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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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.
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.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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.
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.
Supplementary note, figures, tables, video captions and references.
Source data and statistics for Supplementary Fig. 5b.
Source data and statistics for Supplementary Fig. 6b.
Source data and statistics for Supplementary Fig. 8b.
The dynamics of HS-activated eGFP expression.
MRI-guided focused-ultrasound stimulation on a targeted region on the hindlimb of an anaesthetised mouse.
Source data and statistics for Fig. 4c,e.
Source data and statistics for Fig. 5a,c.
Source data and statistics for Fig. 6g.
Source data and statistics for Extended Data Fig. 1b,c.
Source data and statistics for Extended Data Fig. 3b,c.
Source data and statistics for Extended Data Fig. 4c.
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 (2021). https://doi.org/10.1038/s41551-021-00779-w