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Control of the activity of CAR-T cells within tumours via focused ultrasound

Nature Biomedical Engineering volume 5, pages 1336–1347 (2021)Cite this article

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Abstract

Focused ultrasound can deliver energy safely and non-invasively into tissues at depths of centimetres. Here we show that the genetics and cellular functions of chimeric antigen receptor T cells (CAR-T cells) within tumours can be reversibly controlled by the heat generated by short pulses of focused ultrasound via a CAR cassette under the control of a promoter for the heat-shock protein. In mice with subcutaneous tumours, locally injected T cells with the inducible CAR and activated via focused ultrasound guided by magnetic resonance imaging mitigated on-target off-tumour activity and enhanced the suppression of tumour growth, compared with the performance of non-inducible CAR-T cells. Acoustogenetic control of the activation of engineered T cells may facilitate the design of safer cell therapies.

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Fig. 1: Heat-inducible gene activation.
Fig. 2: Heat-inducible CD19CAR expression and functionality in Jurkat and primary T cells.
Fig. 3: MRI-guided FUS-inducible gene activation in phantom and in vivo.
Fig. 4: Tumour suppression by FUS-CAR-T cells in vivo.
Fig. 5: Comparison of on-target off-tumour side effect between standard and FUS-CAR-T-cell therapy.
Fig. 6: Reversible FUS-CAR-T cells.

Data availability

The main data supporting the results of this study are available within the paper and its Supplementary Information. Source data for tumour growth curves are provided with this paper. Other raw data generated during this study are available from the corresponding authors on reasonable request.

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Acknowledgements

This work was supported in part by grants from NIH HL121365, GM125379, GM126016, CA204704 and CA209629 (Y. Wang). We thank F. Couillaud (University of Bordeaux, France) for providing the Hsp template; M. Sadelain (Sloan Kettering Institute, USA) for the PSMA scFv and PSMA constructs and the Nalm-6 cells; E. Dumont and S. Hoarau-Recco (Image Guided Therapy, France) for their most valuable help on the FUS system; and P. Mali for the insightful discussion.

Author information

Authors and Affiliations

  1. Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

    Yiqian Wu, Xin Wang, Jiayi Li, Linshan Zhu, Molly Allen, Yijia Pan, Shu Chien & Yingxiao Wang

  2. Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA, USA

    Yiqian Wu, Yahan Liu, Ziliang Huang, Shu Chien & Yingxiao Wang

  3. Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing, China

    Yahan Liu

  4. State Key Laboratory of Medical Genomics, Department of Haematology, Shanghai Institute of Hematology, National Research Center for Translational Medicine, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zhen Jin

  5. Division of Physical Science, Faculty of Science, Prince of Songkla University, Hat Yai, Thailand

    Praopim Limsakul

  6. Center for Functional MRI, University of California, San Diego, La Jolla, CA, USA

    Robert Bussell, Aaron Jacobson & Thomas Liu

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Contributions

Y. Wu, S.C. and Y. Wang designed the research; Y. Wu, Y.L, Z.H., X.W., Z.J., J.L., P.L., L.Z., M.A., Y.P., R.B. and A.J. performed the research; Y. Wu and Y.L. analysed data; Y. Wu, T. L., S.C. and Y. Wang wrote the manuscript. All authors reviewed the manuscript and approved the final version.

Corresponding authors

Correspondence to Shu Chien or Yingxiao Wang.

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Competing interests

Y. Wang is scientific co-founder of Cell E&G Inc. and Acoustic Cell Therapy Inc. These financial interests do not affect the design, conduct or reporting of this research.

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Peer review information Nature Biomedical Engineering thanks the 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 Effectiveness of FUS-CAR T cells in the single-tumour model.

a, Timeline of the experiment. Fluc+ Nalm-6 tumour cells were injected subcutaneously into NSG mice to generate single tumour model. Ten days after tumour challenge, FUS-inducible CAR T or naive T cells were locally injected at the tumour site, followed by FUS stimulation. b-d, Normalized tumour size (*P = 0.027 at D17, *P = 0.021 at D21) (b), tumour volume (****P = 2.4 × 10−5, ****P < 1.0 × 10−15) (c) and BLI images (d). Tumour size was quantified using the integrated Fluc luminescence intensity of the tumour region and normalized to that of the same tumour on the first measurement. Tumour volume was calculated based on caliper measurement as described in Methods. Two-way ANOVA followed by Sidak’s multiple comparisons test. Data points and error bars represent means of 5 mice ± SEM.

Source data

Extended Data Fig. 2 Functionality of FUS-inducible PSMACAR T cells in vitro.

a, Schematics of transgenes: heat-inducible Cre and lox-stop PSMACAR reporter. b, Representative flow cytometry data of the percentage of double positive T cells after MACS. c, Representative PSMACAR induction by HS. d, Cytotoxicities of the T cells engineered with the transgenes in a against Fluc+ PSMA + PC3 tumour cells at various E:T ratios. From left to right: ****P = 3.09 × 10−6, P = 1.11 × 10−6, P < 1 × 10−15, P < 1 × 10−15, P < 1 × 10−15, P = 3 × 10−15. e, Quantification of IFN-γ and IL-2 cytokine release associated with d. Arrow: cytokine level not detectable. In c to e, CT: without HS. HS: with a continuous 15-min HS. Two-way ANOVA followed by Sidak’s multiple comparisons test. Bar heights and error bars represent means of 3 biological replicate ± SEM.

Extended Data Fig. 3 ‘Tumour only’ in vivo control experiment associated with Fig. 5.

a, Timeline of the ‘tumour only’ control group used as the reference for calculating in vivo cytotoxicity in Fig. 5e. NSG mice were subcutaneously injected with Nalm-6 tumours on both sides and received no other treatment. b–d, Monitoring of tumour aggressiveness: normalized tumour size (b), tumour volume (c) and BLI images (d). Tumour size was quantified using the integrated Fluc luminescence intensity of the tumour region and normalized to that of the same tumour on the first measurement. Tumour volume was calculated based on caliper measurement as described in Methods. No significant difference was detected by mixed-effects analysis followed by Sidak’s multiple comparisons test. Data points and error bars represent means of 4 mice ± SEM. One mouse was sacrificed on D24 according to euthanasia criteria.

Source data

Extended Data Fig. 4 Repeated FUS stimulation in vivo control experiment associated with Fig. 6.

a, Timeline of the experiment. Bilateral tumour-bearing mice received local FUS stimulation without T cell injection at the left tumour on Day 4 and Day 7. The right tumour received no treatment. b,c, Normalized tumour size (b) and BLI images of the tumours on both sides (c). Tumour size was quantified using the integrated Fluc luminescence intensity of the tumour region and normalized to that of the same tumour on the first measurement. Data points and error bars represent means of 4 mice ± SEM.

Source data

Extended Data Fig. 5 Unstimulated reversible FUS-CAR T in vivo control experiment associated with Fig. 6.

a, Timeline of the experiment. Bilateral tumour-bearing mice received local injections of the reversible FUS-CAR (Hsp-CAR) T cells without FUS stimulation at the left tumour (proximal) on Day 4 and Day 7. The right tumour (distal) received no treatment. b,c, Normalized tumour size (*P = 0.011, ****P = 1.4 × 10−5,two-way ANOVA followed by Sidak’s multiple comparisons test) (b) and BLI images of the proximal and distal tumours (c). Tumour size was quantified using the integrated Fluc luminescence intensity of the tumour region and normalized to that of the same tumour on the first measurement. Data points and error bars represent means of 4 mice ± SEM.

Source data

Supplementary information

Supplementary Information

Supplementary note, figures, tables, video captions and references.

Reporting Summary

Peer Review File

Supplementary Dataset 1

Source data and statistics for Supplementary Fig. 5b.

Supplementary Dataset 2

Source data and statistics for Supplementary Fig. 6b.

Supplementary Dataset 3

Source data and statistics for Supplementary Fig. 8b.

Supplementary Video 1

The dynamics of HS-activated eGFP expression.

Supplementary Video 2

MRI-guided focused-ultrasound stimulation on a targeted region on the hindlimb of an anaesthetised mouse.

Source data

Source data for Fig. 4

Source data and statistics for Fig. 4c,e.

Source data for Fig. 5

Source data and statistics for Fig. 5a,c.

Source data for Fig. 6

Source data and statistics for Fig. 6g.

Source data for Extended Data Fig. 1

Source data and statistics for Extended Data Fig. 1b,c.

Source data for Extended Data Fig. 3

Source data and statistics for Extended Data Fig. 3b,c.

Source data for Extended Data Fig. 4

Source data and statistics for Extended Data Fig. 4c.

Source data for Extended Data Fig. 5

Source data and statistics for Extended Data Fig. 5c.

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Wu, Y., Liu, Y., Huang, Z. et al. Control of the activity of CAR-T cells within tumours via focused ultrasound. Nat Biomed Eng 5, 1336–1347 (2021). https://doi.org/10.1038/s41551-021-00779-w

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