK3 for activation. However, recent studies have detected necroptosis in human tumors lacking RIPK3, raising questions about how this form of cell death is triggered.

In this study, the researchers established an in vitro L-MAT (Long-term Mesenchymal-amoeboid Transition) experimental system to mimic the compressive microenvironment in tumors. They found that sustained mechanical compression directly upregulates MLKL transcription; once MLKL expression exceeds the tolerance threshold of tumor cells, RIPK3-independent necroptosis is triggered.

Further experiments showed that the mechanosensitive ion channel PIEZO1 senses compressive signals and directly drives MLKL transcription via the downstream transcription factor STAT1. The accumulated MLKL protein translocates to the cell membrane and subsequently initiates necroptosis.

Notably, MLKL activation suppresses amoeboid migration of MAT tumor cells prior to plasma membrane rupture and necrotic cell death. Activated MLKL disrupts retrograde cortical actin flow and downregulates the expression of migration-related genes, ultimately halting tumor cell motility.

Upon MLKL knockout, tumor cells evade this cell-death checkpoint and retain fast amoeboid migratory properties. A series of in vitro and in vivo models, including Transwell migration assays, microfluidic chips and mouse lung metastasis models, collectively demonstrated that MLKL activation markedly impairs the invasion and metastasis of tumor cells within confined microenvironments.

MLKL Expression in Clinical Tumor Samples

The researchers further analyzed spatial transcriptomic data from clinical tumor samples. In human breast cancer specimens, MLKL expression is high and the PIEZO1-STAT1 pathway is activated in less malignant in situ carcinoma regions, accompanied by abundant CD8⁺ T cell infiltration.

In contrast, MLKL expression is markedly downregulated in highly invasive carcinoma regions, where the PIEZO1-STAT1 axis becomes uncoupled and anti-tumor immune infiltration is diminished. In clinical samples of colorectal cancer liver metastasis, MLKL expression is significantly lower in liver metastatic lesions compared with primary tumors.

These findings suggest that under tumor evolutionary pressure, tumor clones with low MLKL expression and necroptosis resistance are enriched. These clones escape both cell death and immune surveillance, gain enhanced migratory capacity, and ultimately promote malignant progression and distant metastasis.

In summary, this study defines the fate of MAT tumor cells under persistent confinement: these cells undergo RIPK3-independent, MLKL-mediated necroptosis. The PIEZO1-STAT1-MLKL pathway functions as a mechanosensory checkpoint to eliminate highly invasive amoeboid tumor cells; tumor evolution selects for MLKL-low escape clones to facilitate tumor invasion and metastasis.

According to the researchers, this study expands our understanding of the activation modes of necroptosis and provides a theoretical basis for identifying tumor prognostic biomarkers and developing novel anti-metastatic therapeutic targets.