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Acoustic-pressure-driven ultrasonic activation of the mechanosensitive receptor RET and of cell proliferation in colonic tissue

Nature Biomedical Engineering volume 9, pages 742–753 (2025) Cite this article

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Abstract

Ultrasound generates both compressive and shear mechanical forces in soft tissues. However, the specific mechanisms by which these forces activate cellular processes remain unclear. Here we show that low-intensity focused ultrasound can activate the mechanosensitive RET signalling pathway. Specifically, in mouse colon tissues ex vivo and in vivo, focused ultrasound induced RET phosphorylation in colonic crypts cells, which correlated with markers of proliferation and stemness when using hours-long insonication. The activation of the RET pathway is non-thermal, is linearly related to acoustic pressure and is independent of radiation-force-induced shear strain in tissue. Our findings suggest that ultrasound could be used to regulate cell proliferation, particularly in the context of regenerative medicine, and highlight the importance of adhering to current ultrasound-safety regulations for medical imaging.

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Fig. 1: Focused ultrasound induces physical effects that may influence cell fate.
The alternative text for this image may have been generated using AI.
Fig. 2: The activation of the RET mechanosensory receptor by focused ultrasound is pressure dependent.
The alternative text for this image may have been generated using AI.
Fig. 3: Focused ultrasound activates the RET mechanosensory receptor in colonic crypts beyond physiological level and in a short sonication time.
The alternative text for this image may have been generated using AI.
Fig. 4: Focused ultrasound activates cell proliferation in vivo.
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Data availability

The main data supporting the results in this study are available within the paper. All source data for the generation of the figures, are available in Zenodo at https://doi.org/10.5281/zenodo.13903619 (ref. 51).

Code availability

The custom code for the processing of data and figures in Supplementary Video 1 is available in Zenodo at https://doi.org/10.5281/zenodo.13903619 (ref. 51).

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Acknowledgements

The project was supported by the Bettencourt Schueller Foundation and the Agence Nationale de la Recherche (French National Research Agency). Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (Authorization reference APAFIS#10977-201708211557193 v3 given by the national authority, French Ministry of Research, in compliance with international guidelines). M.T. discloses funding support for the research described in this study from Fondation Bettencourt Schueller (Bettencourt Schueller Foundation) – Chair of Physics for Medicine, Agence Nationale de la Recherche (French National Research Agency), 16-CE14-002801. E.F., M.E.F.-S. and E.R. disclose funding support for the research described in this study from Agence Nationale de la Recherche (French National Research Agency) – 16-CE14-002801 and ANR-17-CONV-0005, respectively.

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Author notes

  1. These authors contributed equally: Charlie Demené, Mickael Tanter.

Authors and Affiliations

  1. Institute Physics for Medicine Paris, ESPCI PSL Paris, INSERM U1273, CNRS UMR 8361, Paris Sciences et Lettres University, Paris, France

    Laura Zamfirov, Eliott Teston, Alexandre Dizeux, Thomas Tiennot, Charlie Demené & Mickael Tanter

  2. Mechanics and Genetics of Embryonic and Tumoral Development team, Physics of Cells and Cancer UMR168, INSERM, Université PSL, Sorbonne Université, Institut Curie, Paris, France

    Ngoc-Minh Nguyen, Maria Elena Fernández-Sánchez, Paula Cambronera Ghiglione & Emmanuel Farge

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  1. Laura Zamfirov
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Contributions

C.D. and M.T. designed the study on the wave physics and ultrasound/tissue interaction aspects of this work. E.F. designed the experiments on the mechanobiology and biological aspect of this work with M.E.F.-S., C.D. and M.T.; L.Z., C.D. and M.T. designed the acoustical stimulations and programmed the ultrasound scanner; L.Z., A.D., T.T. and C.D. designed and performed ultrasound and thermal simulations. L.Z. and E.T. set up the in vitro protocol. L.Z., E.T. and C.D. performed the RET experiments in vitro. L.Z., M.E.F.-S. and C.D. performed the RET experiments in vivo. L.Z. performed the Ki67 experiments. N.-M.N., M.E.F.-S. and P.C.G. performed the in vivo LGR5 experiments. L.Z., A.D., E.F., C.D. and M.T. analysed and interpreted the data. L.Z., C.D. and M.T. wrote the manuscript. L.Z., A.D., M.E.F.-S., E.F., C.D. and M.T. edited and corrected the manuscript.

Corresponding authors

Correspondence to Emmanuel Farge, Charlie Demené or Mickael Tanter.

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Nature Biomedical Engineering thanks Phuong Nguyen, Yi-Xian Qin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Peak negative pressure measured as a function of the voltage applied across the transducer.

Hydrophone measurements at perpendicular distance from focal point of the transducer show peak negative pressures in water for different tension at three focusing depths. The maximal peak negative pressure is ~4.7 MPa with a 55V tension at 15-mm focus depth. The corresponding derated pressure using αabs = 0.5 dB.cm−1.MHz−1 has been plotted on the right y axis.

Extended Data Fig. 2 Ultrafast ultrasound imaging of local displacements induced by the ultrasonic transmission in a tissue-mimicking phantom for ultrasonic sequences corresponding to set #4, set #6, and set #3.

Top row: propagation of the shear wave shown at different times after transmission of the acoustic pulse corresponding to set #4 (55 V – 250 µs) at the focal point of the transducer (15 mm). Axial displacement is propagating along the x axis from the focal point in both direction over time. Second, third and fourth row: temporal and spatial behavior of axial shear displacement along the radial direction (left) and maximum transient shear strain (middle), at the focal point of the transducer, have been plotted for set #4 (second row), set #6 (third row), and set #3 (bottom row). Map of the maximum shear reached after transmission of the acoustic pulse (right) has been represented.

Extended Data Fig. 3 Temperature rise at the focal point for 10-min ultrasound stimulation.

The maximal acoustic pressure was used for this simulation. Top, temperature rise after 10 min of ultrasound stimulation with a tension of 55 V and pulse duration of 250 µs with a pulse repetition frequency of 4 Hz. The temperature reaches a constant value 5 min after the beginning of stimulation. Bottom, zoom on the first 20s of the top graph.

Extended Data Fig. 4 Ex vivo sagittal ultrasound imaging of a wild-type colon.

Representative B-mode acoustic image of wild-type mice colon before ultrasound stimulation. B-mode image was used to select ultrasound stimulation target points (9 positions with a 1 mm interspacing at two different depths).

Extended Data Fig. 5 In vivo repartition of pRet-positive crypts after focused ultrasound stimulation.

a. Quantification of the colon crypts that display 1-3 pRet+ cells per crypt and ≥4 pRet+ cells per crypt as a function of 1h ultrasound stimulation (Ctrl - n = 9, US stim - n = 7). Two-way ANOVA and Sidak’s multiple comparisons post hoc test, *** p<0.001, ns non-significant p = 0.35. b. Quantification of the colon crypts that display 1-3 pRet+ cells per crypt and ≥4 pRet+ cells per crypt as a function of 12h ultrasound stimulation (Ctrl - n = 5, US stim - n = 5). Two-way ANOVA and Sidak’s multiple comparisons post hoc test, * p<0.05 (p = 3.4e-2, ** p<0.01 (p = 4.5e-3).

Extended Data Fig. 6 Screening of the mechanical activation of the Src family kinases known to phosphorylate β-catenin Tyr654 and to be insensitive to mechanical stimuli.

a. Fluorescence images of colonic epithelium expressing Tyr1238/Tyr1239 phosphorylated Ron β (pRon), phosphorylated Tyr535 c-Yes (pYes), or Tyr568 and Tyr570 dually phosphorylated c-Kit (pKit) after ultrasound stimulation with parameter set #4 for 1 hour. No phosphorylation of the Tyr1238/1239 site of activation of Ron after ultrasound stimulation was observed. All crypts were negative both in ultrasound stimulated samples (424 total crypts observed, measured in n = 4 mice) and controls (354 total crypts observed, measured in n = 4 mice). No increase of phosphorylation of the Tyr537 site of activation of Yes was observed after ultrasound stimulation. Control samples showed 19.8±4.8% positive crypts (411 total crypts observed, measured in n = 4 mice) and ultrasound stimulated samples showed 20.5±2.4% positive crypts (488 total crypts observed, measured in n = 4 mice). No phosphorylation of the Tyr568/570 site of activation of Kit was observed after ultrasound stimulation. All crypts were negative both in ultrasound stimulated samples (339 total crypts observed, measured in n = 4 mice) and controls (385 total crypts observed, measured in n = 4 mice). Scale bar is 20 μm. b. Quantification of the number of pRon, pYes and pKit positive colonic crypts after 1h of ultrasound stimulation with (Ctrl - n = 4 mice, +1h US stim - n = 4 mice). For pYes positive crypt quantification, unpaired T-test, two-tailed, ns non-significant p = 0.79.

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Supplementary Video 1 (download AVI )

Dynamic measurement of the local displacements and the maximal shear strain induced by the ultrasonic radiation force in each pixel of a tissue-mimicking phantom.

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Zamfirov, L., Nguyen, NM., Fernández-Sánchez, M.E. et al. Acoustic-pressure-driven ultrasonic activation of the mechanosensitive receptor RET and of cell proliferation in colonic tissue. Nat. Biomed. Eng 9, 742–753 (2025). https://doi.org/10.1038/s41551-024-01300-9

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