Review papers with raw data transparency

Quick appraisal

This preprint reports that capillarylike microconstriction of human melanoma cells triggers rapid PIEZO1 activation, calcium influx, transcriptional and epigenetic shifts, and a durable cancer stem celllike/tumorigenic phenotype in survivors; genetic and pharmacologic disruption of PIEZO1 blocks the effect while PIEZO1 activation (Yoda1) phenocopies it Primary

Extended review and critique

Main claim

The authors claim that transient mechanical deformation experienced during capillarylike constriction rapidly activates the mechanosensitive ion channel PIEZO1, producing calcium influx, early phosphorylation of mechanosensitive signalling nodes (NFkB YAP PKC mTOR ERK), chromatin changes and a switch toward a melanoma cancer stem celllike state that increases tumorsphere formation and metastasis; PIEZO1 inhibition or genetic deletion prevents the conversion and PIEZO1 activation alone (Yoda1) is sufficient to induce stemness markers and tumorsphere growth Primary

Why this matters

• Mechanotransduction as a direct driver of cell state plasticity in circulating tumour cells (CTCs) is mechanistically plausible and biologically important because it would increase the fraction of disseminated cells capable of colonization and may explain part of metastasis inefficiency paradox; the authors provide functional genetic pharmacologic and in vivo data consistent with this model In

Strengths

1. Multimodal evidence linking mechanics to signalling transcription and function: live calcium imaging phosphoproteomics RNAseq immunofluorescence tumorsphere assays and in vivo metastasis provide convergent lines of evidence Multimodal
2. Loss and gain of function of PIEZO1 (CRISPR KO and Yoda1) with pharmacologic corroboration supports causality rather than mere association Genetic
3. The microfluidic design mimics physiologically relevant constrictions and shows degree dependent calcium entry consistent with biophysics of membrane tension activating PIEZO1 Microfluidic

Major concerns and limitations

1. Generalisability beyond cell lines and single model The experimental system is dominated by A375-derived melanoma lines and a single in vivo dissemination model; although the authors provide preliminary data in other cell lines the depth is limited. The manuscript acknowledges the need to test patientderived CTCs and other tumour types to confirm conserved biology Generality
2. Physiological realism of the microfluidic model The PDMS device reproduces diameter changes but omits key in vivo variables: blood rheology plasma proteins immune cells platelets endothelial glycocalyx and interstitial shear dynamics; these omissions could alter deformation patterns or signalling (e.g. shear versus squeeze). The authors intentionally did not preactivate endothelium which is a strength for isolating tumourintrinsic effects but weakens claims about in vivo endothelial interactions under realistic inflammatory states Microfluidic
3. Population selection bias due to survival after constriction A measurable fraction of cells die during constriction; the surviving subset is enriched for cells that either tolerated deformation or have preexisting traits; although PIEZO1 signalling appears necessary for the phenotypic switch among survivors the study cannot fully exclude that selection rather than reprogramming contributes to observed enrichment of stemlike markers. The authors present viability data and note immediate loss of viability but attribute the longterm state to reprogramming in survivors; this remains a potential confounder Viability
4. Pharmacology specificity Ruthenium Red is nonselective and GsMTx4 while more selective still affects multiple stretch activated channels; Yoda1 is a known PIEZO1 modulator but offtarget or downstream indirect effects (e.g. via integrin cytoskeleton) cannot be fully excluded without rescue experiments using channeldead mutants or PIEZO1 reexpression in KO backgrounds Pharmacologic
5. Mechanistic depth around chromatin changes The paper reports changes in H3K9ac and H3K9me3 and transcriptional shifts but the mechanistic chain from transient calcium spike to durable epigenetic reprogramming is not fully resolved: which calciumdependent enzymes readers/writers (eg HDACs HATs KDMs DNMTs) are engaged and how long marks persist in vivo require further demonstration Epigenetic

Suggested critical experiments to strengthen claims

1. Rescue experiment reexpressing PIEZO1 in KO cells with wildtype and channeldead mutant alleles to show dependence on PIEZO1 ionconducting activity (not merely scaffolding) for reprogramming.
2. Lineage tracing or barcoding to distinguish selection from induced reprogramming among survivors (e.g. compare barcode diversity prepost constriction to measure selective bottleneck versus state change).
3. Short and long term chromatin profiling ATACseq CUTandTag for H3K9ac H3K9me3 plus assays for calciumdependent chromatin enzymes (eg class IIa HDAC nuclear translocation CaMK IV activation) to connect calcium pulses to durable epigenetic shifts.
4. Use of patientderived CTCs or multiple PDXderived melanoma models to test generality and translational relevance.

Assessment scores

Short bottomline

The preprint offers strong multimodal evidence that capillaryscale mechanical constriction can trigger PIEZO1dependent calcium signalling linked to a stemlike tumorigenic reprogramming in melanoma cells and increases metastatic potential in mice; the data are persuasive but key gaps remain in generality to patientderived cells the distinction between selection versus induced reprogramming pharmacologic specificity and the mechanistic chain from calcium spikes to durable epigenetic reprogramming Authors