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Activatable near-infrared probes for the detection of specific populations of tumour-infiltrating leukocytes in vivo and in urine

Nature Biomedical Engineering volume 7, pages 281–297 (2023)Cite this article

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Abstract

Tracking the immune microenvironment of tumours is essential for understanding the mechanisms behind the effectiveness of cancer immunotherapies. Molecular imaging of tumour-infiltrating leukocytes (TILs) can be used to non-invasively monitor the tumour immune microenvironment, but current imaging agents do not distinguish TILs from leukocytes resident in other tissues. Here we report a library of activatable molecular probes for the imaging, via near-infrared fluorescence, of specific TILs (including M1 macrophages, cytotoxic T lymphocytes and neutrophils) in vivo in real time and also via excreted urine, owing to the probes’ renal clearance. The fluorescence of the probes is activated only in the presence of both tumour and leukocyte biomarkers, which allows for the imaging of populations of specific TILs in mouse models of cancers with sensitivities and specificities similar to those achieved via flow-cytometric analyses of biopsied tumour tissues. We also show that the probes enable the non-invasive evaluation of the immunogenicity of different tumours, the dynamic monitoring of responses to immunotherapies and the accurate prediction of tumour growth under various treatments.

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Fig. 1: Design and mechanism of TAMPs for the specific molecular optical imaging of TILs.
Fig. 2: In vitro characterization of the detection capabilities of TAMPs.
Fig. 3: Superiority of TAMPs compared with single-locked AMPs.
Fig. 4: In vivo real-time NIRF imaging of TILs in 4T1 tumour-bearing mice.
Fig. 5: In vivo real-time NIRF imaging of TILs in CT26 tumour-bearing mice.
Fig. 6: Prediction of cancer immunotherapy by TAMPs.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

K.P. thanks the Singapore Ministry of Education, Academic Research Fund Tier 1 (RG125/19; RT05/20), Academic Research Fund Tier 2 (MOE2018-T2-2-042; MOE-T2EP30220-0010) and the Singapore National Research Foundation (NRF-NRFI07-2021-0005) for financial support.

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Authors and Affiliations

  1. School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore

    Shasha He, Penghui Cheng & Kanyi Pu

  2. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

    Kanyi Pu

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Contributions

K.P. and S.H. conceived and designed the study. S.H. performed the probe-synthesis experiments and the in vitro, in vivo and ex vivo experiments. S.H. and P.C. performed whole-slide imaging experiments. K.P. and S.H. contributed to the analysis and interpretation of the results and to the writing of the paper.

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Correspondence to Kanyi Pu.

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Nature Biomedical Engineering thanks Yingxiao Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Correlation of R-NIRFs with their respective lymphocytes against CT26 tumours.

a–c, absolute number of TILs per million living cells of CT26 tumours after different treatments. Absolute number of (a) M1 macrophages, (b) CTLs and (c) neutrophils per million living cells. aPD-L1 versus saline, M1 macrophages: P = 0.0182; CTLs: P = 0.0143; neutrophils: P = 0.0034. aCD47 versus saline, M1 macrophages: P = 0.0017; CTLs: P = 0.0189; neutrophils: P = 0.0050. aPD-L1/aCD47 versus saline, M1 macrophages: P = 0.0002; CTLs: P = 0.0046; neutrophils: P = 0.0012. d–f, absolute number of biomarker (enzyme) positive non-target and target cells. d, Absolute number of iNOS−Cas-1+ and iNOS+Cas-1+ cells per million living cells. iNOS−Cas-1+ versus iNOS+Cas-1+ cells, saline: P < 0.00001; aPD-L1: P < 0.00001; aCD47: P < 0.00001; aPD-L1/ aCD47: P < 0.00001. e, Absolute number of CD3−GrB+ and CD3+CD8+GrB+ cells per million living cells. CD3−GrB+ versus CD3+CD8+GrB+ cells, saline: P = 0.0073; aPD-L1: P = 0.0001; aCD47: P = 0.0291; aPD-L1/ aCD47: P < 0.00001. f, Absolute number of Ly-6G−NE+ and Ly-6G+NE+ cells per million living cells. Ly-6G−NE+ versus Ly-6G+NE+ cells, saline: P = 0.0003; aPD-L1: P = 0.0029; aCD47: P = 0.0002; aPD-L1/ aCD47: P = 0.0002. g–i, Correlation between numbers of TILs and R-NIRFs of TAMPs in the tumour regions of CT26 tumour-bearing mice after different treatments. Correlation between (g) number of M1 macrophages per million living cells and R-NIRFM1, (h) number of CTLs per million living cells and R-NIRFCTL, and (i) number of neutrophils per million living cells and R-NIRFNE.

Source data

Extended Data Fig. 2 Antitumour efficacy.

Tumour growth curves (n = 6) of (a) 4T1 tumour-bearing mice and (b) CT26 tumour-bearing mice. For 4T1 tumour-bearing mice: aPD-L1 versus saline: P = 0.0005; Oxa versus saline: P < 0.0001; aPD-L1/Oxa versus saline: P < 0.0001. For CT26 tumour-bearing mice: aPD-L1 versus saline: P < 0.0001; aCD47 versus saline: P = 0.0002; aPD-L1/aCD47 versus saline: P < 0.0001. Body weights curves of (c) 4T1 tumour-bearing mice and (d) CT26 tumour-bearing mice. Overall survival curves (n = 6) of (e) 4T1 tumour-bearing mice and (f) CT26 tumour-bearing mice. For 4T1 tumour-bearing mice, aPD-L1/Oxa versus saline: P = 0.0008. For CT26 tumour-bearing mice, aPD-L1/aCD47 versus saline: P = 0.0005. 4T1 tumour-bearing mice were intraperitoneally injected with saline, Oxa (6 mg/kg), aPD-L1 (10 mg/kg) and aPD-L1/Oxa at day 0, 2 and 4. CT26 tumour-bearing mice were intraperitoneally injected with saline, aCD47 (10 mg/kg), aPD-L1 (10 mg/kg) and aPD-L1/aCD47 at day 0, 2 and 4.

Source data

Extended Data Fig. 3 Correlation between R-NIRFs and tumours.

a, Correlation between urinary R-NIRFs of TAMPs and in vivo R-NIRFs of TAMPs in tumour regions of tumour-bearing mice with different treatments. b, Correlation between TILs and urinary R-NIRFs of TAMPs of tumour-bearing mice with different treatments.

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He, S., Cheng, P. & Pu, K. Activatable near-infrared probes for the detection of specific populations of tumour-infiltrating leukocytes in vivo and in urine. Nat. Biomed. Eng 7, 281–297 (2023). https://doi.org/10.1038/s41551-023-01009-1

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