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Multiplexed imaging in oncology

Abstract

In oncology, technologies for clinical molecular imaging are used to diagnose patients, establish the efficacy of treatments and monitor the recurrence of disease. Multiplexed methods increase the number of disease-specific biomarkers that can be detected simultaneously, such as the overexpression of oncogenic proteins, aberrant metabolite uptake and anomalous blood perfusion. The quantitative localization of each biomarker could considerably increase the specificity and the accuracy of technologies for clinical molecular imaging to facilitate granular diagnoses, patient stratification and earlier assessments of the responses to administered therapeutics. In this Review, we discuss established techniques for multiplexed imaging and the most promising emerging multiplexing technologies applied to the imaging of isolated tissues and cells and to non-invasive whole-body imaging. We also highlight advances in radiology that have been made possible by multiplexed imaging.

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Fig. 1: Combinations of biomarkers for optimal multiplexed imaging.
Fig. 2: Imaging modalities, arranged according to resolution and multiplexing capability.
Fig. 3: Cell-based multiplexed imaging techniques.
Fig. 4: Multiplexed clinical imaging modalities.
Fig. 5: Intraoperative multiplexed fluorescence imaging.
Fig. 6: Optoacoustic imaging techniques discriminate between multiplexed signals.
Fig. 7: Raman imaging with SERS nanoparticles.

References

  1. Heinzmann, K., Carter, L. M., Lewis, J. S. & Aboagye, E. O. Multiplexed imaging for diagnosis and therapy. Nat. Biomed. Eng. 1, 697–713 (2017).

    Article  PubMed  Google Scholar 

  2. Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883–892 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Janku, F. Tumor heterogeneity in the clinic: is it a real problem? Ther. Adv. Med. Oncol. 6, 43–51 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Giedt, R. J. et al. Single-cell barcode analysis provides a rapid readout of cellular signaling pathways in clinical specimens. Nat. Commun. 9, 4550 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Haun, J. B. et al. Micro-NMR for rapid molecular analysis of human tumor samples. Sci. Transl. Med. 3, 71ra16 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Nathan, E. Frenk et al. High-content biopsies facilitate molecular analyses and do not increase complication rates in patients with advanced solid tumors. JCO Precis. Oncol. 1, 1–9 (2017).

    Google Scholar 

  7. Kodack, D. P. et al. Primary patient-derived cancer cells and their potential for personalized cancer patient care. Cell Rep. 21, 3298–3309 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Whitley, M. J. et al. A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer. Sci. Transl. Med. 8, 320ra324 (2016).

    Article  CAS  Google Scholar 

  9. Liao, L. J., Lo, W. C., Hsu, W. L., Cheng, P. W. & Wang, C. P. Assessment of pain score and specimen adequacy for ultrasound-guided fine-needle aspiration biopsy of thyroid nodules. J. Pain. Res 11, 61–66 (2018).

    Article  PubMed  Google Scholar 

  10. Umkehrer, C. et al. Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters. Nat. Biotechnol. https://doi.org/10.1038/s41587-020-0614-0 (2020).

  11. Ullal, A. V. et al. Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates. Sci. Transl. Med. 6, 219ra219 (2014).

    Article  CAS  Google Scholar 

  12. Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Moffitt, J. R. et al. High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proc. Natl Acad. Sci. USA 113, 14456–14461 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang, G., Moffitt, J. R. & Zhuang, X. Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy. Sci. Rep. 8, 4847 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Wu, X., Mao, S., Ying, Y., Krueger, C. J. & Chen, A. K. Progress and challenges for live-cell imaging of genomic loci using CRISPR-based platforms. Genomics Proteom. Bioinform. https://doi.org/10.1016/j.gpb.2018.10.001 (2019).

  16. Im, H. et al. Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone. Proc. Natl Acad. Sci. USA 112, 5613–5618 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pathania, D. et al. Holographic assessment of lymphoma tissue (HALT) for global oncology field applications. Theranostics 6, 1603–1610 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Im, H. et al. Design and clinical validation of a point-of-care device for the diagnosis of lymphoma via contrast-enhanced microholography and machine learning. Nat. Biomed. Eng. 2, 666–674 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Min, J. et al. Computational optics enables breast cancer profiling in point-of-care settings. ACS Nano 12, 9081–9090 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fereidouni, F. et al. Microscopy with ultraviolet surface excitation for rapid slide-free histology. Nat. Biomed. Eng. 1, 957–966 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Orringer, D. A. et al. Rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated Raman scattering microscopy. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-016-0027 (2017).

  22. Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-017-0084 (2017).

  23. Lin, J. R. et al. Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. eLife https://doi.org/10.7554/eLife.31657 (2018).

  24. Gerdes, M. J. et al. Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proc. Natl Acad. Sci. USA 110, 11982–11987 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tanaka, N. et al. Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-020-0576-z (2020).

  27. Richardson, D. S. & Lichtman, J. W. Clarifying tissue clearing. Cell 162, 246–257 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Liebmann, T. et al. Three-dimensional study of Alzheimer’s disease hallmarks using the iDISCO clearing method. Cell Rep. 16, 1138–1152 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cuccarese, M. F. et al. Heterogeneity of macrophage infiltration and therapeutic response in lung carcinoma revealed by 3D organ imaging. Nat. Commun. 8, 14293 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Spraggins, J. M. et al. Next-generation technologies for spatial proteomics: integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis. Proteomics 16, 1678–1689 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Castellino, S., Groseclose, M. R. & Wagner, D. MALDI imaging mass spectrometry: bridging biology and chemistry in drug development. Bioanalysis 3, 2427–2441 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Giesen, C. et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. Methods 11, 417–422 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Angelo, M. et al. Multiplexed ion beam imaging of human breast tumors. Nat. Med. 20, 436–442 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Shen, C. et al. 2D and 3D CT radiomics features prognostic performance comparison in non-small cell lung cancer. Transl. Oncol. 10, 886–894 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Echegaray, S. et al. A rapid segmentation-insensitive “digital biopsy” method for radiomic feature extraction: method and pilot study using CT images of non-small cell lung cancer. Tomography 2, 283–294 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Coursey, C. A. et al. Dual-energy multidetector CT: how does it work, what can it tell us, and when can we use it in abdominopelvic imaging? Radiographics 30, 1037–1055 (2010).

    Article  PubMed  Google Scholar 

  37. McCollough, C. H., Leng, S., Yu, L. & Fletcher, J. G. Dual- and multi-energy CT: principles, technical approaches, and clinical applications. Radiology 276, 637–653 (2015).

    Article  PubMed  Google Scholar 

  38. Yeh, B. M. et al. Opportunities for new CT contrast agents to maximize the diagnostic potential of emerging spectral CT technologies. Adv. Drug Deliv. Rev. 113, 201–222 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Beels, L. et al. Dose-length product of scanners correlates with DNA damage in patients undergoing contrast CT. Eur. J. Radiol. 81, 1495–1499 (2012).

    Article  PubMed  Google Scholar 

  40. Pathe, C. et al. The presence of iodinated contrast agents amplifies DNA radiation damage in computed tomography. Contrast Media Mol. Imaging 6, 507–513 (2011).

    Article  CAS  PubMed  Google Scholar 

  41. Piechowiak, E. I., Peter, J. F., Kleb, B., Klose, K. J. & Heverhagen, J. T. Intravenous iodinated contrast agents amplify DNA radiation damage at CT. Radiology 275, 692–697 (2015).

    Article  PubMed  Google Scholar 

  42. Rothkamm, K., Balroop, S., Shekhdar, J., Fernie, P. & Goh, V. Leukocyte DNA damage after multi-detector row CT: a quantitative biomarker of low-level radiation exposure. Radiology 242, 244–251 (2007).

    Article  PubMed  Google Scholar 

  43. Momose, A., Takeda, T., Itai, Y. & Hirano, K. Phase-contrast X-ray computed tomography for observing biological soft tissues. Nat. Med. 2, 473–475 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Baran, P. et al. Optimization of propagation-based X-ray phase-contrast tomography for breast cancer imaging. Phys. Med. Biol. 62, 2315–2332 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Symons, R. et al. Photon-counting CT for simultaneous imaging of multiple contrast agents in the abdomen: an in vivo study. Med. Phys. 44, 5120–5127 (2017).

    Article  PubMed  Google Scholar 

  46. Trueb, P., Zambon, P. & Broennimann, C. Assessment of the spectral performance of hybrid photon counting X-ray detectors. Med. Phys. 44, e207–e214 (2017).

    Article  PubMed  Google Scholar 

  47. Taguchi, K. & Iwanczyk, J. S. Vision 20/20: single photon counting x-ray detectors in medical imaging. Med. Phys. 40, 100901 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Carter, L. M., Poty, S., Sharma, S. K. & Lewis, J. S. Preclinical optimization of antibody-based radiopharmaceuticals for cancer imaging and radionuclide therapy—model, vector, and radionuclide selection. J. Labelled Comp. Radiopharm. https://doi.org/10.1002/jlcr.3612 (2018).

  49. Cornelis, F. H. et al. Long-half-life (89)Zr-labeled radiotracers can guide percutaneous biopsy within the PET/CT suite without reinjection of radiotracer. J. Nucl. Med. 59, 399–402 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Phillips, E. et al. Clinical translation of an ultrasmall inorganic optical-PET imaging nanoparticle probe. Sci. Transl. Med. 6, 260ra149 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Black, N. F., McJames, S. & Kadrmas, D. J. Rapid multi-tracer PET tumor imaging with F-FDG and secondary shorter-lived tracers. IEEE Trans. Nucl. Sci. 56, 2750–2758 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kadrmas, D. J., Rust, T. C. & Hoffman, J. M. Single-scan dual-tracer FLT+FDG PET tumor characterization. Phys. Med. Biol. 58, 429–449 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Weissleder, R., Schwaiger, M. C., Gambhir, S. S. & Hricak, H. Imaging approaches to optimize molecular therapies. Sci. Transl. Med. 8, 355ps316 (2016).

    Article  CAS  Google Scholar 

  54. Black, K. C. et al. Dual-radiolabeled nanoparticle SPECT probes for bioimaging. Nanoscale 7, 440–444 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Sharir, T. & Slomka, P. Dual-isotope myocardial perfusion SPECT imaging: past, present, and future. J. Nucl. Cardiol. https://doi.org/10.1007/s12350-017-0966-0 (2017).

    Article  PubMed  Google Scholar 

  56. Berg, E., Roncali, E., Kapusta, M., Du, J. & Cherry, S. R. A combined time-of-flight and depth-of-interaction detector for total-body positron emission tomography. Med. Phys. 43, 939–950 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Zhang, X., Zhou, J., Cherry, S. R., Badawi, R. D. & Qi, J. Quantitative image reconstruction for total-body PET imaging using the 2-meter long EXPLORER scanner. Phys. Med. Biol. 62, 2465–2485 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Cherry, S. R. et al. Total-body PET: maximizing sensitivity to create new opportunities for clinical research and patient care. J. Nucl. Med. 59, 3–12 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Wibmer, A. G., Hricak, H., Ulaner, G. A. & Weber, W. Trends in oncologic hybrid imaging. Eur. J. Hybrid. Imaging 2, 1 (2018).

    Article  PubMed  Google Scholar 

  60. Sanguedolce, F. et al. Baseline multiparametric MRI for selection of prostate cancer patients suitable for active surveillance: which features matter? Clin. Genitourin. Cancer https://doi.org/10.1016/j.clgc.2017.10.020 (2017).

  61. Kesch, C. et al. Multiparametric MRI fusion-guided biopsy for the diagnosis of prostate cancer. Curr. Opin. Urol. https://doi.org/10.1097/mou.0000000000000461 (2017).

  62. Brembilla, G. et al. Preoperative multiparametric MRI of the prostate for the prediction of lymph node metastases in prostate cancer patients treated with extended pelvic lymph node dissection. Eur. Radiol. https://doi.org/10.1007/s00330-017-5229-6 (2017).

  63. Ma, D. et al. Magnetic resonance fingerprinting. Nature 495, 187–192 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. European Society of, R. Magnetic resonance fingerprinting - a promising new approach to obtain standardized imaging biomarkers from MRI. Insights Imaging 6, 163–165 (2015).

    Article  Google Scholar 

  65. Harisinghani, M. G. et al. Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N. Engl. J. Med. 348, 2491–2499 (2003).

    Article  PubMed  Google Scholar 

  66. Kircher, M. F. et al. In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic T-lymphocyte trafficking to tumors. Cancer Res. 63, 6838–6846 (2003).

    CAS  PubMed  Google Scholar 

  67. Miller, M. A., Arlauckas, S. & Weissleder, R. Prediction of anti-cancer nanotherapy efficacy by imaging. Nanotheranostics 1, 296–312 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Weissleder, R., Saini, S., Stark, D. D., Wittenberg, J. & Ferrucci, J. T. Dual-contrast MR imaging of liver cancer in rats. AJR Am. J. Roentgenol. 150, 561–566 (1988).

    Article  CAS  PubMed  Google Scholar 

  69. Anderson, C. E. et al. Dual contrast - magnetic resonance fingerprinting (DC-MRF): a platform for simultaneous quantification of multiple MRI contrast agents. Sci. Rep. 7, 8431 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Hurd, R. E., Yen, Y. F., Chen, A. & Ardenkjaer-Larsen, J. H. Hyperpolarized 13C metabolic imaging using dissolution dynamic nuclear polarization. J. Magn. Reson. Imaging 36, 1314–1328 (2012).

    Article  PubMed  Google Scholar 

  71. Nelson, S. J. et al. Metabolic imaging of patients with prostate cancer using hyperpolarized [1-(1)(3)C]pyruvate. Sci. Transl. Med. 5, 198ra108 (2013).

  72. Miloushev, V. Z. et al. Metabolic Imaging of the Human Brain with Hyperpolarized 13C Pyruvate Demonstrates 13C Lactate Production in Brain Tumor Patients. Cancer Res. https://doi.org/10.1158/0008-5472.can-18-0221 (2018).

  73. Wilson, D. M. et al. Multi-compound polarization by DNP allows simultaneous assessment of multiple enzymatic activities in vivo. J. Magn. Reson. 205, 141–147 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Klippel, S., Freund, C. & Schroder, L. Multichannel MRI labeling of mammalian cells by switchable nanocarriers for hyperpolarized xenon. Nano Lett. 14, 5721–5726 (2014).

    Article  CAS  PubMed  Google Scholar 

  75. Koch, M. & Ntziachristos, V. Advancing surgical vision with fluorescence imaging. Annu. Rev. Med. 67, 153–164 (2016).

    Article  CAS  PubMed  Google Scholar 

  76. Yun, S. H. & Kwok, S. J. J. Light in diagnosis, therapy and surgery. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-016-0008 (2017).

  77. Kobayashi, H. et al. Simultaneous multicolor imaging of five different lymphatic basins using quantum dots. Nano Lett. 7, 1711–1716 (2007).

    Article  CAS  PubMed  Google Scholar 

  78. Erogbogbo, F. et al. In vivo targeted cancer imaging, sentinel lymph node mapping and multi-channel imaging with biocompatible silicon nanocrystals. ACS Nano 5, 413–423 (2011).

    Article  CAS  PubMed  Google Scholar 

  79. Behrooz, A. et al. Multispectral open-air intraoperative fluorescence imaging. Opt. Lett. 42, 2964–2967 (2017).

    Article  PubMed  Google Scholar 

  80. Keating, J. et al. Identification of breast cancer margins using intraoperative near-infrared imaging. J. Surg. Oncol. 113, 508–514 (2016).

    Article  CAS  PubMed  Google Scholar 

  81. Keating, J. J. et al. Intraoperative molecular imaging of lung adenocarcinoma can identify residual tumor cells at the surgical margins. Mol. Imaging Biol. 18, 209–218 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Zeng, C. et al. Intraoperative identification of liver cancer microfoci using a targeted near-infrared fluorescent probe for imaging-guided surgery. Sci. Rep. 6, 21959 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. van den Berg, N. S., Buckle, T., KleinJan, G. H., van der Poel, H. G. & van Leeuwen, F. W. B. Multispectral fluorescence imaging during robot-assisted laparoscopic sentinel node biopsy: a first step towards a fluorescence-based anatomic roadmap. Eur. Urol. 72, 110–117 (2017).

    Article  PubMed  Google Scholar 

  84. Miampamba, M. et al. Sensitive in vivo visualization of breast cancer using ratiometric protease-activatable fluorescent imaging agent, AVB-620. Theranostics 7, 3369–3386 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Lamberts, L. E. et al. Tumor-specific uptake of fluorescent Bevacizumab-IRDye800CW microdosing in patients with primary breast cancer: a phase I feasibility study. Clin. Cancer Res. 23, 2730–2741 (2017).

    Article  CAS  PubMed  Google Scholar 

  86. Carney, B., Kossatz, S. & Reiner, T. Molecular imaging of PARP. J. Nucl. Med. 58, 1025–1030 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. van Dam, G. M. et al. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat. Med. 17, 1315–1319 (2011).

    Article  PubMed  CAS  Google Scholar 

  88. Stummer, W. et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 7, 392–401 (2006).

    Article  CAS  PubMed  Google Scholar 

  89. Georges, J. F. et al. Delta-aminolevulinic acid-mediated photodiagnoses in surgical oncology: a historical review of clinical trials. Front. Surg. 6, 45 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  90. Haider, S. A., Lim, S., Kalkanis, S. N. & Lee, I. Y. The impact of 5-aminolevulinic acid on extent of resection in newly diagnosed high grade gliomas: a systematic review and single institutional experience. J. Neurooncol. 141, 507–515 (2019).

    Article  PubMed  Google Scholar 

  91. Lanahan, C. R. et al. Real-time, intraoperative detection of residual breast cancer in lumpectomy cavity margins using the LUM imaging system: results of a feasibility study. Cancer Res. 78 (4 Suppl.), abstr. P2-12-05 (2018).

  92. Mohan, J. F. et al. Imaging the emergence and natural progression of spontaneous autoimmune diabetes. Proc. Natl Acad. Sci. USA 114, E7776–E7785 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Wang, Y. W., Reder, N. P., Kang, S., Glaser, A. K. & Liu, J. T. C. Multiplexed optical imaging of tumor-directed nanoparticles: a review of imaging systems and approaches. Nanotheranostics 1, 369–388 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  94. Ntziachristos, V. & Razansky, D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT). Chem. Rev. 110, 2783–2794 (2010).

    Article  CAS  PubMed  Google Scholar 

  95. Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. Nat. Methods 7, 603–614 (2010).

    Article  CAS  PubMed  Google Scholar 

  96. Stoffels, I. et al. Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging. Sci. Transl. Med. 7, 317ra199 (2015).

    Article  PubMed  CAS  Google Scholar 

  97. Neuschmelting, V., Lockau, H., Ntziachristos, V., Grimm, J. & Kircher, M. F. Lymph node micrometastases and in-transit metastases from melanoma: in vivo detection with multispectral optoacoustic imaging in a mouse model. Radiology 280, 137–150 (2016).

    Article  PubMed  Google Scholar 

  98. Schwarz, M., Buehler, A., Aguirre, J. & Ntziachristos, V. Three-dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo. J. Biophoton. 9, 55–60 (2016).

    Article  CAS  Google Scholar 

  99. Neuschmelting, V. et al. WST11 vascular targeted photodynamic therapy effect monitoring by multispectral optoacoustic tomography (MSOT) in mice. Theranostics 8, 723–734 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Johnson, S. P., Ogunlade, O., Lythgoe, M. F., Beard, P. & Pedley, R. B. Longitudinal photoacoustic imaging of the pharmacodynamic effect of vascular targeted therapy on tumors. Clin. Cancer Res. 25, 7436–7447 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Reshetnyak, Y. K. Imaging tumor acidity: pH-low insertion peptide probe for optoacoustic tomography. Clin. Cancer Res. 21, 4502–4504 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Xie, B. et al. Optoacoustic detection of early therapy-induced tumor cell death using a targeted imaging agent. Clin. Cancer Res. 23, 6893–6903 (2017).

    Article  CAS  PubMed  Google Scholar 

  103. Yin, W. et al. Tumor specific liposomes improve detection of pancreatic adenocarcinoma in vivo using optoacoustic tomography. J. Nanobiotechnol. 13, 90 (2015).

    Article  CAS  Google Scholar 

  104. Banala, S. et al. Quinone-fused porphyrins as contrast agents for photoacoustic imaging. Chem. Sci. 8, 6176–6181 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Roberts, S. A. et al. Sonophore-enhanced nanoemulsions for optoacoustic imaging of cancer. Chem. Sci. https://doi.org/10.1039/C8SC01706A (2018).

  106. Aguirre, J. et al. Precision assessment of label-free psoriasis biomarkers with ultra-broadband optoacoustic mesoscopy. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-017-0068 (2017).

  107. Cheng, J. X. & Xie, X. S. Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine. Science 350, aaa8870 (2015).

    Article  PubMed  CAS  Google Scholar 

  108. Fu, D., Yang, W. & Xie, X. S. Label-free imaging of neurotransmitter acetylcholine at neuromuscular junctions with stimulated raman scattering. J. Am. Chem. Soc. 139, 583–586 (2017).

    Article  CAS  PubMed  Google Scholar 

  109. Lu, F. K. et al. Label-free DNA imaging in vivo with stimulated Raman scattering microscopy. Proc. Natl Acad. Sci. USA 112, 11624–11629 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Zhang, R. R. & Kuo, J. S. Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy. Neurosurgery 78, N9–N11 (2016).

    Article  PubMed  Google Scholar 

  111. Freudiger, C. W. et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322, 1857–1861 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Evans, C. L. et al. Chemically-selective imaging of brain structures with CARS microscopy. Opt. Express 15, 12076–12087 (2007).

    Article  CAS  PubMed  Google Scholar 

  113. Andreou, C., Kishore, S. A. & Kircher, M. F. Surface-enhanced Raman spectroscopy: a new modality for cancer imaging. J. Nucl. Med. 56, 1295–1299 (2015).

    Article  CAS  PubMed  Google Scholar 

  114. Xia, Q., Chen, Z., Zhou, Y. & Liu, R. Near-infrared organic fluorescent nanoparticles for long-term monitoring and photodynamic therapy of cancer. Nanotheranostics 3, 156–165 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  115. Reichel, D., Tripathi, M., Butte, P., Saouaf, R. & Perez, J. M. Tumor-activatable clinical nanoprobe for cancer imaging. Nanotheranostics 3, 196–211 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  116. Wei, L. et al. Fabrication of positively charged fluorescent polymer nanoparticles for cell imaging and gene delivery. Nanotheranostics 2, 157–167 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  117. Li, J. et al. Two-color-based nanoflares for multiplexed micrornas imaging in live cells. Nanotheranostics 2, 96–105 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  118. Choi, D. et al. Iodinated echogenic glycol chitosan nanoparticles for X-ray CT/US dual imaging of tumor. Nanotheranostics 2, 117–127 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  119. Pallaoro, A., Braun, G. B. & Moskovits, M. Biotags based on surface-enhanced Raman can be as bright as fluorescence tags. Nano Lett. 15, 6745–6750 (2015).

    Article  CAS  PubMed  Google Scholar 

  120. Andreou, C. et al. Imaging of liver tumors using surface-enhanced raman scattering nanoparticles. ACS Nano 10, 5015–5026 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Harmsen, S. et al. Rational design of a chalcogenopyrylium-based surface-enhanced resonance Raman scattering nanoprobe with attomolar sensitivity. Nat. Commun. 6, 6570 (2015).

    Article  CAS  PubMed  Google Scholar 

  122. Harmsen, S. et al. Surface-enhanced resonance Raman scattering nanostars for high-precision cancer imaging. Sci. Transl. Med. 7, 271ra277 (2015).

    Article  CAS  Google Scholar 

  123. Nayak, T. R. et al. Tissue factor-specific ultra-bright SERRS nanostars for Raman detection of pulmonary micrometastases. Nanoscale 9, 1110–1119 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Ye, L. et al. Comparing semiconductor nanocrystal toxicity in pregnant mice and non-human primates. Nanotheranostics 3, 54–65 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  125. Karabeber, H. et al. Guiding brain tumor resection using surface-enhanced Raman scattering nanoparticles and a hand-held Raman scanner. ACS Nano 8, 9755–9766 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Kircher, M. F. et al. A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle. Nat. Med. 18, 829–834 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Zavaleta, C. L. et al. Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy. Proc. Natl Acad. Sci. USA 106, 13511–13516 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Oseledchyk, A., Andreou, C., Wall, M. A. & Kircher, M. F. Folate-targeted surface-enhanced resonance raman scattering nanoprobe ratiometry for detection of microscopic ovarian cancer. ACS Nano 11, 1488–1497 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Wang, Y. W. et al. Raman-encoded molecular imaging with topically applied SERS nanoparticles for intraoperative guidance of lumpectomy. Cancer Res. 77, 4506–4516 (2017).

    Article  CAS  PubMed  Google Scholar 

  130. Wang, Y. W. et al. Multiplexed molecular imaging of fresh tissue surfaces enabled by convection-enhanced topical staining with SERS-coded nanoparticles. Small 12, 5612–5621 (2016).

    Article  PubMed  CAS  Google Scholar 

  131. Nicolson, F. et al. Non-invasive in vivo imaging of cancer using Surface-Enhanced Spatially Offset Raman Spectroscopy (SESORS). Theranostics 9, 5899–5913 (2019).

  132. Bohndiek, S. E. et al. A small animal Raman instrument for rapid, wide-area, spectroscopic imaging. Proc. Natl Acad. Sci. USA 110, 12408–12413 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Thomas, G. et al. Evaluating feasibility of an automated 3-dimensional scanner using Raman spectroscopy for intraoperative breast margin assessment. Sci. Rep. 7, 13548 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Garai, E. et al. A real-time clinical endoscopic system for intraluminal, multiplexed imaging of surface-enhanced Raman scattering nanoparticles. PLoS ONE 10, e0123185 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  135. Thakor, A. S. et al. The fate and toxicity of Raman-active silica-gold nanoparticles in mice. Sci. Transl. Med. 3, 79ra33 (2011).

    Article  PubMed  CAS  Google Scholar 

  136. Dubey, R. D. et al. Novel hyaluronic acid conjugates for dual nuclear imaging and therapy in CD44-expressing tumors in mice in vivo. Nanotheranostics 1, 59–79 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  137. Zhang, S., Gupta, S., Fitzgerald, T. J. & Bogdanov, A. A.Jr. Dual radiosensitization and anti-STAT3 anti-proliferative strategy based on delivery of gold nanoparticle—oligonucleotide nanoconstructs to head and neck cancer cells. Nanotheranostics 2, 1–11 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  138. Zhang, Q. et al. Construction of multifunctional Fe3O4-MTX@HBc nanoparticles for MR imaging and photothermal therapy/chemotherapy. Nanotheranostics 2, 87–95 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  139. Liu, R., Tang, J., Xu, Y., Zhou, Y. & Dai, Z. Nano-sized indocyanine green J-aggregate as a one-component theranostic agent. Nanotheranostics 1, 430–439 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  140. Liu, L., Ruan, Z., Yuan, P., Li, T. & Yan, L. Oxygen self-sufficient amphiphilic polypeptide nanoparticles encapsulating BODIPY for potential near infrared imaging-guided photodynamic therapy at low energy. Nanotheranostics 2, 59–69 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  141. Lin, S. Y., Huang, R. Y., Liao, W. C., Chuang, C. C. & Chang, C. W. Multifunctional PEGylated albumin/IR780/iron oxide nanocomplexes for cancer photothermal therapy and MR imaging. Nanotheranostics 2, 106–116 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  142. Gupta, M. K. et al. Recent strategies to design vascular theranostic nanoparticles. Nanotheranostics 1, 166–177 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  143. Thurber, G. M., Figueiredo, J. L. & Weissleder, R. Multicolor fluorescent intravital live microscopy (FILM) for surgical tumor resection in a mouse xenograft model. PLoS ONE 4, e8053 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  144. Herzog, E. et al. Optical imaging of cancer heterogeneity with multispectral optoacoustic tomography. Radiology 263, 461–468 (2012).

    Article  PubMed  Google Scholar 

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Acknowledgements

The work of R.W. was supported by the National Institute of Health grants R33CA202064, UH2CA202637, RO1CA204019, RO1CA206890 and UO1CA206997. The work of M.F.K. was supported by the National Institute of Health grants R01EB017748 and R01CA222836, a Pershing Square Sohn Prize, and the Parker Institute for Cancer Immunotherapy. This work is dedicated to the memory of Moritz F. Kircher, our colleague, student, mentor and friend.

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Correspondence to Ralph Weissleder.

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R.W. has received consultancy payments from ModeRNA, Tarveda Pharmaceuticals, Alivio Therapeutics and Accure Health, and is a shareholder of T2Biosystems, Lumicell and Accure Health. All patents associated with R.W. have been assigned to and are handled by the Massachusetts General Hospital. M.F.K is a co-founder of RIO Imaging, which did not contribute to this manuscript. All patents associated with M.F.K. have been assigned to and are handled by Stanford University or Memorial Sloan Kettering Cancer Center, respectively. C.A. declares no competing interests.

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Andreou, C., Weissleder, R. & Kircher, M.F. Multiplexed imaging in oncology. Nat. Biomed. Eng 6, 527–540 (2022). https://doi.org/10.1038/s41551-022-00891-5

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