Intraoperative diagnostics and elimination of residual microtumours with plasmonic nanobubbles

Journal name:
Nature Nanotechnology
Volume:
11,
Pages:
525–532
Year published:
DOI:
doi:10.1038/nnano.2015.343
Received
Accepted
Published online

Abstract

Failure of cancer surgery to intraoperatively detect and eliminate microscopic residual disease (MRD) causes lethal recurrence and metastases, and the removal of important normal tissues causes excessive morbidity. Here, we show that a plasmonic nanobubble (PNB), a non-stationary laser pulse-activated nanoevent, intraoperatively detects and eliminates MRD in the surgical bed. PNBs were generated in vivo in head and neck cancer cells by systemically targeting tumours with gold colloids and locally applying near-infrared, low-energy short laser pulses, and were simultaneously detected with an acoustic probe. In mouse models, between 3 and 30 residual cancer cells and MRD (undetectable with current methods) were non-invasively detected up to 4 mm deep in the surgical bed within 1 ms. In resectable MRD, PNB-guided surgery prevented local recurrence and delivered 100% tumour-free survival. In unresectable MRD, PNB nanosurgery improved survival twofold compared with standard surgery. Our results show that PNB-guided surgery and nanosurgery can rapidly and precisely detect and remove MRD in simple intraoperative procedures.

At a glance

Figures

  1. Mechanism of PNB diagnostics of residual microtumours and cancer cells in vivo.
    Figure 1: Mechanism of PNB diagnostics of residual microtumours and cancer cells in vivo.

    a, Systemic delivery of gold conjugates to the tumour via their leaky vasculature. b, Accumulation of gold conjugates by receptors of cancer cells (gold shown with white arrows in illustrative scanning electron microscopy images41). c, Intracellular clustering of gold conjugates via receptor-mediated endocytosis (illustrative transmission electron microscopy images27). A gold cluster, on exposure to a single laser pulse of low fluence, selectively generates a PNB only in cancer cells; normal cells with non-specifically internalized single gold nanoparticles do not generate PNBs because of the higher threshold of PNB generation. d, The acoustic signal of a PNB (illustrative red time response) reports even a single cancer cell in solid tissue, but not normal cells (illustrative green time response).

  2. PNBs report even single cancer cells in vitro in transparent medium and in tissue.
    Figure 2: PNBs report even single cancer cells in vitro in transparent medium and in tissue.

    ac, Simultaneously detected optical scattering and acoustic time responses to single laser pulses (782 nm, 30 ps) applied to individual HNSCC cancer cells in transparent medium: PNB-positive optical time response in a gold conjugate-pretreated cell (red) and PNB-negative time response in an intact (not pretreated with gold) cell (black) (a); acoustic time response in gold-pretreated (red) and intact (black) cells (b); amplitude of the bipolar PNB-specific spike in the acoustic time response as a function of PNB lifetime obtained in the same cells under the variable-fluence laser pulse (10–66 mJ cm–2), where the black horizontal line shows the level of the noise and background, and the dashed red line shows the linear fit above the detection threshold (c). d, Experimental setup with an endoscope (grey), injection needle (silver) and the acoustic sensor (gold) applied to chicken breast (scale bar, 10 mm). e, Acoustic time responses before (black) and after injection (red) of three gold conjugate-pretreated cells at a tissue depth of 1 mm. f, Diagnostic Index (relative increase in the amplitude of the test time response versus that of the reference, cancer-free time response) as a function of the number of gold conjugate-pretreated cells injected at a tissue depth of 1 mm (filled symbols) or 3–4 mm (open symbols). Data are mean ± standard error. Error bars are based on six measurements under identical conditions.

  3. Intraoperative non-invasive detection of cancer cells in a surgical bed in vivo with a single laser pulse.
    Figure 3: Intraoperative non-invasive detection of cancer cells in a surgical bed in vivo with a single laser pulse.

    Laser pulse: 782 nm, 30 ps, 70 mJ cm–2. a, Experimental setup with an endoscope, cell injection needle and the acoustic sensor applied to a surgical bed (scale bar, 5 mm). b, Acoustic time responses before (black) and after (red) injection of three gold conjugate-pretreated cancer cells at a tissue depth of 1 mm. c, Acoustic time responses before (black) and after (red) injection of ten gold conjugate-pretreated cancer cells at a tissue depth of 1 mm. d, Acoustic time responses before (black) and after (red) injection of ten intact (untreated with gold conjugates) cancer cells at a tissue depth of 1 mm. e, Diagnostic Index (relative increase in the amplitude of the test time response versus that of the reference, cancer-free time response) as a function of the number of injected cells for gold conjugate-pretreated (red) and intact (black) cancer cells at a depth of 1 mm. Data are mean ± standard error. Error bars are based on six measurements under identical conditions.

  4. Biodistribution and toxicity in vivo after systemic administration of gold conjugates reveal a safe and efficient accumulation of gold nanoparticles in a tumour.
    Figure 4: Biodistribution and toxicity in vivo after systemic administration of gold conjugates reveal a safe and efficient accumulation of gold nanoparticles in a tumour.

    a, Biodistribution for anti-human (solid red, Panitumumab antibody) and anti-mouse (hashed grey, ab231 antibody) gold conjugates with antibody against epidermal growth factor receptor (EGFR) in nude (gold-Panitumumab conjugates) and normal mice (gold-ab234 conjugates) (n = 3). bg, Histological analysis of organs obtained from intact (bd) and gold conjugate-injected (eg) animals 72 h after administration of gold conjugates: liver (b,e), lung (c,f), kidney (d,g) (scale bar, 100 µm). h, Bodyweight as a function of time for intact mice (black) and mice after systemic injection of gold conjugates with Panitumumab antibody (red). Data are mean ± standard error. Error bars are based on six mice per group.

  5. PNBs intraoperatively detect MRD in a surgical bed and guide its resection in real time with standard surgery.
    Figure 5: PNBs intraoperatively detect MRD in a surgical bed and guide its resection in real time with standard surgery.

    a, Image of the surgical bed after primary surgery. T, MRD-positive location in the nest of the primary tumour; R, MRD-negative location with normal tissue. b, Acoustic time responses to single laser pulses (782 nm, 30 ps, 70 mJ cm–2) obtained immediately after primary surgery at the ‘T’ location of possible MRD (red) and at the MRD-negative location ‘R’ (black). c, Image of the surgical bed after PNB-guided surgery. d, Acoustic time responses obtained after PNB-guided surgery in the location of the secondary resections (green) and in the initially MRD-negative location (black).

  6. PNBs improve surgical outcome in both resectable and unresectable MRDs.
    Figure 6: PNBs improve surgical outcome in both resectable and unresectable MRDs.

    The animal group-averaged metrics of local recurrent tumours after standard surgery with resectable MRD (blue, n = 6), PNB-guided surgery of resectable MRD (green, n = 5) and PNB nanosurgery of unresectable MRD (red, n = 6) show a significant improvement in the outcome in both resectable and unresectable cases when the surgery is enhanced with PNBs. a, Tumour volume. b, Animal survival. c, Histograms of the Diagnostic Index obtained in MRD-positive (red) and -negative (black) locations after standard surgery and for MRD-positive locations after PNB-guided surgery (green). d, Recurrent tumour volumes plotted for the group-averaged Diagnostic Indices after standard (blue) and PNB-guided (green) surgery show the prognostic potential of PNBs to intraoperatively predict tumour recurrence. Data are mean ± standard error. Error bars are based on six (blue and red) or five (green) mice per group.

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Author information

  1. These authors contributed equally to this work

    • Ekaterina Y. Lukianova-Hleb &
    • Yoo-Shin Kim

Affiliations

  1. Department of BioSciences at Rice, Rice University, Houston, Texas 77005, USA

    • Ekaterina Y. Lukianova-Hleb &
    • Dmitri O. Lapotko
  2. Department of Translational Imaging, Methodist Hospital Research Institute, Houston, Texas 77030, USA

    • Yoo-Shin Kim &
    • Brian E. O'Neill
  3. N.N. Alexandrov National Cancer Centre of Belarus, Minsk 223040, Belarus

    • Ihor Belatsarkouski
  4. Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    • Ann M. Gillenwater

Contributions

E.Y.L.H. conducted PNB experiments, prepared the figures and wrote the manuscript. Y.S.K. conducted the animal experiments and collected animal data. I.B., A.M.G., D.O.L., B.E.O. and E.Y.L.H. discussed the experimental design and results, and clinical applications of the technology. B.E.O. contributed to the conceptual experimental design and organized the animal handling and monitoring. D.O.L. developed the technology and research strategy, designed the experimental setup and wrote the manuscript.

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The authors declare no competing financial interests.

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